http://2011.igem.org/wiki/index.php?title=Special:Contributions/Nickpv&feed=atom&limit=50&target=Nickpv&year=&month=2011.igem.org - User contributions [en]2024-03-29T13:46:13ZFrom 2011.igem.orgMediaWiki 1.16.0http://2011.igem.org/Team:UNIPV-Pavia/FreezerTeam:UNIPV-Pavia/Freezer2011-11-09T13:30:19Z<p>Nickpv: </p>
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Freezer Management<br />
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<p>For a detailed correspondence between our BioBrick "wiki names" and submitted BioBrick codes take a look <a href="https://2011.igem.org/Team:UNIPV-Pavia/Parts/Submitted#freezer" >here</a>.<br />
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<table class="data"><br />
<tr><br />
<td class="row"><b>Biobrick name</b></td><br />
<td class="row"><b>Description</b></td><br />
<td class="row"><b>Store</b></td><br />
<td class="row"><b>Length</b></td><br />
<td class="row"><b>Strain</b></td><br />
<td class="row"><b>Vector</b></td><br />
<td class="row"><b>Quality Control</b></td><br />
<td class="row"><b>Creation Date</b></td><br />
</tr><br />
<br />
<tr><br />
<td class="row"><a href="http://partsregistry.org/Part:BBa_K081022">BBa_K081022</a></td><br />
<td class="row">Plambda-RBS30-luxR-T(B1006)-pLux</td><br />
<td class="row">BioBricks 2011</td><br />
<td class="row">969<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (MIT)</td><br />
<td class="row">29/06/2011</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row"><a href="http://partsregistry.org/Part:BBa_C0060">BBa_C0060</a></td><br />
<td class="row">(aiiA) autoinducer inactivation enzyme</td><br />
<td class="row">BioBricks 2011</td><br />
<td class="row">789<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (MIT)</td><br />
<td class="row">29/06/2011</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row"><a href="http://partsregistry.org/Part:BBa_B0015">BBa_B0015</a></td><br />
<td class="row">(TT) double terminator</td><br />
<td class="row">2009</td><br />
<td class="row">129</td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1AK3</td><br />
<td class="row">Seq ok (MIT)</td><br />
<td class="row">FREEZER 2009 Dist</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row"><a href="http://partsregistry.org/Part:BBa_R0040">BBa_R0040</a></td><br />
<td class="row">(pTet) TetR repressible promoter</td><br />
<td class="row">2009</td><br />
<td class="row">54</td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (MIT)</td><br />
<td class="row">FREEZER 2009 Dist</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row"><a href="http://partsregistry.org/Part:BBa_C0061">BBa_C0061</a></td><br />
<td class="row">(luxI) autoinducer synthetase for AHL</td><br />
<td class="row">BioBricks 2011</td><br />
<td class="row">618<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (MIT)</td><br />
<td class="row">30/06/2011</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row"><a href="http://partsregistry.org/Part:BBa_I13501">BBa_I13501</a></td><br />
<td class="row">(mRFP-TT) Screening plasmid intermediate</td><br />
<td class="row">requested from the Registry</td><br />
<td class="row">843<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1AK3</td><br />
<td class="row">Seq ok (MIT)</td><br />
<td class="row">FREEZER Requested 2011</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row"><a href="http://partsregistry.org/Part:BBa_B0030">BBa_B0030</a></td><br />
<td class="row">RBS30 (eff=0.6)</td><br />
<td class="row">2009</td><br />
<td class="row">15</td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (MIT)</td><br />
<td class="row">FREEZER 2009 Dist</td><br />
</tr><br />
<br />
<br />
<br />
<tr><br />
<td class="row"><a href="http://partsregistry.org/Part:BBa_B0031">BBa_B0031</a></td><br />
<td class="row">RBS31 (eff=0.07) derivative of BBa_0030</td><br />
<td class="row">2009</td><br />
<td class="row">14</td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (MIT)</td><br />
<td class="row">FREEZER 2009 Dist</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row"><a href="http://partsregistry.org/Part:BBa_B0032">BBa_B0032</a></td><br />
<td class="row">RBS32 (eff=0.3) / also used as non-fluorescent control in TECAN measurements (RBS)</td><br />
<td class="row">2009</td><br />
<td class="row">13</td><br />
<td class="row">E. coli TOP10 / E. coli MGZ1</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (MIT)</td><br />
<td class="row">FREEZER 2009 Dist</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row"><a href="http://partsregistry.org/Part:BBa_B0034">BBa_B0034</a></td><br />
<td class="row">RBS34 (eff=1)</td><br />
<td class="row">2009</td><br />
<td class="row">12</td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (MIT)</td><br />
<td class="row">FREEZER 2009 Dist</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row"><a href="http://partsregistry.org/Part:BBa_I13521">BBa_I13521</a></td><br />
<td class="row">(pTet-RBS34-RFP-TT) Ptet mRFP Constitutive on mRFP</td><br />
<td class="row">2010</td><br />
<td class="row">923<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A3</td><br />
<td class="row">Seq ok (MIT)</td><br />
<td class="row">FREEZER 2010 Dist</td><br />
</tr><br />
<br />
<br />
<br />
<tr><br />
<td class="row"><a href="http://partsregistry.org/Part:BBa_C0261">BBa_C0261</a></td><br />
<td class="row">(RBS34-luxI) AHL-making Enzyme, luxI (+RBS)</td><br />
<td class="row">BioBricks 2011</td><br />
<td class="row">661<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (MIT)</td><br />
<td class="row">07/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row"><a href="http://partsregistry.org/Part:BBa_I13507">BBa_I13507</a></td><br />
<td class="row">(RBS34-mRFP-TT) Screening plasmid intermediate</td><br />
<td class="row">2009</td><br />
<td class="row">861<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (MIT)</td><br />
<td class="row">FREEZER 2009 Dist</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row"><a href="http://partsregistry.org/Part:pSB4C5">pSB4C5</a></td><br />
<td class="row">Low copy BioBrick standard vector - chloramphenicol antibiotic resistance</td><br />
<td class="row">2009</td><br />
<td class="row">3221</td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Seq ok (MIT)</td><br />
<td class="row">FREEZER 2009 Dist</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row"><a href="http://partsregistry.org/Part:pSB3K3">pSB3K3</a></td><br />
<td class="row">Low to medium copy BioBrick standard vector - kanamycin resistance</td><br />
<td class="row">BioBricks 2011</td><br />
<td class="row">2750</td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB3K3</td><br />
<td class="row">Seq ok (MIT)</td><br />
<td class="row">30/06/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row"><a href="http://partsregistry.org/Part:BBa_K300005">BBa_K3000005</a></td><br />
<td class="row">GFP (Silver Standard Prefix) with terminator</td><br />
<td class="row">2010</td><br />
<td class="row">854</td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1C3</td><br />
<td class="row">Seq ok (BMR Gemonics)</td><br />
<td class="row">FREEZER 2010 Ligations</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row"><a href="http://partsregistry.org/Part:BBa_K112808">BBa_K112808</a> in <a href="http://partsregistry.org/Part:pSB4C5">pSB4C5</a></td><br />
<td class="row">Enterobacteria phage T4 Lysis Device - no promoter / also used as non-fluorescent control in TECAN measurements (ENTERO4C5)</td><br />
<td class="row">2009</td><br />
<td class="row">1785</td><br />
<td class="row">E. coli TOP10 / E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Seq ok (BMR Gemonics)</td><br />
<td class="row">FREEZER 2009 Ligations</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">T9002</td><br />
<td class="row"><a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a></td><br />
<td class="row">2009</td><br />
<td class="row">1945</td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (MIT)</td><br />
<td class="row">2009 Registry Distribution</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row">ENTERO-RBS</td><br />
<td class="row"><a href="http://partsregistry.org/Part:BBa_K112808">BBa_K112808</a> in <a href="http://partsregistry.org/Part:pSB4C5">pSB4C5</a> cotransformed with <a href="http://partsregistry.org/Part:BBa_B0032">BBa_B0032</a> / also used as non-fluorescent control in TECAN measurements</td><br />
<td class="row">2011</td><br />
<td class="row">1785 - 13</td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2 - pSB4C5</td><br />
<td class="row">Seq ok</td><br />
<td class="row">FREEZER 2011 Ligations</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">T9002-ENTERO</td><br />
<td class="row"><a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cotransformed with <a href="http://partsregistry.org/Part:BBa_K112808">BBa_K112808</a> in <a href="http://partsregistry.org/Part:pSB4C5">pSB4C5</a> / used as measurement device in TECAN measurements</td><br />
<td class="row">2011</td><br />
<td class="row">1945 - 1785</td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2 - pSB4C5</td><br />
<td class="row">Seq ok</td><br />
<td class="row">FREEZER 2011 Ligations</td><br />
</tr><br />
<br />
</table><br />
<br />
<br><br />
<br><br />
<br />
<table class="data"><br />
<tr><br />
<td class="row"><b>Biobrick name</b></td><br />
<td class="row"><b>Description</b></td><br />
<td class="row"><b>Ligation</b></td><br />
<td class="row"><b>Length</b></td><br />
<td class="row"><b>Strain</b></td><br />
<td class="row"><b>Vector</b></td><br />
<td class="row"><b>Quality Control</b></td><br />
<td class="row"><b>Creation Date</b></td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E1-2</td><br />
<td class="row">aiiA-TT</td><br />
<td class="row">C0060(E-S)+B0015(E-X)</td><br />
<td class="row">951<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1AK3</td><br />
<td class="row">Length OK</td><br />
<td class="row">07/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E2-2</td><br />
<td class="row">RBS30-LuxI</td><br />
<td class="row">C0061(X-P)+B0030(S-P)</td><br />
<td class="row">664<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Length OK</td><br />
<td class="row">07/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E3-1</td><br />
<td class="row">RBS31-LuxI</td><br />
<td class="row">C0061(X-P)+B0031(S-P)</td><br />
<td class="row">663<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Length OK</td><br />
<td class="row">07/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E4-2</td><br />
<td class="row">RBS32-LuxI</td><br />
<td class="row">C0061(X-P)+B0032(S-P)</td><br />
<td class="row">662<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Length OK</td><br />
<td class="row">07/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E5-2</td><br />
<td class="row">RBS30-mRFP-TT</td><br />
<td class="row">I13501(X-P)+B0030(S-P)</td><br />
<td class="row">864<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Length OK</td><br />
<td class="row">07/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E6-1</td><br />
<td class="row">RBS31-mRFP-TT</td><br />
<td class="row">I13501(X-P)+B0031(S-P)</td><br />
<td class="row">863<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Length OK</td><br />
<td class="row">07/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E7-2</td><br />
<td class="row">RBS32-mRFP-TT</td><br />
<td class="row">I13501(X-P)+B0032(S-P)</td><br />
<td class="row">862<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Length OK</td><br />
<td class="row">07/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E8-3</td><br />
<td class="row">Plambda-RBS30-luxR-T(B1006)-pLux</td><br />
<td class="row">K081022(E-P)+4C5(E-P)</td><br />
<td class="row">969<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Length OK</td><br />
<td class="row">07/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E9-2</td><br />
<td class="row">RBS30-aiiA-TT</td><br />
<td class="row">E1(X-P)+B0030(S-P)</td><br />
<td class="row">972<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">13/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E10-1</td><br />
<td class="row">RBS31-aiiA-TT</td><br />
<td class="row">E1(X-P)+B0031(S-P)</td><br />
<td class="row">971<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">13/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E11-1</td><br />
<td class="row">RBS32-aiiA-TT</td><br />
<td class="row">E1(X-P)+B0032(S-P)</td><br />
<td class="row">970<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">14/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E12-1</td><br />
<td class="row">RBS34-aiiA-TT</td><br />
<td class="row">E1(X-P)+ B0034(S-P)</td><br />
<td class="row">969<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">14/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E13-1</td><br />
<td class="row">pTet-RBS30-LuxI</td><br />
<td class="row">E2(X-P)+R0040(S-P)</td><br />
<td class="row">726<sup>*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">13/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E14</td><br />
<td class="row">pTet-RBS31-LuxI</td><br />
<td class="row">E3(X-P)+R0040(S-P)</td><br />
<td class="row">725<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row"></td><br />
<td class="row">FAILED</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E15</td><br />
<td class="row">pTet-RBS32-LuxI</td><br />
<td class="row">E4(X-P)+R0040(S-P)</td><br />
<td class="row">724<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1A2</td><br />
<td class="row"></td><br />
<td class="row">FAILED</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E16-1</td><br />
<td class="row">pTet-RBS34-LuxI</td><br />
<td class="row">C0261(X-P)+R0040(S-P)</td><br />
<td class="row">723<sup>*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">13/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E17-2</td><br />
<td class="row">Plambda-RBS30-luxR-T(B1006)-pLux-RBS30-mRFP-TT</td><br />
<td class="row">E5(X-P)+E8(S-P)</td><br />
<td class="row">1841<sup>*</sup></td><br />
<td class="row">E. coli TOP10/ E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">13/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E18-2</td><br />
<td class="row">Plambda-RBS30-luxR-T(B1006)-pLux-RBS31-mRFP-TT</td><br />
<td class="row">E6(X-P)+E8(S-P)</td><br />
<td class="row">1840<sup>*</sup></td><br />
<td class="row">E. coli TOP10/ E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">13/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E19-2</td><br />
<td class="row">Plambda-RBS30-luxR-T(B1006)-pLux-RBS32-mRFP-TT</td><br />
<td class="row">E7(X-P)+E8(S-P)</td><br />
<td class="row">1839<sup>*</sup></td><br />
<td class="row">E. coli TOP10/ E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">13/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E20-2</td><br />
<td class="row">Plambda-RBS30-luxR-T(B1006)-pLux-RBS34-mRFP-TT</td><br />
<td class="row">I13507(X-P)+E8(S-P)</td><br />
<td class="row">1838<sup>*</sup></td><br />
<td class="row">E. coli TOP10/ E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">13/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E21-1</td><br />
<td class="row">pTet-RBS30-mRFP-TT</td><br />
<td class="row">E5(X-P)+R0040(S-P)</td><br />
<td class="row">926<sup>*</sup></td><br />
<td class="row">E. coli TOP10/ E. coli MGZ1</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">14/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E22-2</td><br />
<td class="row">pTet-RBS31-mRFP-TT</td><br />
<td class="row">E6(X-P)+R0040(S-P)</td><br />
<td class="row">925<sup>*</sup></td><br />
<td class="row">E. coli TOP10/ E. coli MGZ1</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">13/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E23-1</td><br />
<td class="row">pTet-RBS32-mRFP-TT</td><br />
<td class="row">E7(X-P)+R0040(S-P)</td><br />
<td class="row">924<sup>*</sup></td><br />
<td class="row">E. coli TOP10/ E. coli MGZ1</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">14/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row"><br />
<a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0040">BBa_R0040</a> in <a href="http://partsregistry.org/wiki/index.php/Part:BBa_J61002">BBa_J61002</a></td><br />
<td class="row">pTet in BBa_J61002 plasmid</td><br />
<td class="row">BBa_R0040(S-P)+BBa_J23101(S-P)</td><br />
<td class="row">923<sup>*</sup></td><br />
<td class="row">E. coli TOP10/ E. coli MGZ1</td><br />
<td class="row">BBa_J61002</td><br />
<td class="row">Length OK</td><br />
<td class="row">15/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E24-2</td><br />
<td class="row">pTet-RBS30-aiiA-TT</td><br />
<td class="row">E9(X-P)+R0040(S-P)</td><br />
<td class="row">1034<sup>*</sup></td><br />
<td class="row">E. coli TOP10/ E. coli MGZ1</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">21/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E25-1</td><br />
<td class="row">pTet-RBS31-aiiA-TT</td><br />
<td class="row">E10(X-P)+R0040(S-P)</td><br />
<td class="row">1033<sup>*</sup></td><br />
<td class="row">E. coli TOP10/ E. coli MGZ1</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">21/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E26-2</td><br />
<td class="row">pTet-RBS32-aiiA-TT</td><br />
<td class="row">E11(X-P)+R0040(S-P)</td><br />
<td class="row">1032<sup>*</sup></td><br />
<td class="row">E. coli TOP10/ E. coli MGZ1</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">21/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E27-2</td><br />
<td class="row">pTet-RBS34-aiiA-TT</td><br />
<td class="row">E12(X-P)+R0040(S-P)</td><br />
<td class="row">1031<sup>*</sup></td><br />
<td class="row">E. coli TOP10/ E. coli MGZ1</td><br />
<td class="row">pSB1A2</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">21/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E28-1</td><br />
<td class="row">pTet-RBS30-luxI in pSB4C5</td><br />
<td class="row">E13(E-P)+pSB4C5(E-P)</td><br />
<td class="row">726<sup>*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Bad Seq (BMR Genomics)</td><br />
<td class="row">21/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E29</td><br />
<td class="row">pTet-RBS31-luxI in pSB4C5</td><br />
<td class="row">E14(E-P)+pSB4C5(E-P)</td><br />
<td class="row">725<sup>*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row"></td><br />
<td class="row">FAILED</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E30</td><br />
<td class="row">pTet-RBS32-luxI in pSB4C5</td><br />
<td class="row">E15(E-P)+pSB4C5(E-P)</td><br />
<td class="row">724<sup>*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row"></td><br />
<td class="row">FAILED</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E31-1</td><br />
<td class="row">pTet-RBS34-luxI in pSB4C5</td><br />
<td class="row">E16(E-P)+pSB4C5(E-P)</td><br />
<td class="row">723<sup>*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">21/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E32-2</td><br />
<td class="row">pTet-RBS30-mRFP-TT in pSB4C5</td><br />
<td class="row">E21(E-P)+pSB4C5(E-P)</td><br />
<td class="row">926<sup>*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">21/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E33-2</td><br />
<td class="row">pTet-RBS31-mRFP-TT in pSB4C5</td><br />
<td class="row">E22(E-P)+pSB4C5(E-P)</td><br />
<td class="row">925<sup>*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">21/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E34-1</td><br />
<td class="row">pTet-RBS32-mRFP-TT in pSB4C5</td><br />
<td class="row">E23(E-P)+pSB4C5(E-P)</td><br />
<td class="row">924<sup>*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">21/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E35-2</td><br />
<td class="row">pTet-RBS34-mRFP-TT in pSB4C5</td><br />
<td class="row">I13521(E-P)+pSB4C5(E-P)</td><br />
<td class="row">923<sup>*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">21/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E36</td><br />
<td class="row">pTet-J61002 in pSB4C5</td><br />
<td class="row">pTet-J61002(E-P)+pSB4C5(E-P)</td><br />
<td class="row">923<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Length ok</td><br />
<td class="row">25/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E37-2</td><br />
<td class="row">pTet-RBS30-aiiA-TT in pSB4C5</td><br />
<td class="row">E24(E-P)+pSB4C5(E-P)</td><br />
<td class="row">1034<sup>*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">29/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E38-1</td><br />
<td class="row">pTet-RBS31-aiiA-TT in pSB4C5</td><br />
<td class="row">E25(E-P)+pSB4C5(E-P)</td><br />
<td class="row">1033<sup>*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">29/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E39-1</td><br />
<td class="row">pTet-RBS32-aiiA-TT in pSB4C5</td><br />
<td class="row">E26(E-P)+pSB4C5(E-P)</td><br />
<td class="row">1032<sup>*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">29/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E40-2</td><br />
<td class="row">pTet-RBS34-aiiA-TT in pSB4C5</td><br />
<td class="row">E27(E-P)+pSB4C5(E-P)</td><br />
<td class="row">1031<sup>*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">29/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E41N-1</td><br />
<td class="row">pTet-RBS31-luxI in pSB4C5</td><br />
<td class="row">E36(S-P)+E3(X-P)</td><br />
<td class="row">725<sup>*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">04/08/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E42-1</td><br />
<td class="row">pTet-RBS32-luxI in pSB4C5</td><br />
<td class="row">E36(S-P)+ E4(X-P)</td><br />
<td class="row">724<sup>*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">29/07/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E43-3</td><br />
<td class="row">pTet-RBS30-luxI in pSB4C5</td><br />
<td class="row">E36(S-P)+ E2(X-P)</td><br />
<td class="row">726<sup>*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">25/08/2011</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row">E44-1</td><br />
<td class="row">pTet-RBS34-mRFP-TT in pSB1C3</td><br />
<td class="row"><a href="http://partsregistry.org/wiki/index.php/Part:BBa_R0040">BBa_R0040</a> in <a href="http://partsregistry.org/wiki/index.php/Part:BBa_J61002">BBa_J61002</a> (E-P) + pSB1C3 (E-P)</td><br />
<td class="row">923<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1C3</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">04/09/2011</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row">J101-E5</td><br />
<td class="row">J23101-RBS30-mRFP-TT in pSB4C5</td><br />
<td class="row">BBa_J23101(S-P)+ E5(S-P)</td><br />
<td class="row">905*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Length OK</td><br />
<td class="row">02/08/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">J101-31</td><br />
<td class="row">J23101-RBS31-mRFP-TT in pSB4C5</td><br />
<td class="row">BBa_J23101(S-P)+ E6(S-P)</td><br />
<td class="row">904*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Length OK</td><br />
<td class="row">02/08/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">J101-E7</td><br />
<td class="row">J23101-RBS32-mRFP-TT in pSB4C5</td><br />
<td class="row">BBa_J23101(S-P)+ E7(S-P)</td><br />
<td class="row">903*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Length OK</td><br />
<td class="row">02/08/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">J101-4C5</td><br />
<td class="row">J23101-RBS34-mRFP-TT in pSB4C5</td><br />
<td class="row">BBa_J23101(E-P)+ pSB4C5(E-P)</td><br />
<td class="row">902*</sup></td><br />
<td class="row">E. coli MGZ1</td><br />
<td class="row">pSB4C5</td><br />
<td class="row">Length OK</td><br />
<td class="row">02/08/2011</td><br />
</tr><br />
<br />
<br />
<br />
<br />
<tr><br />
<td class="row">E3O-1C3-3</td><br />
<td class="row">RBS31-LuxI</td><br />
<td class="row">E3-1(E-P)+ pSB1C3 (E-P)</td><br />
<td class="row">663<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1C3</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">07/09/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E4N-1C3-3</td><br />
<td class="row">RBS32-LuxI</td><br />
<td class="row">E4-2(E-P)+ pSB1C3 (E-P)</td><br />
<td class="row">662<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1C3</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">11/09/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E5-1C3</td><br />
<td class="row">RBS30-mRFP-TT</td><br />
<td class="row">E5-2(E-P)+ pSB1C3 (E-P)</td><br />
<td class="row">864<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1C3</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">29/08/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E6-1C3</td><br />
<td class="row">RBS31-mRFP-TT</td><br />
<td class="row">E6-1(E-P)+ pSB1C3 (E-P)</td><br />
<td class="row">863<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1C3</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">29/08/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E7-1C3</td><br />
<td class="row">RBS32-mRFP-TT</td><br />
<td class="row">E7-2(E-P)+ pSB1C3 (E-P)</td><br />
<td class="row">862<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1C3</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">29/08/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E9-1C3</td><br />
<td class="row">RBS30-aiiA-TT</td><br />
<td class="row">E9-2(E-P)+ pSB1C3 (E-P)</td><br />
<td class="row">972<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1C3</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">29/08/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E10-1C3</td><br />
<td class="row">RBS31-aiiA-TT</td><br />
<td class="row">E10-1(E-P)+ pSB1C3 (E-P)</td><br />
<td class="row">971<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1C3</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">29/08/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">E11-1C3</td><br />
<td class="row">RBS32-aiiA-TT</td><br />
<td class="row">E11-1(E-P)+ pSB1C3 (E-P)</td><br />
<td class="row">970<sup>*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1C3</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">29/08/2011</td><br />
</tr><br />
<br />
<br />
<br />
<tr><br />
<td class="row">J101-E5-1C3</td><br />
<td class="row">J23101-RBS30-mRFP-TT</td><br />
<td class="row">J101-E5 (E-P) + pSB1C3(E-P)</td><br />
<td class="row">905*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1C3</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">29/08/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">J101-31-C3-1</td><br />
<td class="row">J23101-RBS31-mRFP-TT</td><br />
<td class="row">J101-31 (E-P) + pSB1C3(E-P)</td><br />
<td class="row">904*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1C3</td><br />
<td class="row">Seq ok (BMR Genomics)</td><br />
<td class="row">04/09/2011</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">J101-E7-1C3-1</td><br />
<td class="row">J23101-RBS32-mRFP-TT</td><br />
<td class="row">J101-E7 (E-P) + pSB1C3(E-P)</td><br />
<td class="row">903*</sup></td><br />
<td class="row">E. coli TOP10</td><br />
<td class="row">pSB1C3</td><br />
<td class="row">Seq OK (BMR Gemonics)</td><br />
<td class="row">29/08/2011</td><br />
</tr><br />
<br />
<br />
</table><br />
</center><br />
<br />
<br />
<p><br />
<div align="center"><sup>*</sup> The sequences of these parts may contain at least a 25 bp <a href="http://partsregistry.org/Help:Barcodes">barcode</a>.</div><br />
<br />
<br />
</html><br />
<br />
{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Project/ResultsTeam:UNIPV-Pavia/Project/Results2011-09-21T18:01:25Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<br />
<br />
<html><br />
<h2 class="art-postheader"><br />
Results<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#assembly"><span class="tocnumber">1</span> <span class="toctext">Part assembly</span></a> <br />
<li class="toclevel-1"><a href="#characterization"><span class="tocnumber">1</span> <span class="toctext">Characterization of basic modules</span></a> <br />
<ul><br />
<li class="toclevel-2"><a href="#promoters"><span class="tocnumber">2.1</span> <span class="toctext">Promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#enzymes"><span class="tocnumber">2.2</span> <span class="toctext">Characterization of the activity of the enzymes AiiA and LuxI</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.3</span> <span class="toctext">Characterization of RBS efficiency</span></a></li><br />
</ul><br />
<li class="toclevel-1"><a href="#growth"><span class="tocnumber">3</span> <span class="toctext">Identification of bacterial growth parameters</span></a></li><br />
<li class="toclevel-1"><a href="#HSL"><span class="tocnumber">4</span> <span class="toctext">Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</span></a></li><br />
<li class="toclevel-1"><a href="#t9002"><span class="tocnumber">5</span> <span class="toctext">Characterization of BBa_T9002 biosensor</span></a></li><br />
</ul></td></tr></table><br />
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<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C. For the cloning of the parts, <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#TOP10'><em>E. coli</em> TOP10</a> was used. <br />
</em><br />
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<a name='assembly'></a><h1>Parts assembly</h1><br />
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All the parts have been cloned with success. The part name, plasmids and quality controls are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Freezer'>Freezer section</a>. <br />
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<a name='characterization'></a><h1>Characterization of basic modules</h1><br />
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<!--------pTet and pLux-----------><br />
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<a name='promoters'></a><h2>Characterization of promoters pTet and pLux</h2><br />
<br />
<p align='justify'><br />
<p>Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection.</p><br />
<p>The assembled RBSs are:</p><br />
<br><br />
<div align='center'><br />
<table class='data'><br />
<tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr><br />
<tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr><br />
<tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr><br />
<tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr><br />
<tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr><br />
</table></div><br />
<br><br />
<div align="justify"><p>For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.</p><br />
<p>The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. <br />
RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP).</p><br />
<p>For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.</p><br />
</p><br />
<p>Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least squares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs:</p><p></p><br />
<p><ol><ul><li><b><br />
RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity</p><br />
</li><p><li><b><br />
RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity</p><br />
</li><p><li><br />
<b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and it is representative of the position of linear region</p><br />
</li><br />
<p><li><br />
<b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</div></li></p><br />
</ul></ol><br />
</p><br />
<br />
<br />
<p align='justify'><br />
The estimated parameters for the Hill functions of pLux are summarized in the table below. For more details on parameter estimation, see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>model section</a>. <br />
</p><br />
<br />
<table class='data' width='100%'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Lux</sub></sub> [(AUr/min)/cell]</b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Lux</sub></sub> [-]</b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Lux</sub></sub> [-]</b></td><br />
<td class="row"><b>k<sub>p<sub>Lux</sub></sub> [ng/ml]</b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">438 [10]</td><br />
<td class="row">0.05 [>100]</td><br />
<td class="row">2 [47]</td><br />
<td class="row">1.88 [27]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">9.8 [7]</td><br />
<td class="row">0.11 [57]</td><br />
<td class="row">1.2 [29]</td><br />
<td class="row">1.5 [26]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">206 [3]</td><br />
<td class="row">0 [>>100]</td><br />
<td class="row">1.36 [10]</td><br />
<td class="row">1.87 [9]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">1105 [6]</td><br />
<td class="row">0.02 [>100]</td><br />
<td class="row">1.33 [19]</td><br />
<td class="row">2.34 [18]</td><br />
</tr><br />
</table><br />
<div align="center">Data are provided as average [CV%].</div><br><br />
<br />
<p>The operative parameters are summarized in the table below:</p></div><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>4.28</td><td class='row'>0.20</td><td class='row'>1.08</td><td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>4.93</td><td class='row'>0.55</td><td class='row'>0.25</td><td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>9.49</td><td class='row'>0.02</td><td class='row'>0.47</td><td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>21.53</td><td class='row'>0.51</td><td class='row'>0.53</td><td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/5/50/E17_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/5/50/E17_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/c/c4/E18_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c4/E18_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/7/78/E19_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/78/E19_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/5/55/E20_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/5/55/E20_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<p>The protocols for the characterization of p<sub>Tet</sub> promoter are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#pTet_protocol'>p<sub>Tet</sub> measurement section</a>.</p><br />
<p><br />
Here we have characterized its transcriptional strength as a function of aTc induction (ng/ul) for different RBSs. Three different induction curves were obtained and are reported in figure:</p></div><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<br />
<br />
<table width='100%'><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/9/99/E34_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/9/99/E34_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/e/e4/E35_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e4/E35_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<p align='justify'><br />
The estimated parameters of the Hill curves described in the figures are summarized in the table below:<br />
</p><br />
<br><br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub> [(AUr/min)/cell]</b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub> [-]</b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub> [-]</b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub> [nM]</b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">230.67 [3.7]</td><br />
<td class="row">0.028 [91.61]</td><br />
<td class="row">4.61 [23.73]</td><br />
<td class="row">8.75 [4.16]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">55.77 [12]</td><br />
<td class="row">1.53E-11 [>>100]</td><br />
<td class="row">4.98 [57.62]</td><br />
<td class="row">7.26 [14.98]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">120 [5.95]</td><br />
<td class="row">0.085 [40.6]</td><br />
<td class="row">24.85 [47.6]</td><br />
<td class="row">9 [5.43]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br><br><br />
<br />
<br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [ng/ml]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [ng/ml]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>1.53</td><td class='row'>~0</td><td class='row'>7.95</td><td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>3.16</td><td class='row'>~0</td><td class='row'>6.7</td><td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>2.73</td><td class='row'>0.23</td><td class='row'>8.96</td><td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
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<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!--------AiiA and LuxI-----------><br />
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<a name='enzymes'></a><h2>Characterization of enzymes AiiA and LuxI</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002'>modelling section</a>) using the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>p<sub>Tet</sub>-RBSx-LuxI-TT</a> measurement systems. <br />
<br><br />
The parameters V<sub>max</sub>, k<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>p<sub>Tet</sub>-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
<br />
<div align="justify"><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div></div><br />
<br />
<br />
<div align="justify"><p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p></div><br />
<br />
<br />
<div align=center"><br />
<table align="center" class='data'><br />
<tr><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>k<sub>M,LuxI</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>3.56*10<sup>-9</sup></td><br />
<td class='row'>6.87*10<sup>3</sup></td><br />
<td class='row'> 87 </td><br />
<td class='row'>8.5</td><br />
<td class='row'>ND</td><br />
<td class='row'> 252 </td><br />
</tr></table><br />
</div><br />
<br> <br />
</p><br />
</p><br />
<br />
<p align='justify'><br />
The provided parameters k<sub>M</sub> and V<sub>max</sub> represent the enzymatic activity of LuxI, described by our model. They must not be confused with the operative parameters of the Michaelis-Menten relation. <br />
These synthetic parameters have a great importance, since they can be used in more complicated models in order to predict the behavior of complex circuits.<br />
</p><br />
<br />
<p align='justify'><br />
The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity.<br />
</p><br />
<br />
<p align='justify'><br />
The parameters k<sub>cat</sub>, k<sub>M,AiiA</sub> and &alpha;<sub>RBSx</sub> would have been estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>p<sub>Tet</sub>-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
Unfortunately, their estimation revealed difficult.<br><br />
In the first experiments with the measurement system <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a> in LOW-COPY at pH=7 no degradation of HSL was observed. The collected data are shown in the figure below. HSL degradation is identical in the measurement system and in the negative control after 21 hours.<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<div align="justify"><br />
Experiments on these parts gave us the opportunity to characterize only the activity of the enzyme in <em>E. COLI</em> TOP10 in high copy number plasmid, providing only some information about the order of magnitude of the model parameters, which has been designed to work in <em>E. COLI</em> MGZ1 in low copy number plasmid.<br />
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<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!--------------------------------><br />
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<!--------------RBS---------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
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<br />
<a name='rbs'></a><h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
<br />
RBSs were used for the fine tuning of CTRL+E. Different experimental conditions were assayed.<br />
<br><br><br />
<br />
Estimated efficiencies in pSB4C5 plasmid with -RBSx-mRFP-TT coding sequence under the control of the specified promoter:<br />
<br><br><br />
<br />
<div align="center"><table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>p<sub>Lux</sub></sub></b></td><br />
<td class='row'><b>eff<sub>p<sub>Tet</sub></sub></b></td><br />
<td class='row'><b>eff<sub>J23101</sub></b></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>0.40</td><td class='row'>1.6814</td><td class='row'>2.45</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.01</td><td class='row'>ND</td><td class='row'>0.04</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.19</td><td class='row'>0.4193</td><td class='row'>0.40</td><td class='row'>0,3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table></div><br />
<br><br><br />
Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT, with GeneX=mRFP, AiiA or LuxI:<br />
<br><br><br />
<br />
<div align="center"><table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>mRFP</sub></b></td><br />
<td class='row'><b>eff<sub>AiiA</sub></b></td><br />
<td class='row'><b>eff<sub>LuxI</sub></b></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>1.72</td><td class='row'>0.53</td><td class='row'>0.45</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.03</td><td class='row'>0.83</td><td class='row'>0.028</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.37</td><td class='row'>0.50</td><td class='row'>N.D.</td><td class='row'>0.3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table></div><br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-----------growth---------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<a name='growth'></a><h2>Identification of bacterial growth parameters</h2><br />
<br />
<div class="listcircle"><br />
<p align='justify'><br />
<br />
The bacterial growth curve has been modelled as a logistic curve and is represented by the following equation:<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br><br />
N(0)=n<sub>0</sub><br />
<br><br><br />
<br />
where &mu; represents the growth rate of the cells (<em>E. coli</em> MGZ1 in M9 supplemented medium) and N<sub>max</sub> represents the maximum number of cells in the well. For a detailed description of the parameters, see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>modelling section</a>. For details on parameters identification, see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#N'>identification section.</a> <br />
The growth curves in all the performed experiments are measured in O.D.<sub>600</sub>. Since the <em>N</em> species in the model is expressed in <em>cell number</em>, a conversion factor has been estimated. The conversion factor <b>K<sub>O.D.toC.F.U.</sub></b> has been estimated as follows.<br />
<ol><ul><br />
<li>Two cultures C1 and C2 (MGZ1 cells) were grown in 1ml M9 medium till saturation (ON liquid culture, 37°C, 220 rpm).</li><br />
<li>Next morning, both C1 and C2 were diluted in M9 medium with a final volume of 1ml with the following dilution factors:<br />
<ul><li>1:1</li><li>1:10</li><li>1:100</li><li>1:1000</li><br />
</ul> in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm.<br />
</li><br />
<li>After 1 hour, O.D.<sub>600</sub> was measured using TECAN microplate reader (don't forget to measure a M9 sample for blanking!)<br><br />
<em>NB: from now on, cultures must be placed in ice to stop cell growth.</em></li><br />
<li>At the same time, proper dilution of the cultures were plated on LB agar plates.<br><br />
<em>NB: All the dilutions are performed moving 100 &mu;l of culture in previously ice-chilled 900 &mu;l fresch M9. 100 &mu;l of the final dilution are plated (It still represents a 1:10 dilution!)</em></li><br />
<li>Plates were grown overnight and next morning C.F.U. were counted.</li><br />
<li>C.F.U. values were corrected by the dilution factor and a linear regression (N vs O.D.<sub>600</sub>) was performed in order to evaluate the conversion factor <b>K<sub>O.D.toC.F.U.</sub></b>. </li><br />
<li><b>K<sub>O.D.toC.F.U.</sub></b> was used as conversion factor to multiply the O.D.<sub>600</sub> value of saturation in the growth curves (~0,5). </li><br />
<br />
</ul></ol><br />
<br />
<p>The results are summarized in the table and in the figure below: </p><br />
<br />
<center><br />
<table class='data'><tr><td class='row'><b> Culture </b></td><td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><td class='row'><b> C.F.U. </b></td><td class='row'><b> Dilution Factor (10^) </b></td><td class='row'><b> N </b></td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,367950002 </td><td class='row'> 0,758004416 </td><td class='row'> 990 </td><td class='row'> 5 </td><td class='row'> 990000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,044649998 </td><td class='row'> 0,091982322 </td><td class='row'> 141 </td><td class='row'> 6 </td><td class='row'> 1410000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,004799999 </td><td class='row'> 0,009888356 </td><td class='row'> 136 </td><td class='row'> 5 </td><td class='row'> 136000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,000549998 </td><td class='row'> 0,001133037 </td><td class='row'> 20 </td><td class='row'> 6 </td><td class='row'> 200000000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,54840003 </td><td class='row'> 1,129744917 </td><td class='row'> 165 </td><td class='row'> 4 </td><td class='row'> 16500000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,058200002 </td><td class='row'> 0,119896339 </td><td class='row'> 23 </td><td class='row'> 5 </td><td class='row'> 23000000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,008700002 </td><td class='row'> 0,017922652 </td><td class='row'> 251 </td><td class='row'> 3 </td><td class='row'> 2510000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,000100002 </td><td class='row'> 0,000206011 </td><td class='row'> 24 </td><td class='row'> 4 </td><td class='row'> 2400000 </td> </tr><br />
</table></center><br><br />
</p><br />
<br />
<center><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"><br />
</center><br />
<br />
<p>The estimation of &mu; parameter was performed by determining the slope of the logarithmic curve of O.D.<sub>600</sub> in exponential phase. Exponential phase was determined by visual inspection as the linear phase of the logarithmic curve of O.D.<sub>600</sub>. <br />
<br><br><br />
The estimated parameters are summarized in the table below:<br />
<br><br />
</p><br />
<center><br />
<table width='50%' class='data'><tr><br />
<td class='row'><b>N<sub>max</sub> [cell number]</b></td><br />
<td class='row'><b>&mu; [min<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><td class='row'>1*10<sup>9</sup></td><br />
<td class='row'>0.004925</td><br />
</tr><br />
</table><br />
</center><br />
<p>&nbsp;</p><br />
<p>The reported value of &mu; corresponds to a doubling time of 142 minutes.<br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------HSL---------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<a name='HSL'></a><h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'><br />
In order to estimate the spontaneous degradation rate of HSL in M9 medium and in a culture of MGZ1 cells as a function of pH, two simple tests have been performed.<br><br />
Two different M9 media were prepared, one with the nominal pH (7.0) and one with pH=6.0.<br><br />
These media, now named respectively M9<sub>pH 7</sub> and M9<sub>pH 6</sub>, were added with a known concentration of HSL (100 nM) and then incubated at 37°C, 220 rpm (NB: the media were not infected with any culture but the standard growth conditions were reproduced). The amount of HSL present in the medium was assayed through the BBa_T9002 biosensor at 4 time points: <br><br />
<ul><br />
<li>t=0 h;</li><br />
<li>t=1 h;</li><br />
<li>t=2 h;</li><br />
<li>t=4 h;</li><br />
<li>t=8 h;</li><br />
</ul><br />
<p>The obtained time series of HSL amounts were processed to evaluate the time constant governing the dynamic of HSL degradation, supposing an exponential decay. The results are reported in the table below: </p><br />
<br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><br />
</td><br />
<td class='row'><br />
<b>t<sub>1/2</sub><sup>*</sup> [h]</b><br />
</td><td class='row'><br />
<b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b><br />
</td><br />
</tr><br />
<tr><td class='row'><b>M9<sub>pH 6</sub></b><br />
</td><br />
<td class='row'>32<br />
</td><br />
<td class='row'>0.022<br />
</td><br />
</tr><br />
<tr><td class='row'><b>M9<sub>pH 7</sub></b><br />
</td><br />
<td class='row'>8<br />
</td><br />
<td class='row'>0.087<br />
</td><br />
</tr><br />
</table><br />
</center><br />
<p><br />
The described experiment was repeated with a MGZ1 culture in order to evaluate the effect of culture on HSL stability. The estimated values are reported in the table below:</p><br />
<br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><br />
</td><br />
<td class='row'><br />
<b>t<sub>1/2</sub><sup>*</sup> [h]</b><br />
</td><td class='row'><br />
<b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b><br />
</td><br />
</tr><br />
<tr><td class='row'><b>Culture<sub>pH 6</sub></b><br />
</td><br />
<td class='row'>&#8734;<br />
</td><br />
<td class='row'>0<br />
</td><br />
</tr><br />
<tr><td class='row'><b>Culture<sub>pH 7</sub></b><br />
</td><br />
<td class='row'>19<br />
</td><br />
<td class='row'>0.037<br />
</td><br />
</tr><br />
</table><br />
</center><br />
<br />
<p>It is evident from the reported data that the spontaneous degradation of HSL is negligible when CTRL+E is implemented in MGZ1 cells grown in M9 medium with pH=6.0 and pH=7.0.<br><br />
Thus in the simulations we set &gamma;<sub>HSL</sub>=0; <br />
<br />
</p><br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-------------t9002--------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='t9002'></a><h2>Characterization of BBa_T9002 biosensor</h2><br />
As described in the <a href="https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002">modelling section</a>, BioBrick <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> is an HSL biosensor, which provides a non linear relationship between HSL input and S<sub>cell</sub> output. More precisely, the characteristic sigmoidal curve requires synthetic parameters for its accurate identification. These are the minimum and maximum values, the swtich point (i.e., the curve inflection point), and the upper and lower boundaries of linearity. This biosensor revealed greatly reliable, providing measurement repeatability and minimal experimental noise. Referring to its activation formula, the calibration curve is shown below.<br><br><br />
<br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage" width="47%" height="50%"></a></div></div><br />
<br><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage" width="100%"></a></div></div><br />
<br />
<br />
<center><br />
<table width='50%' class='data'><tr><br />
<td class='row'><b>Minimum [S<sub>cell</sub>]</b></td><br />
<td class='row'><b>Maximum [S<sub>cell</sub>]</b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Lower boundary of linearity [nM]</b></td><br />
<td class='row'><b>Upper boundary of linearity [nM]</b></td><br />
</tr><br />
<br />
<tr><br />
<td class='row'>17.31</td><br />
<td class='row'>739.4</td><br />
<td class='row'>1.39</td><br />
<td class='row'>0.38</td><br />
<td class='row'>5.07</td><br />
<br />
</tr><br />
</table><br />
</center><br />
<br><br><br />
In order to determine the threshold sensitivity of T9002 biosensor, experiments were performed with several HSL inductions minimally interspaced in the region of low detectability. Hypothesizing that the inducer is 1:20 diluted (as for all of our tests), the minimum detectable HSL concentration is 3 nM.<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
</div><br />
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</html><br />
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<br />
{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Project/ResultsTeam:UNIPV-Pavia/Project/Results2011-09-21T17:57:26Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<br />
<br />
<html><br />
<h2 class="art-postheader"><br />
Results<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<br />
<br />
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<!--------------------------------><br />
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<!-----------MENU-----------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br><br />
<a name='indice'></a><br />
<br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#assembly"><span class="tocnumber">1</span> <span class="toctext">Part assembly</span></a> <br />
<li class="toclevel-1"><a href="#characterization"><span class="tocnumber">1</span> <span class="toctext">Characterization of basic modules</span></a> <br />
<ul><br />
<li class="toclevel-2"><a href="#promoters"><span class="tocnumber">2.1</span> <span class="toctext">Promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#enzymes"><span class="tocnumber">2.2</span> <span class="toctext">Characterization of the activity of the enzymes AiiA and LuxI</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.3</span> <span class="toctext">Characterization of RBS efficiency</span></a></li><br />
</ul><br />
<li class="toclevel-1"><a href="#growth"><span class="tocnumber">3</span> <span class="toctext">Identification of bacterial growth parameters</span></a></li><br />
<li class="toclevel-1"><a href="#HSL"><span class="tocnumber">4</span> <span class="toctext">Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</span></a></li><br />
<li class="toclevel-1"><a href="#t9002"><span class="tocnumber">5</span> <span class="toctext">Characterization of BBa_T9002 biosensor</span></a></li><br />
</ul></td></tr></table><br />
</div><br />
<script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script><br />
<br />
<br />
<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C. For the cloning of the parts, <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#TOP10'><em>E. coli</em> TOP10</a> was used. <br />
</em><br />
<br><br />
<br><br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-----------ASSEMBLY-------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<br />
<a name='assembly'></a><h1>Parts assembly</h1><br />
<br />
All the parts have been cloned with success. The part name, plasmids and quality controls are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Freezer'>Freezer section</a>. <br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-------CHARACTERIZATION---------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='characterization'></a><h1>Characterization of basic modules</h1><br />
<br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------pTet and pLux-----------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<a name='promoters'></a><h2>Characterization of promoters pTet and pLux</h2><br />
<br />
<p align='justify'><br />
<p>Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection.</p><br />
<p>The assembled RBSs are:</p><br />
<br><br />
<div align='center'><br />
<table class='data'><br />
<tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr><br />
<tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr><br />
<tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr><br />
<tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr><br />
<tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr><br />
</table></div><br />
<br><br />
<div align="justify"><p>For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.</p><br />
<p>The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. <br />
RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP).</p><br />
<p>For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.</p><br />
</p><br />
<p>Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least squares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs:</p><p></p><br />
<p><ol><ul><li><b><br />
RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity</p><br />
</li><p><li><b><br />
RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity</p><br />
</li><p><li><br />
<b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and it is representative of the position of linear region</p><br />
</li><br />
<p><li><br />
<b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</div></li></p><br />
</ul></ol><br />
</p><br />
<br />
<br />
<p align='justify'><br />
The estimated parameters for the Hill functions of pLux are summarized in the table below. For more details on parameter estimation, see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>model section</a>. <br />
</p><br />
<br />
<table class='data' width='100%'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Lux</sub></sub> [(AUr/min)/cell]</b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Lux</sub></sub> [-]</b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Lux</sub></sub> [-]</b></td><br />
<td class="row"><b>k<sub>p<sub>Lux</sub></sub> [ng/ml]</b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">438 [10]</td><br />
<td class="row">0.05 [>100]</td><br />
<td class="row">2 [47]</td><br />
<td class="row">1.88 [27]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">9.8 [7]</td><br />
<td class="row">0.11 [57]</td><br />
<td class="row">1.2 [29]</td><br />
<td class="row">1.5 [26]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">206 [3]</td><br />
<td class="row">0 [>>100]</td><br />
<td class="row">1.36 [10]</td><br />
<td class="row">1.87 [9]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">1105 [6]</td><br />
<td class="row">0.02 [>100]</td><br />
<td class="row">1.33 [19]</td><br />
<td class="row">2.34 [18]</td><br />
</tr><br />
</table><br />
<div align="center">Data are provided as average [CV%].</div><br><br />
<br />
<p>The operative parameters are summarized in the table below:</p></div><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>4.28</td><td class='row'>0.20</td><td class='row'>1.08</td><td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>4.93</td><td class='row'>0.55</td><td class='row'>0.25</td><td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>9.49</td><td class='row'>0.02</td><td class='row'>0.47</td><td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>21.53</td><td class='row'>0.51</td><td class='row'>0.53</td><td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/5/50/E17_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/5/50/E17_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/c/c4/E18_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c4/E18_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/7/78/E19_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/78/E19_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/5/55/E20_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/5/55/E20_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<p>The protocols for the characterization of p<sub>Tet</sub> promoter are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#pTet_protocol'>p<sub>Tet</sub> measurement section</a>.</p><br />
<p><br />
Here we have characterized its transcriptional strength as a function of aTc induction (ng/ul) for different RBSs. Three different induction curves were obtained and are reported in figure:</p></div><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<br />
<br />
<table width='100%'><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/9/99/E34_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/9/99/E34_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/e/e4/E35_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e4/E35_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<p align='justify'><br />
The estimated parameters of the Hill curves described in the figures are summarized in the table below:<br />
</p><br />
<br><br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub> [(AUr/min)/cell]</b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub> [-]</b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub> [-]</b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub> [nM]</b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">230.67 [3.7]</td><br />
<td class="row">0.028 [91.61]</td><br />
<td class="row">4.61 [23.73]</td><br />
<td class="row">8.75 [4.16]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">55.77 [12]</td><br />
<td class="row">1.53E-11 [>>100]</td><br />
<td class="row">4.98 [57.62]</td><br />
<td class="row">7.26 [14.98]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">120 [5.95]</td><br />
<td class="row">0.085 [40.6]</td><br />
<td class="row">24.85 [47.6]</td><br />
<td class="row">9 [5.43]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br><br><br />
<br />
<br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [ng/ml]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [ng/ml]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>1.53</td><td class='row'>~0</td><td class='row'>7.95</td><td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>3.16</td><td class='row'>~0</td><td class='row'>6.7</td><td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>2.73</td><td class='row'>0.23</td><td class='row'>8.96</td><td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------AiiA and LuxI-----------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='enzymes'></a><h2>Characterization of enzymes AiiA and LuxI</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002'>modelling section</a>) using the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>p<sub>Tet</sub>-RBSx-LuxI-TT</a> measurement systems. <br />
<br><br />
The parameters V<sub>max</sub>, k<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>p<sub>Tet</sub>-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
<br />
<div align="justify"><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div></div><br />
<br />
<br />
<div align="justify"><p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p></div><br />
<br />
<br />
<div align=center"><br />
<table align="center" class='data'><br />
<tr><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>k<sub>M,LuxI</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>3.56*10<sup>-9</sup></td><br />
<td class='row'>6.87*10<sup>3</sup></td><br />
<td class='row'> 87 </td><br />
<td class='row'>8.5</td><br />
<td class='row'>ND</td><br />
<td class='row'> 252 </td><br />
</tr></table><br />
</div><br />
<br> <br />
</p><br />
</p><br />
<br />
<p align='justify'><br />
The provided parameters k<sub>M</sub> and V<sub>max</sub> represent the enzymatic activity of LuxI, described by our model. They must not be confused with the operative parameters of the Michaelis-Menten relation. <br />
These synthetic parameters have a great importance, since they can be used in more complicated models in order to predict the behavior of complex circuits.<br />
</p><br />
<br />
<p align='justify'><br />
The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity.<br />
</p><br />
<br />
<p align='justify'><br />
The parameters k<sub>cat</sub>, k<sub>M,AiiA</sub> and &alpha;<sub>RBSx</sub> would have been estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>p<sub>Tet</sub>-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
Unfortunately, their estimation revealed difficult.<br><br />
In the first experiments with the measurement system <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a> in LOW-COPY at pH=7 no degradation of HSL was observed. The collected data are shown in the figure below. HSL degradation is identical in the measurement system and in the negative control after 21 hours.<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<div align="justify"><br />
Experiments on these parts gave us the opportunity to characterize only the activity of the enzyme in <em>E. COLI</em> TOP10 in high copy number plasmid, providing only some information about the order of magnitude of the model parameters, which has been designed to work in <em>E. COLI</em> MGZ1 in low copy number plasmid.<br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------RBS---------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='rbs'></a><h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-----------growth---------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<a name='growth'></a><h2>Identification of bacterial growth parameters</h2><br />
<br />
<div class="listcircle"><br />
<p align='justify'><br />
<br />
The bacterial growth curve has been modelled as a logistic curve and is represented by the following equation:<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br><br />
N(0)=n<sub>0</sub><br />
<br><br><br />
<br />
where &mu; represents the growth rate of the cells (<em>E. coli</em> MGZ1 in M9 supplemented medium) and N<sub>max</sub> represents the maximum number of cells in the well. For a detailed description of the parameters, see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>modelling section</a>. For details on parameters identification, see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#N'>identification section.</a> <br />
The growth curves in all the performed experiments are measured in O.D.<sub>600</sub>. Since the <em>N</em> species in the model is expressed in <em>cell number</em>, a conversion factor has been estimated. The conversion factor <b>K<sub>O.D.toC.F.U.</sub></b> has been estimated as follows.<br />
<ol><ul><br />
<li>Two cultures C1 and C2 (MGZ1 cells) were grown in 1ml M9 medium till saturation (ON liquid culture, 37°C, 220 rpm).</li><br />
<li>Next morning, both C1 and C2 were diluted in M9 medium with a final volume of 1ml with the following dilution factors:<br />
<ul><li>1:1</li><li>1:10</li><li>1:100</li><li>1:1000</li><br />
</ul> in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm.<br />
</li><br />
<li>After 1 hour, O.D.<sub>600</sub> was measured using TECAN microplate reader (don't forget to measure a M9 sample for blanking!)<br><br />
<em>NB: from now on, cultures must be placed in ice to stop cell growth.</em></li><br />
<li>At the same time, proper dilution of the cultures were plated on LB agar plates.<br><br />
<em>NB: All the dilutions are performed moving 100 &mu;l of culture in previously ice-chilled 900 &mu;l fresch M9. 100 &mu;l of the final dilution are plated (It still represents a 1:10 dilution!)</em></li><br />
<li>Plates were grown overnight and next morning C.F.U. were counted.</li><br />
<li>C.F.U. values were corrected by the dilution factor and a linear regression (N vs O.D.<sub>600</sub>) was performed in order to evaluate the conversion factor <b>K<sub>O.D.toC.F.U.</sub></b>. </li><br />
<li><b>K<sub>O.D.toC.F.U.</sub></b> was used as conversion factor to multiply the O.D.<sub>600</sub> value of saturation in the growth curves (~0,5). </li><br />
<br />
</ul></ol><br />
<br />
<p>The results are summarized in the table and in the figure below: </p><br />
<br />
<center><br />
<table class='data'><tr><td class='row'><b> Culture </b></td><td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><td class='row'><b> C.F.U. </b></td><td class='row'><b> Dilution Factor (10^) </b></td><td class='row'><b> N </b></td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,367950002 </td><td class='row'> 0,758004416 </td><td class='row'> 990 </td><td class='row'> 5 </td><td class='row'> 990000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,044649998 </td><td class='row'> 0,091982322 </td><td class='row'> 141 </td><td class='row'> 6 </td><td class='row'> 1410000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,004799999 </td><td class='row'> 0,009888356 </td><td class='row'> 136 </td><td class='row'> 5 </td><td class='row'> 136000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,000549998 </td><td class='row'> 0,001133037 </td><td class='row'> 20 </td><td class='row'> 6 </td><td class='row'> 200000000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,54840003 </td><td class='row'> 1,129744917 </td><td class='row'> 165 </td><td class='row'> 4 </td><td class='row'> 16500000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,058200002 </td><td class='row'> 0,119896339 </td><td class='row'> 23 </td><td class='row'> 5 </td><td class='row'> 23000000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,008700002 </td><td class='row'> 0,017922652 </td><td class='row'> 251 </td><td class='row'> 3 </td><td class='row'> 2510000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,000100002 </td><td class='row'> 0,000206011 </td><td class='row'> 24 </td><td class='row'> 4 </td><td class='row'> 2400000 </td> </tr><br />
</table></center><br><br />
</p><br />
<br />
<center><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"><br />
</center><br />
<br />
<p>The estimation of &mu; parameter was performed by determining the slope of the logarithmic curve of O.D.<sub>600</sub> in exponential phase. Exponential phase was determined by visual inspection as the linear phase of the logarithmic curve of O.D.<sub>600</sub>. <br />
<br><br><br />
The estimated parameters are summarized in the table below:<br />
<br><br />
</p><br />
<center><br />
<table width='50%' class='data'><tr><br />
<td class='row'><b>N<sub>max</sub> [cell number]</b></td><br />
<td class='row'><b>&mu; [min<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><td class='row'>1*10<sup>9</sup></td><br />
<td class='row'>0.004925</td><br />
</tr><br />
</table><br />
</center><br />
<p>&nbsp;</p><br />
<p>The reported value of &mu; corresponds to a doubling time of 142 minutes.<br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------HSL---------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<a name='HSL'></a><h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'><br />
In order to estimate the spontaneous degradation rate of HSL in M9 medium and in a culture of MGZ1 cells as a function of pH, two simple tests have been performed.<br><br />
Two different M9 media were prepared, one with the nominal pH (7.0) and one with pH=6.0.<br><br />
These media, now named respectively M9<sub>pH 7</sub> and M9<sub>pH 6</sub>, were added with a known concentration of HSL (100 nM) and then incubated at 37°C, 220 rpm (NB: the media were not infected with any culture but the standard growth conditions were reproduced). The amount of HSL present in the medium was assayed through the BBa_T9002 biosensor at 4 time points: <br><br />
<ul><br />
<li>t=0 h;</li><br />
<li>t=1 h;</li><br />
<li>t=2 h;</li><br />
<li>t=4 h;</li><br />
<li>t=8 h;</li><br />
</ul><br />
<p>The obtained time series of HSL amounts were processed to evaluate the time constant governing the dynamic of HSL degradation, supposing an exponential decay. The results are reported in the table below: </p><br />
<br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><br />
</td><br />
<td class='row'><br />
<b>t<sub>1/2</sub><sup>*</sup> [h]</b><br />
</td><td class='row'><br />
<b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b><br />
</td><br />
</tr><br />
<tr><td class='row'><b>M9<sub>pH 6</sub></b><br />
</td><br />
<td class='row'>32<br />
</td><br />
<td class='row'>0.022<br />
</td><br />
</tr><br />
<tr><td class='row'><b>M9<sub>pH 7</sub></b><br />
</td><br />
<td class='row'>8<br />
</td><br />
<td class='row'>0.087<br />
</td><br />
</tr><br />
</table><br />
</center><br />
<p><br />
The described experiment was repeated with a MGZ1 culture in order to evaluate the effect of culture on HSL stability. The estimated values are reported in the table below:</p><br />
<br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><br />
</td><br />
<td class='row'><br />
<b>t<sub>1/2</sub><sup>*</sup> [h]</b><br />
</td><td class='row'><br />
<b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b><br />
</td><br />
</tr><br />
<tr><td class='row'><b>Culture<sub>pH 6</sub></b><br />
</td><br />
<td class='row'>&#8734;<br />
</td><br />
<td class='row'>0<br />
</td><br />
</tr><br />
<tr><td class='row'><b>Culture<sub>pH 7</sub></b><br />
</td><br />
<td class='row'>19<br />
</td><br />
<td class='row'>0.037<br />
</td><br />
</tr><br />
</table><br />
</center><br />
<br />
<p>It is evident from the reported data that the spontaneous degradation of HSL is negligible when CTRL+E is implemented in MGZ1 cells grown in M9 medium with pH=6.0 and pH=7.0.<br><br />
Thus in the simulations we set &gamma;<sub>HSL</sub>=0; <br />
<br />
</p><br />
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<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-------------t9002--------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='t9002'></a><h2>Characterization of BBa_T9002 biosensor</h2><br />
As described in the <a href="https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002">modelling section</a>, BioBrick <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> is an HSL biosensor, which provides a non linear relationship between HSL input and S<sub>cell</sub> output. More precisely, the characteristic sigmoidal curve requires synthetic parameters for its accurate identification. These are the minimum and maximum values, the swtich point (i.e., the curve inflection point), and the upper and lower boundaries of linearity. This biosensor revealed greatly reliable, providing measurement repeatability and minimal experimental noise. Referring to its activation formula, the calibration curve is shown below.<br><br><br />
<br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage" width="47%" height="50%"></a></div></div><br />
<br><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage" width="100%"></a></div></div><br />
<br />
<br />
<center><br />
<table width='50%' class='data'><tr><br />
<td class='row'><b>Minimum [S<sub>cell</sub>]</b></td><br />
<td class='row'><b>Maximum [S<sub>cell</sub>]</b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Lower boundary of linearity [nM]</b></td><br />
<td class='row'><b>Upper boundary of linearity [nM]</b></td><br />
</tr><br />
<br />
<tr><br />
<td class='row'>17.31</td><br />
<td class='row'>739.4</td><br />
<td class='row'>1.39</td><br />
<td class='row'>0.38</td><br />
<td class='row'>5.07</td><br />
<br />
</tr><br />
</table><br />
</center><br />
<br><br><br />
In order to determine the threshold sensitivity of T9002 biosensor, experiments were performed with several HSL inductions minimally interspaced in the region of low detectability. Hypothesizing that the inducer is 1:20 diluted (as for all of our tests), the minimum detectable HSL concentration is 3 nM.<br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Parts/CharacterizedTeam:UNIPV-Pavia/Parts/Characterized2011-09-21T17:57:20Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<html><br />
<a name='indice'></a><br />
<h2 class="art-postheader"><br />
Characterized Parts<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br><br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#new"><span class="tocnumber">1</span> <span class="toctext">New Parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#J101"><span class="tocnumber">1.1</span> <span class="toctext">J23101x series with different RBSs</span></a></li><br />
<li class="toclevel-2"><a href="#pTetLuxI"><span class="tocnumber">1.2</span> <span class="toctext">LuxI Expression cassette driven by aTc</span></a></li><br />
<li class="toclevel-2"><a href="#pTetAiiA"><span class="tocnumber">1.3</span> <span class="toctext">AiiA Expression cassette driven by aTc</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Existing"><span class="tocnumber">2</span> <span class="toctext">Characterization of existing parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#note"><span class="tocnumber">2.1</span> <span class="toctext">Notes on promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.2</span> <span class="toctext">RBSs from the community collection</span></a></li><br />
<li class="toclevel-2"><a href="#pLux"><span class="tocnumber">2.3</span> <span class="toctext">p<sub>Lux</sub> - a 3OC<sub>6</sub>-HSL-in PoPs-out device</span></a></li><br />
<li class="toclevel-2"><a href="#pTet"><span class="tocnumber">2.4</span> <span class="toctext">p<sub>Tet</sub> - BBa_R0040</span></a></li><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.5</span> <span class="toctext">LuxI - BBa_C0061</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.6</span> <span class="toctext">AiiA - BBa_C0060</span></a></li><br />
<li class="toclevel-2"><a href="#K999"><span class="tocnumber">2.7</span> <span class="toctext">pSB1C3 vector with mRFP between S and P for easy cloning</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Improvement"><span class="tocnumber">3</span> <span class="toctext">Sequence debugging</span></a></li><br />
</ul></td></tr></table><br />
</div><br><br />
<br />
<br />
<br />
<br><br><br />
<br />
<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part: pSB4C5">pSB4C5</a>. <br />
</em><br />
<br><br />
<br><br />
<br />
<br />
<div class="listcircle"><br />
<a name='new'></a><h1>New parts</h1><br />
<a name='J101'></a><h2>J23101x series</h2><br />
<br />
<ol type='1'><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> (wiki name: J101-E5 ) J101-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> (wiki name: J101-31 ) J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> (wiki name: J101-E7 ) J101-RBS32-mRFP-TT </li><br />
</ol><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- J23101x description --><br />
<!-- ----------------- --><br />
<p align='justify'><br />
<div align="justify"><p>BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in '<a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>data analysis</a>' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene.</p><br />
<p>The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P. This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts.</p><br />
<p>Salis et al. [Nat Biotec, 2009] stated that <em> 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels'</em> and again <em>'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.'</em></p><br />
<br />
<p>For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS. In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.</p><br />
<br />
<p><br><em><b>NB</b>: in the RPU computation, the J23101-RBS34-mRFP-TT (<A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_J23101">BBa_J23101</a>) in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency.</em></p></div><br />
<br />
<table align='center'><tr><td width='50%' ><br />
<table class='data'><br />
<tr><br />
<td class='row'><b>J23101 promoter with RBS</b><br />
</td><br />
<td class='row'><b>S<sub>cell</sub></b><br />
</td><br />
<td class='row'><b>R.P.U.s</b><br />
</td><br />
</tr><br />
<tr><td class='row'>RBS30<br />
</td><br />
<td class='row'>122 [13.23]</td><br />
<td class='row'>2.45 [0.27]</td><br />
</tr><br />
<tr><td class='row'>RBS31<br />
</td><br />
<td class='row'>2 [0.25]</td><br />
<td class='row'>0.04 [0.01]</td><br />
</tr><br />
<tr><td class='row'>RBS32<br />
</td><br />
<td class='row'>20 [1.7]</td><br />
<td class='row'>0.40 [0.03]</td><br />
</tr><br />
<tr><td class='row'>RBS34<br />
</td><br />
<td class='row'>50 [1.87]</td><br />
<td class='row'>1 [0.02]</td><br />
</tr><br />
</table></td><br />
<div align="center">Data are provided as average [Standard error].</div><br><br />
<td width='50%' ><br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="image"><img alt="RPUs of J23101 promoter with different RBSs" src="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="thumbimage" width="80%"></a></div></div><br />
</td><br />
</tr></table><br />
<br><br><br />
<div align="justify">On the other hand, if the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.</div><br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!-- ----------------- --><br />
<!-- pTetLuxI description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetLuxI'></a><h2>LuxI expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> (wiki name: E13 ) pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> (wiki name: E14 ) pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> (wiki name: E15 ) pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI </li></ul></ol><br />
<br />
<br><br><br />
<p align='justify'><em>Though these parts don't have a transcriptional terminator, they have been characterized in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a>, that contains the BBa_B0054 terminator. This choice is motivated by the need to reproduce the exact experimental context of the final circuit, as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Solution#Circuit_design'>solution section</a>. <br />
</em></p><br />
<br><br><br />
<p align='justify'><br />
LuxI has been characterized in terms of enzymatic activity under the regulation of p<sub>Tet</sub> promoter. <br />
</p><br />
<p align='justify'><br />
K<sub>M,LuxI</sub> and V<sub>max</sub> parameters representing its activity have been estimated and the promoter strength (represented by a synthetic parameter &alpha;<sub>pTet</sub> for every pTet-RBS combination) at full induction (100 ng/ml) has been estimated too with a simultaneous fitting of the available data. <br />
<br />
</p><br />
<br />
<br />
<p align='justify'><br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002'>modelling section</a>). <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system describing the behavior of this measurement circuit is reported here: </p><br />
<br />
<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg" class="thumbimage" height="70%" width="68%"></a></div></div><br />
<br />
<br />
<br />
<p align='justify'><br />
Since the measurement systems are only assayed in the exponential growth phase, in the equation (3) N &lt; N<sub>max</sub> is assumed. <br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known and summarized in the table below:</p><br />
<br />
<div align="center"><br />
<table align="center" class='data' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.004925</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
</div><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, k<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>pTet-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
<br />
<div align="justify"><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div></div><br />
<br />
<br />
<div align="justify"><p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p></div><br />
<br />
<br />
<div align="center"><br />
<table align="center" class='data'><br />
<tr><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>k<sub>M,LuxI</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>3.56*10<sup>-9</sup></td><br />
<td class='row'>6.87*10<sup>3</sup></td><br />
<td class='row'>87</td><br />
<td class='row'>8.5</td><br />
<td class='row'>ND</td><br />
<td class='row'>252</td><br />
</tr></table><br />
</div><br />
<br> <br />
</p><br />
</p><br />
<br />
<br />
</p><br />
<br />
<br />
<p align='justify'><br />
The collected data have been used to identify the parameters of our model. Despite the data-poor context, the model predictions fit the experimental data, thus demonstrating that the equation that models the HSL synthesis by LuxI is a good approximation of real processes. <br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
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<br />
<!-- ----------------- --><br />
<!-- pTetAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetAiiA'></a><h2>AiiA expression cassette driven by aTc-inducible p<sub>Tet</sub> promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> (wiki name: E24 ) p<sub>Tet</sub>-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> (wiki name: E25 ) p<sub>Tet</sub>-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) p<sub>Tet</sub>-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) p<sub>Tet</sub>-RBS34-AiiA-TT </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<p aling="justify"><br />
The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity.<br />
Similar to LuxI, a system of differential equations (referring to <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>) has been derived. <br />
</p><br />
<br />
<div style='text-align:justify'><br />
<div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg" class="thumbimage" height="70%" width="68%"></a><br />
</div></div><br />
<br />
<p align='justify'><br />
The parameters k<sub>cat</sub>, k<sub>M,AiiA</sub> and &alpha;<sub>RBSx</sub> would have been estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>p<sub>Tet</sub>-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
Unfortunately, their estimation revealed difficult.<br><br />
In the first experiments with the measurement system <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a> in LOW-COPY at pH=7 no degradation of HSL was observed. The collected data are shown in the figure below. HSL degradation is identical in the measurement system and in the negative control after 21 hours.<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<div align="justify">Further investigation on the enzyme were performed in more suitable conditions, to evaluate its intrinsic activity. AiiA activity was investigated in HIGH COPY plasmid in <em>E. coli</em> TOP10, in order to understand if the enzyme worked. In this case, a significant difference in degradation between p<sub>Tet</sub>-RBSx-AiiA-TT and the negative control was observed, also just after 7 hours. That was the proof of the good functioning (in HIGH COPY) of the enzyme.</div><br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<div align="justify"><br />
Experiments on these parts gave us the opportunity to characterize only the activity of the enzyme in <em>E. COLI</em> TOP10 in high copy number plasmid, providing only some information about the order of magnitude of the model parameters, which has been designed to work in <em>E. COLI</em> MGZ1 in low copy number plasmid.</div><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<a name='Existing'></a><h1>Existing parts</h1><br />
<br />
<a name='note'></a><h2>Notes for promoter characterization</h2><br />
<p align='justify'><br />
<p>Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection.</p><br />
<p>The assembled RBSs are:</p><br />
<br><br />
<div align='center'><br />
<table class='data'><br />
<tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr><br />
<tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr><br />
<tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr><br />
<tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr><br />
<tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr><br />
</table></div><br />
<br><br />
<div align="justify"><p>For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.</p><br />
<p>The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. <br />
RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP).</p><br />
<p>For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.</p><br />
</p><br />
<p>Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least squares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs:</p><p></p><br />
<p><ol><ul><li><b><br />
RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity</p><br />
</li><p><li><b><br />
RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity</p><br />
</li><p><li><br />
<b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and it is representative of the position of linear region</p><br />
</li><br />
<p><li><br />
<b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</div></li></p><br />
</ul></ol><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --RBS description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='rbs'></a><h2>RBSs</h2><br />
<p align='justify'><br />
<p><div align="justify">The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of protein produced. <br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element.</p><br />
<p>The evaluation of RBS efficiency can be performed in a very intuitive fashion:</p><br />
<ol><br />
<li>select the RBSs you want to study</li><br />
<li>assemble them in a Promoter - XX - Coding sequence circuit</li><br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/d/d1/Vettore_base.jpg" class="thumbimage" width="60%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<li>measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS (<a href='http://partsregistry.org/wiki/index.php/Part: BBa_B0034'>BBa_B0034</a>). </li></div></ol><br><br />
<br />
<br />
<div align="justify"><p>This simple measurement system allows the quantification of RBS efficiency depending on the experimental context (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments.</p><br />
<p>To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.</p><br />
<p>In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, p<sub>Tet</sub>, p<sub>Lux</sub>). The system output was measured and the RBS efficiency evaluated. The results are summarized in the table below:</div></p><br />
<br><br />
<div align="center"><table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>p<sub>Lux</sub></sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>p<sub>Tet</sub></sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>J23101</sub></b><sup>**</sup></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>0.40</td><td class='row'>1.6814</td><td class='row'>2.45</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.01</td><td class='row'>ND</td><td class='row'>0.04</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.19</td><td class='row'>0.4193</td><td class='row'>0.40</td><td class='row'>0,3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table></div><br />
<br />
<br><br />
<div align="justify">On the other hand, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (p<sub>Tet</sub>-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below:</div><br><br />
<br />
<div align="center"><table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>mRFP</sub></b><sup>**</sup></td><br />
<td class='row'><b>eff<sub>AiiA</sub></b><sup>***</sup></td><br />
<td class='row'><b>eff<sub>LuxI</sub><sup>****</sup></b></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>1.72</td><td class='row'>0.53</td><td class='row'>0.45</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.03</td><td class='row'>0.83</td><td class='row'>0.028</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.37</td><td class='row'>0.50</td><td class='row'>N.D.</td><td class='row'>0.3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table></div><br />
<br><br />
<p align='justify'><br />
<div align="justify"><p><sup>*</sup> The RBS efficiency for inducible devices expressing mRFP was estimated as the ratio of the AUCs (Area under the curve) of the induction curve of the system with the studied RBS and the B0034 reference: AUC<sub>P, RBSx</sub>/AUC<sub>P, B0034</sub></p><br />
<p><sup>**</sup> The RBS efficiency for constitutive promoters expressing mRFP was computed as the ratio between S<sub>cell<sub>P, RBSx</sub></sub>/S<sub>cell<sub>P, B0034</sub></sub></p><br />
<p><sup>***</sup> The RBS efficiency for p<sub>Tet</sub> promoter driving the expression of AiiA enzyme was computed as the ratio &alpha;<sub>p<sub>Tet</sub>, RBSx</sub>/&alpha;<sub>p<sub>Tet</sub>, B0034</sub> estimated for the measurement system p<sub>Tet</sub>-RBSx-AiiA-TT. &alpha;<sub>p<sub>Tet</sub></sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#AiiA'>here</a>. p<sub>Tet</sub> was tested at full induction (100 ng/ml).</p><br />
<p><sup>****</sup> The RBS efficiency for promoters driving the expression of LuxI was computed as the ratio &alpha;<sub>p<sub>Tet</sub>, RBSx</sub>/&alpha;<sub>p<sub>Tet</sub>, B0034</sub> estimated from the measurement systems p<sub>Tet</sub>-RBSx-LuxI. &alpha;<sub>p<sub>Tet</sub></sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#LuxI'>here</a>. p<sub>Tet</sub> was tested at full induction (100 ng/ml).</p></div><br />
<br />
<br><br />
</p><br />
The parts we used to characterize the RBSs are listed here:<br />
<ol><br />
<ul><li>mRFP expression with different promoters</li><br />
<ul><br />
<li>J23101</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> J101-RBS30-RFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> J101-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_J23101 "> BBa_J23101 </a> J101-RBS34-mRFP-TT </li><br />
</ul><br />
<li>p<sub>Tet</sub></li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> p<sub>Tet</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
<li>p<sub>Lux</sub></li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
</ul><br />
<li>p<sub>Tet</sub> driving the expression of different genes</li><br />
<ul><li>mRFP</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> p<sub>Tet</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
<li>AiiA</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> p<sub>Tet</sub>-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> p<sub>Tet</sub>-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> p<sub>Tet</sub>-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> p<sub>Tet</sub>-RBS34-AiiA-TT </li><br />
</ul><br />
<li>LuxI</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> p<sub>Tet</sub>-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> p<sub>Tet</sub>-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> p<sub>Tet</sub>-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> p<sub>Tet</sub>-RBS34-LuxI </li><br />
</ul></ul><br />
</ul></ol><br />
<br />
<br />
</p><br />
<p align='justify'><br />
The results reported in the table suggest that the RBS efficiency ranking is not always maintained. In particular, for the different promoters driving the expression of mRFP, the ranking of the declared efficiencies is maintained for p<sub>Lux</sub>, but not for p<sub>Tet</sub> and J23101. The RBS B0030 results to be the most efficient for both J23101 and p<sub>Tet</sub>, but not for p<sub>Lux</sub> (NB: this effect might be due to an effective non-modularity of RBS, but also to saturating phenomena occurring for this very strong promoter at full induction). RBS B0031 always shows a very low efficiency, while B0032 an intermediate efficiency between B0031 and the stronger RBSs B0030 and B0034. <br><br />
For what concerns the encoded gene variation, more significant differences can be observed. RBS B0030 has the higher efficiency only for mRFP, while the values for AiiA and LuxI are similar (~0.5). Unexpectedly, the weak RBS B0031 has a higher efficiency with AiiA gene (0.83), while with mRFP and LuxI. With B0032 no activity was observed for LuxI, while for AiiA and mRFP the results are quite consistent with the one reported above. <br />
</p><br />
<p align='justify'><br />
These results are encouraging: though the partial non-modularity of RBS with the encoded gene is confirmed, the hypothesis of modularity with the promoter is to some extent confirmed. Three classes of efficiencies were identified:<br />
<ul><br />
<li>low efficiency RBS (B0031)</li><br />
<li>medium efficiency RBS (B0032)</li><br />
<li>high efficiency RBSs (B0030, B0034)</li><br />
</ul> <br />
</p><br />
<br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --pLux description- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<a name='pLux'></a><h2>p<sub>Lux</sub> promoter</h2><br />
<p align='justify'><br />
<br />
<br />
</p><br />
<ol><ul><br />
<br />
<br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> (wiki name: E17 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> (wiki name: E18 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> (wiki name: E19 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> (wiki name: E20 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS34-mRFP-TT </li></ul><br />
</ol><br />
<br />
<p align='justify'><br />
The estimated parameters for the Hill functions are summarized in the table below. For more details on parameter estimation, see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>model section</a>. <br />
</p><br />
<br />
<table class='data' width='100%'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Lux</sub></sub> [(AUr/min)/cell]</b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Lux</sub></sub> [-]</b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Lux</sub></sub> [-]</b></td><br />
<td class="row"><b>k<sub>p<sub>Lux</sub></sub> [ng/ml]</b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">438 [10]</td><br />
<td class="row">0.05 [>100]</td><br />
<td class="row">2 [47]</td><br />
<td class="row">1.88 [27]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">9.8 [7]</td><br />
<td class="row">0.11 [57]</td><br />
<td class="row">1.2 [29]</td><br />
<td class="row">1.5 [26]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">206 [3]</td><br />
<td class="row">0 [>>100]</td><br />
<td class="row">1.36 [10]</td><br />
<td class="row">1.87 [9]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">1105 [6]</td><br />
<td class="row">0.02 [>100]</td><br />
<td class="row">1.33 [19]</td><br />
<td class="row">2.34 [18]</td><br />
</tr><br />
</table><br />
<div align="center">Data are provided as average [CV%].</div><br><br />
<br />
<br />
<div align="justify"><br />
<p>From this table, it is evident that, whilst &alpha;<sub>p<sub>Lux</sub></sub> assumes significantly different values for different RBSs, &eta;<sub>pLux</sub> and k<sub>pLux</sub> assume very similar values. This result shows that RBS variation modulates the amplitude of Hill function, not affecting the shape of the curve. The four induction curves result to be the same Hill function modulated in amplitude by a parameter, that is the estimated RBS efficiency for this measurement system.</p><br />
<p>These results are quite encouraging, because suggest that, given the non-modular behavior of RBS dpending on the encoded gene, the RBS has a modular behaviour respect to the promoter.</p><br />
<p>The operative parameters are summarized in the table below:</p></div><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>4.28</td><td class='row'>0.20</td><td class='row'>1.08</td><td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>4.93</td><td class='row'>0.55</td><td class='row'>0.25</td><td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>9.49</td><td class='row'>0.02</td><td class='row'>0.47</td><td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>21.53</td><td class='row'>0.51</td><td class='row'>0.53</td><td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<p align='justify'>These operative parameters provide useful information on the behavior of this 3OC6-HSL inducible device. RPU<sub>max</sub> assumes very different values in terms of RPUs.<br />
This can't be explained by RBS modulation, since RPUs have been evaluated by normalizing S<sub>cell</sub> of p<sub>Lux</sub>-RBSx for the one of J23101-RBSx. It is evident that some nonlinear effect on maximum strength, maybe due to saturation phenomena on protein expression, occur. <br />
The same RPUs should be observed for every RBS, since the normalization by the standard reference used for RPUs computation should eliminate the RBS contribute. Here different RPUs are observed, maybe due to nonlinear RBS behavior or to saturation phenomena occurring with this very strong promoter. The switch point and linear boundaries are quite constant in all the cases, showing that the linear region of this system is not affected by RBS changes.</p><br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/5/50/E17_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/5/50/E17_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/c/c4/E18_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c4/E18_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/7/78/E19_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/78/E19_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/5/55/E20_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/5/55/E20_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!-- ----------------- --><br />
<!-- -pTet description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='pTet'></a><h2>p<sub>Tet</sub> promoter</h2><br />
<br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> (wiki name: E21 ) p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> (wiki name: E22 ) p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) p<sub>Tet</sub>-RBS32-mRFP-TT<br />
</li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT <br />
</li></ul><br />
</ul><br />
</ol><br />
<br />
<div align="justify"><br />
<p>The protocols for the characterization of p<sub>Tet</sub> promoter are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#pTet_protocol'>p<sub>Tet</sub> measurement section</a>.</p><br />
<p>The data collected from the mRFP measurement systems were processed as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'> data analysis section</a>. The induction curves were obtained by fitting a Hill function as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>modelling section</a> and the estimated <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>parameters</a> for p<sub>Tet</sub> are reported in the pictures and in table below. </p><br />
<p>This promoter is widely studied and characterized usually using the strong RBS BBa_B0034. Here we have characterized its transcriptional strength as a function of aTc induction (ng/ml) for different RBSs. Three different induction curves were obtained and are reported in figure:</p></div><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<br />
<br />
<!-- <td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/9/93/E33_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/9/93/E33_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td> --><br />
<br />
<table width='100%'><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/9/99/E34_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/9/99/E34_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/e/e4/E35_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e4/E35_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<p align='justify'><br />
The estimated parameters of the Hill curves described in the figures are summarized in the table below:<br />
</p><br />
<br><br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub> [(AUr/min)/cell]</b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub> [-]</b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub> [-]</b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub> [nM]</b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">230.67 [3.7]</td><br />
<td class="row">0.028 [91.61]</td><br />
<td class="row">4.61 [23.73]</td><br />
<td class="row">8.75 [4.16]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">55.77 [12]</td><br />
<td class="row">1.53E-11 [>>100]</td><br />
<td class="row">4.98 [57.62]</td><br />
<td class="row">7.26 [14.98]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">120 [5.95]</td><br />
<td class="row">0.085 [40.6]</td><br />
<td class="row">24.85 [47.6]</td><br />
<td class="row">9 [5.43]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br><br><br />
<br />
<p align='justify'><br />
The measurement system p<sub>Tet</sub>-B0031-mRFP-TT couldn't be assayed because its fluorescence output is under the detectability threshold of our measurement instrument. For this reason, the parameters of the corresponding Hill curve couldn't be estimated and are reported as 'Not Determined' ND. <br />
</p><br />
<br />
<div align='justify'><br />
<p>&alpha; parameter (representing the maximum trascriptional rate in the studied range of induction) varies as expected with the RBS variation and also the &delta; and &eta; parameters are quite different among the RBS variations.</p><br />
<p>The k<sub>p<sub>Tet</sub></sub> parameter is quite constant among the RBS variations, thus suggesting that in this case the RBS variation doesn't substantially affect the switch point of the Hill curve, even if the amplitude and the maximum slope are not quite maintained (for the &eta; parameter, maybe fitting problems). <br />
<br />
</p><br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p></div><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [ng/ml]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [ng/ml]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>1.53</td><td class='row'>~0</td><td class='row'>7.95</td><td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>3.16</td><td class='row'>~0</td><td class='row'>6.7</td><td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>2.73</td><td class='row'>0.23</td><td class='row'>8.96</td><td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
From these parameters, it is evident that whilst the switch-point is almost maintained for all the RBSs, the linear boundaries are similar for RBS30 and RBS32 but for RBS34 are moved on the right of one order of magnitude. <br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!-- ----------------- --><br />
<!-- --LuxI description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='LuxI'></a><h2>LuxI gene - BBa_C0061</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002'>modelling section</a>) using the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>p<sub>Tet</sub>-RBSx-LuxI-TT</a> measurement systems. <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here: </p><br />
<br />
<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg" class="thumbimage" height="70%" width="68%"></a></div></div><br />
<br />
<br />
<br />
<p align='justify'><br />
Since the measurement systems are only assayed in the exponential growth phase, in the equation (3) N &lt; <N<sub>max</sub> is assumed. <br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known and summarized in the table below:</p><br />
<br />
<div align="center"><br />
<table align="center" class='data' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.004925</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
</div><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, k<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>p<sub>Tet</sub>-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
<br />
<div align="justify"><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div></div><br />
<br />
<br />
<div align="justify"><p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p></div><br />
<br />
<br />
<div align=center"><br />
<table align="center" class='data'><br />
<tr><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>k<sub>M,LuxI</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>3.56*10<sup>-9</sup></td><br />
<td class='row'>6.87*10<sup>3</sup></td><br />
<td class='row'> 87 </td><br />
<td class='row'>8.5</td><br />
<td class='row'>ND</td><br />
<td class='row'> 252 </td><br />
</tr></table><br />
</div><br />
<br> <br />
</p><br />
</p><br />
<br />
<p align='justify'><br />
The provided parameters k<sub>M</sub> and V<sub>max</sub> represent the enzymatic activity of LuxI, described by our model. They must not be confused with the operative parameters of the Michaelis-Menten relation. <br />
These synthetic parameters have a great importance, since they can be used in more complicated models in order to predict the behavior of complex circuits.<br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!-- ----------------- --><br />
<!-- --AiiA description-- --><br />
<!-- ----------------- --><br />
<br />
<br />
<a name='AiiA'></a><h2>AiiA gene - BBa_C0060</h2><br />
<br />
<p align='justify'><br />
The activity of AiiA enzyme has been evaluated by testing the measurement systems <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a>. <br />
Similar to LuxI, a system of differential equations (referring to <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>) has been derived. <br />
</p><br />
<br />
<div style='text-align:justify'><br />
<div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg" class="thumbimage" height="70%" width="68%"></a><br />
</div></div><br />
<br />
<p align='justify'><br />
The parameters k<sub>cat</sub>, k<sub>M,AiiA</sub> and &alpha;<sub>RBSx</sub> would have been estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>p<sub>Tet</sub>-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
Unfortunately, their estimation revealed difficult.<br><br />
In the first experiments with the measurement system <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a> in LOW-COPY at pH=7 no degradation of HSL was observed. The collected data are shown in the figure below. HSL degradation is identical in the measurement system and in the negative control after 21 hours.<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<div align="justify">Further investigation on the enzyme were performed in more suitable conditions, to evaluate its intrinsic activity. AiiA activity was investigated in HIGH COPY plasmid in <em>E. coli</em> TOP10, in order to understand if the enzyme worked. In this case, a significant difference in degradation between p<sub>Tet</sub>-RBSx-AiiA-TT and the negative control was observed, also just after 7 hours. That was the proof of the good functioning (in HIGH COPY) of the enzyme.</div><br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<div align="justify"><br />
Since the enzyme activity had been assessed, some suitable working conditions were investigated for AiiA. Since our studies on HSL stability for different pHs has shown that its stability improves at pH=6.0, this experimental condition was used to test once again MGZ1 strain with the system expressing AiiA in low copy number plasmid. <br />
<br />
Unfortunately, we had some problems with the biosensor, BBa_T9002, that didn't work as usual, resulting in a calibration curve modified. So, k<sub>M,AiiA</sub> and k<sub>cat</sub> were impossible to be estimated from these data. We decided to estimate them from experimental data coming from Tecan tests preformed in <em>E.COLI</em> TOP10, where aiiA gene was cloned in a high copy numebr plasmid, pSB1A2, downstream p<sub>Tet</sub> with different RBSs; in this way we could have some semi-quantitative information about the order of magnitude of these parameters. Taking into account the different copy number, we tried to simulate our model behavior with reasonable values for AiiA parameters.</div><br />
<br><br />
<br />
</p><br />
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<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!-- ----------------- --><br />
<!-- ---pSB1C3-mRFP-- --><br />
<!-- ----------------- --><br />
<br />
<a name='K999'></a><h2>pSB1C3 plasmid with mRFP between S and P bearing p<sub>Tet</sub> (easy-to-clone)</h2><br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/e/ee/BBa_I13507.jpg" class="thumbimage" width="50%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<div align="justify">This vector was designed and realized in order to facilitate the cloning of coding sequences downstream of the strong promoter p<sub>Tet</sub>. This vector was assembled by ligating S-P excided mRFP coding sequence from BBa_J61002 and ligating it in BBa_R0040 cut with S and P. Thus, the resulting vector contains mRFP between S and P. p<sub>Tet</sub> can be easily excided (E-P) and moved in the desired vector (E-P) and then the desired coding sequence can be easily assembled by digesting S-P the vector and X-P the coding sequence, thus obtaining a final part that is standard10-compatible.</div><br><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<a name='Improvement'></a><h1>Existing parts: sequence debugging</h1><br />
<br />
<div align="justify"><br />
<br />
<ol><ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516021 "> BBa_K516021 </a> (wiki name: E10 ) RBS31-AiiA-TT Rebuilt existing part from BBa_I13914 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516022 "> BBa_K516022 </a> (wiki name: E11 ) RBS32-AiiA-TT Rebuilt existing part from BBa_I13912 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516030 "> BBa_K516030 </a> (wiki name: E5 ) RBS30-mRFP-TT Rebuilt existing part from BBa_S04180 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516032 "> BBa_K516032 </a> (wiki name: E7 ) RBS32-mRFP-TT Rebuilt existing part from BBa_ J133000 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) p<sub>Tet</sub>-RBS34-LuxI Rebuilt existing part from BBa_S03623 (DNA available, only 2008 kit, inconsistent) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) p<sub>Tet</sub>-RBS32-AiiA-TT Rebuilt existing part from BBa_ J22071 (2008 only, Bad sequencing) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) p<sub>Tet</sub>-RBS34-AiiA-TT Rebuilt existing part from BBa_K077047 (Part deleted) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) p<sub>Tet</sub>-RBS32-mRFP-TT Rebuilt existing part from BBa_I20252 (DNA planning) </li></ul></ol><br />
</div><br />
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<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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</html><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Parts/CharacterizedTeam:UNIPV-Pavia/Parts/Characterized2011-09-21T17:47:51Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<html><br />
<a name='indice'></a><br />
<h2 class="art-postheader"><br />
Characterized Parts<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br><br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#new"><span class="tocnumber">1</span> <span class="toctext">New Parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#J101"><span class="tocnumber">1.1</span> <span class="toctext">J23101x series with different RBSs</span></a></li><br />
<li class="toclevel-2"><a href="#pTetLuxI"><span class="tocnumber">1.2</span> <span class="toctext">LuxI Expression cassette driven by aTc</span></a></li><br />
<li class="toclevel-2"><a href="#pTetAiiA"><span class="tocnumber">1.3</span> <span class="toctext">AiiA Expression cassette driven by aTc</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Existing"><span class="tocnumber">2</span> <span class="toctext">Characterization of existing parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#note"><span class="tocnumber">2.1</span> <span class="toctext">Notes on promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.2</span> <span class="toctext">RBSs from the community collection</span></a></li><br />
<li class="toclevel-2"><a href="#pLux"><span class="tocnumber">2.3</span> <span class="toctext">p<sub>Lux</sub> - a 3OC<sub>6</sub>-HSL-in PoPs-out device</span></a></li><br />
<li class="toclevel-2"><a href="#pTet"><span class="tocnumber">2.4</span> <span class="toctext">p<sub>Tet</sub> - BBa_R0040</span></a></li><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.5</span> <span class="toctext">LuxI - BBa_C0061</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.6</span> <span class="toctext">AiiA - BBa_C0060</span></a></li><br />
<li class="toclevel-2"><a href="#K999"><span class="tocnumber">2.7</span> <span class="toctext">pSB1C3 vector with mRFP between S and P for easy cloning</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Improvement"><span class="tocnumber">3</span> <span class="toctext">Sequence debugging</span></a></li><br />
</ul></td></tr></table><br />
</div><br><br />
<br />
<br />
<br />
<br><br><br />
<br />
<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part: pSB4C5">pSB4C5</a>. <br />
</em><br />
<br><br />
<br><br />
<br />
<br />
<div class="listcircle"><br />
<a name='new'></a><h1>New parts</h1><br />
<a name='J101'></a><h2>J23101x series</h2><br />
<br />
<ol type='1'><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> (wiki name: J101-E5 ) J101-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> (wiki name: J101-31 ) J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> (wiki name: J101-E7 ) J101-RBS32-mRFP-TT </li><br />
</ol><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- J23101x description --><br />
<!-- ----------------- --><br />
<p align='justify'><br />
<div align="justify"><p>BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in '<a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>data analysis</a>' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene.</p><br />
<p>The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P. This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts.</p><br />
<p>Salis et al. [Nat Biotec, 2009] stated that <em> 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels'</em> and again <em>'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.'</em></p><br />
<br />
<p>For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS. In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.</p><br />
<br />
<p><br><em><b>NB</b>: in the RPU computation, the J23101-RBS34-mRFP-TT (<A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_J23101">BBa_J23101</a>) in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency.</em></p></div><br />
<br />
<table align='center'><tr><td width='50%' ><br />
<table class='data'><br />
<tr><br />
<td class='row'><b>J23101 promoter with RBS</b><br />
</td><br />
<td class='row'><b>S<sub>cell</sub></b><br />
</td><br />
<td class='row'><b>R.P.U.s</b><br />
</td><br />
</tr><br />
<tr><td class='row'>RBS30<br />
</td><br />
<td class='row'>122 [13.23]</td><br />
<td class='row'>2.45 [0.27]</td><br />
</tr><br />
<tr><td class='row'>RBS31<br />
</td><br />
<td class='row'>2 [0.25]</td><br />
<td class='row'>0.04 [0.01]</td><br />
</tr><br />
<tr><td class='row'>RBS32<br />
</td><br />
<td class='row'>20 [1.7]</td><br />
<td class='row'>0.40 [0.03]</td><br />
</tr><br />
<tr><td class='row'>RBS34<br />
</td><br />
<td class='row'>50 [1.87]</td><br />
<td class='row'>1 [0.02]</td><br />
</tr><br />
</table></td><br />
<div align="center">Data are provided as average [Standard error].</div><br><br />
<td width='50%' ><br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="image"><img alt="RPUs of J23101 promoter with different RBSs" src="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="thumbimage" width="80%"></a></div></div><br />
</td><br />
</tr></table><br />
<br><br><br />
<div align="justify">On the other hand, if the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.</div><br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!-- ----------------- --><br />
<!-- pTetLuxI description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetLuxI'></a><h2>LuxI expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> (wiki name: E13 ) pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> (wiki name: E14 ) pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> (wiki name: E15 ) pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI </li></ul></ol><br />
<br />
<br><br><br />
<p align='justify'><em>Though these parts don't have a transcriptional terminator, they have been characterized in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a>, that contains the BBa_B0054 terminator. This choice is motivated by the need to reproduce the exact experimental context of the final circuit, as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Solution#Circuit_design'>solution section</a>. <br />
</em></p><br />
<br><br><br />
<p align='justify'><br />
LuxI has been characterized in terms of enzymatic activity under the regulation of p<sub>Tet</sub> promoter. <br />
</p><br />
<p align='justify'><br />
K<sub>M,LuxI</sub> and V<sub>max</sub> parameters representing its activity have been estimated and the promoter strength (represented by a synthetic parameter &alpha;<sub>pTet</sub> for every pTet-RBS combination) at full induction (100 ng/ml) has been estimated too with a simultaneous fitting of the available data. <br />
<br />
</p><br />
<br />
<br />
<p align='justify'><br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002'>modelling section</a>). <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system describing the behavior of this measurement circuit is reported here: </p><br />
<br />
<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg" class="thumbimage" height="70%" width="68%"></a></div></div><br />
<br />
<br />
<br />
<p align='justify'><br />
Since the measurement systems are only assayed in the exponential growth phase, in the equation (3) N &lt; N<sub>max</sub> is assumed. <br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known and summarized in the table below:</p><br />
<br />
<div align="center"><br />
<table align="center" class='data' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.004925</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
</div><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, k<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>pTet-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
<br />
<div align="justify"><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div></div><br />
<br />
<br />
<div align="justify"><p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p></div><br />
<br />
<br />
<div align="center"><br />
<table align="center" class='data'><br />
<tr><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>k<sub>M,LuxI</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>3.56*10<sup>-9</sup></td><br />
<td class='row'>6.87*10<sup>3</sup></td><br />
<td class='row'>87</td><br />
<td class='row'>8.5</td><br />
<td class='row'>ND</td><br />
<td class='row'>252</td><br />
</tr></table><br />
</div><br />
<br> <br />
</p><br />
</p><br />
<br />
<br />
</p><br />
<br />
<br />
<p align='justify'><br />
The collected data have been used to identify the parameters of our model. Despite the data-poor context, the model predictions fit the experimental data, thus demonstrating that the equation that models the HSL synthesis by LuxI is a good approximation of real processes. <br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
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<br />
<!-- ----------------- --><br />
<!-- pTetAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetAiiA'></a><h2>AiiA expression cassette driven by aTc-inducible p<sub>Tet</sub> promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> (wiki name: E24 ) p<sub>Tet</sub>-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> (wiki name: E25 ) p<sub>Tet</sub>-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) p<sub>Tet</sub>-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) p<sub>Tet</sub>-RBS34-AiiA-TT </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity.<br />
<br />
Similar to LuxI, a system of differential equations (referring to <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>) has been derived. <br />
</p><br />
<br />
<div style='text-align:justify'><br />
<div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg" class="thumbimage" height="70%" width="68%"></a><br />
</div></div><br />
<br />
<p align='justify'><br />
The parameters k<sub>cat</sub>, k<sub>M,AiiA</sub> and &alpha;<sub>RBSx</sub> would have been estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>p<sub>Tet</sub>-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
Unfortunately, their estimation revealed difficult.<br><br />
In the first experiments with the measurement system <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a> in LOW-COPY at pH=7 no degradation of HSL was observed. The collected data are shown in the figure below. HSL degradation is identical in the measurement system and in the negative control after 21 hours.<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<div align="justify">Further investigation on the enzyme were performed in more suitable conditions, to evaluate its intrinsic activity. AiiA activity was investigated in HIGH COPY plasmid in <em>E. coli</em> TOP10, in order to understand if the enzyme worked. In this case, a significant difference in degradation between p<sub>Tet</sub>-RBSx-AiiA-TT and the negative control was observed, also just after 7 hours. That was the proof of the good functioning (in HIGH COPY) of the enzyme.</div><br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<div align="justify"><br />
Experiments on these parts gave us the opportunity to characterize only the activity of the enzyme in <em>E. COLI</em> TOP10 in high copy number plasmid, providing only some information about the order of magnitude of the model parameters, which has been designed to work in <em>E. COLI</em> MGZ1 in low copy number plasmid.<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<a name='Existing'></a><h1>Existing parts</h1><br />
<br />
<a name='note'></a><h2>Notes for promoter characterization</h2><br />
<p align='justify'><br />
<p>Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection.</p><br />
<p>The assembled RBSs are:</p><br />
<br><br />
<div align='center'><br />
<table class='data'><br />
<tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr><br />
<tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr><br />
<tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr><br />
<tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr><br />
<tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr><br />
</table></div><br />
<br><br />
<div align="justify"><p>For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.</p><br />
<p>The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. <br />
RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP).</p><br />
<p>For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.</p><br />
</p><br />
<p>Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least squares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs:</p><p></p><br />
<p><ol><ul><li><b><br />
RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity</p><br />
</li><p><li><b><br />
RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity</p><br />
</li><p><li><br />
<b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and it is representative of the position of linear region</p><br />
</li><br />
<p><li><br />
<b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</div></li></p><br />
</ul></ol><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --RBS description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='rbs'></a><h2>RBSs</h2><br />
<p align='justify'><br />
<p><div align="justify">The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of protein produced. <br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element.</p><br />
<p>The evaluation of RBS efficiency can be performed in a very intuitive fashion:</p><br />
<ol><br />
<li>select the RBSs you want to study</li><br />
<li>assemble them in a Promoter - XX - Coding sequence circuit</li><br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/d/d1/Vettore_base.jpg" class="thumbimage" width="60%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<li>measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS (<a href='http://partsregistry.org/wiki/index.php/Part: BBa_B0034'>BBa_B0034</a>). </li></div></ol><br><br />
<br />
<br />
<div align="justify"><p>This simple measurement system allows the quantification of RBS efficiency depending on the experimental context (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments.</p><br />
<p>To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.</p><br />
<p>In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, p<sub>Tet</sub>, p<sub>Lux</sub>). The system output was measured and the RBS efficiency evaluated. The results are summarized in the table below:</div></p><br />
<br><br />
<div align="center"><table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>p<sub>Lux</sub></sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>p<sub>Tet</sub></sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>J23101</sub></b><sup>**</sup></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>0.40</td><td class='row'>1.6814</td><td class='row'>2.45</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.01</td><td class='row'>ND</td><td class='row'>0.04</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.19</td><td class='row'>0.4193</td><td class='row'>0.40</td><td class='row'>0,3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table></div><br />
<br />
<br><br />
<div align="justify">On the other hand, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (p<sub>Tet</sub>-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below:</div><br><br />
<br />
<div align="center"><table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>mRFP</sub></b><sup>**</sup></td><br />
<td class='row'><b>eff<sub>AiiA</sub></b><sup>***</sup></td><br />
<td class='row'><b>eff<sub>LuxI</sub><sup>****</sup></b></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>1.72</td><td class='row'>0.53</td><td class='row'>0.45</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.03</td><td class='row'>0.83</td><td class='row'>0.028</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.37</td><td class='row'>0.50</td><td class='row'>N.D.</td><td class='row'>0.3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table></div><br />
<br><br />
<p align='justify'><br />
<div align="justify"><p><sup>*</sup> The RBS efficiency for inducible devices expressing mRFP was estimated as the ratio of the AUCs (Area under the curve) of the induction curve of the system with the studied RBS and the B0034 reference: AUC<sub>P, RBSx</sub>/AUC<sub>P, B0034</sub></p><br />
<p><sup>**</sup> The RBS efficiency for constitutive promoters expressing mRFP was computed as the ratio between S<sub>cell<sub>P, RBSx</sub></sub>/S<sub>cell<sub>P, B0034</sub></sub></p><br />
<p><sup>***</sup> The RBS efficiency for p<sub>Tet</sub> promoter driving the expression of AiiA enzyme was computed as the ratio &alpha;<sub>p<sub>Tet</sub>, RBSx</sub>/&alpha;<sub>p<sub>Tet</sub>, B0034</sub> estimated for the measurement system p<sub>Tet</sub>-RBSx-AiiA-TT. &alpha;<sub>p<sub>Tet</sub></sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#AiiA'>here</a>. p<sub>Tet</sub> was tested at full induction (100 ng/ml).</p><br />
<p><sup>****</sup> The RBS efficiency for promoters driving the expression of LuxI was computed as the ratio &alpha;<sub>p<sub>Tet</sub>, RBSx</sub>/&alpha;<sub>p<sub>Tet</sub>, B0034</sub> estimated from the measurement systems p<sub>Tet</sub>-RBSx-LuxI. &alpha;<sub>p<sub>Tet</sub></sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#LuxI'>here</a>. p<sub>Tet</sub> was tested at full induction (100 ng/ml).</p></div><br />
<br />
<br><br />
</p><br />
The parts we used to characterize the RBSs are listed here:<br />
<ol><br />
<ul><li>mRFP expression with different promoters</li><br />
<ul><br />
<li>J23101</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> J101-RBS30-RFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> J101-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_J23101 "> BBa_J23101 </a> J101-RBS34-mRFP-TT </li><br />
</ul><br />
<li>p<sub>Tet</sub></li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> p<sub>Tet</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
<li>p<sub>Lux</sub></li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
</ul><br />
<li>p<sub>Tet</sub> driving the expression of different genes</li><br />
<ul><li>mRFP</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> p<sub>Tet</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
<li>AiiA</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> p<sub>Tet</sub>-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> p<sub>Tet</sub>-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> p<sub>Tet</sub>-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> p<sub>Tet</sub>-RBS34-AiiA-TT </li><br />
</ul><br />
<li>LuxI</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> p<sub>Tet</sub>-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> p<sub>Tet</sub>-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> p<sub>Tet</sub>-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> p<sub>Tet</sub>-RBS34-LuxI </li><br />
</ul></ul><br />
</ul></ol><br />
<br />
<br />
</p><br />
<p align='justify'><br />
The results reported in the table suggest that the RBS efficiency ranking is not always maintained. In particular, for the different promoters driving the expression of mRFP, the ranking of the declared efficiencies is maintained for p<sub>Lux</sub>, but not for p<sub>Tet</sub> and J23101. The RBS B0030 results to be the most efficient for both J23101 and p<sub>Tet</sub>, but not for p<sub>Lux</sub> (NB: this effect might be due to an effective non-modularity of RBS, but also to saturating phenomena occurring for this very strong promoter at full induction). RBS B0031 always shows a very low efficiency, while B0032 an intermediate efficiency between B0031 and the stronger RBSs B0030 and B0034. <br><br />
For what concerns the encoded gene variation, more significant differences can be observed. RBS B0030 has the higher efficiency only for mRFP, while the values for AiiA and LuxI are similar (~0.5). Unexpectedly, the weak RBS B0031 has a higher efficiency with AiiA gene (0.83), while with mRFP and LuxI. With B0032 no activity was observed for LuxI, while for AiiA and mRFP the results are quite consistent with the one reported above. <br />
</p><br />
<p align='justify'><br />
These results are encouraging: though the partial non-modularity of RBS with the encoded gene is confirmed, the hypothesis of modularity with the promoter is to some extent confirmed. Three classes of efficiencies were identified:<br />
<ul><br />
<li>low efficiency RBS (B0031)</li><br />
<li>medium efficiency RBS (B0032)</li><br />
<li>high efficiency RBSs (B0030, B0034)</li><br />
</ul> <br />
</p><br />
<br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --pLux description- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<a name='pLux'></a><h2>p<sub>Lux</sub> promoter</h2><br />
<p align='justify'><br />
<br />
<br />
</p><br />
<ol><ul><br />
<br />
<br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> (wiki name: E17 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> (wiki name: E18 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> (wiki name: E19 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> (wiki name: E20 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS34-mRFP-TT </li></ul><br />
</ol><br />
<br />
<p align='justify'><br />
The estimated parameters for the Hill functions are summarized in the table below. For more details on parameter estimation, see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>model section</a>. <br />
</p><br />
<br />
<table class='data' width='100%'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Lux</sub></sub> [(AUr/min)/cell]</b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Lux</sub></sub> [-]</b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Lux</sub></sub> [-]</b></td><br />
<td class="row"><b>k<sub>p<sub>Lux</sub></sub> [ng/ml]</b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">438 [10]</td><br />
<td class="row">0.05 [>100]</td><br />
<td class="row">2 [47]</td><br />
<td class="row">1.88 [27]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">9.8 [7]</td><br />
<td class="row">0.11 [57]</td><br />
<td class="row">1.2 [29]</td><br />
<td class="row">1.5 [26]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">206 [3]</td><br />
<td class="row">0 [>>100]</td><br />
<td class="row">1.36 [10]</td><br />
<td class="row">1.87 [9]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">1105 [6]</td><br />
<td class="row">0.02 [>100]</td><br />
<td class="row">1.33 [19]</td><br />
<td class="row">2.34 [18]</td><br />
</tr><br />
</table><br />
<div align="center">Data are provided as average [CV%].</div><br><br />
<br />
<br />
<div align="justify"><br />
<p>From this table, it is evident that, whilst &alpha;<sub>p<sub>Lux</sub></sub> assumes significantly different values for different RBSs, &eta;<sub>pLux</sub> and k<sub>pLux</sub> assume very similar values. This result shows that RBS variation modulates the amplitude of Hill function, not affecting the shape of the curve. The four induction curves result to be the same Hill function modulated in amplitude by a parameter, that is the estimated RBS efficiency for this measurement system.</p><br />
<p>These results are quite encouraging, because suggest that, given the non-modular behavior of RBS dpending on the encoded gene, the RBS has a modular behaviour respect to the promoter.</p><br />
<p>The operative parameters are summarized in the table below:</p></div><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>4.28</td><td class='row'>0.20</td><td class='row'>1.08</td><td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>4.93</td><td class='row'>0.55</td><td class='row'>0.25</td><td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>9.49</td><td class='row'>0.02</td><td class='row'>0.47</td><td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>21.53</td><td class='row'>0.51</td><td class='row'>0.53</td><td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<p align='justify'>These operative parameters provide useful information on the behavior of this 3OC6-HSL inducible device. RPU<sub>max</sub> assumes very different values in terms of RPUs.<br />
This can't be explained by RBS modulation, since RPUs have been evaluated by normalizing S<sub>cell</sub> of p<sub>Lux</sub>-RBSx for the one of J23101-RBSx. It is evident that some nonlinear effect on maximum strength, maybe due to saturation phenomena on protein expression, occur. <br />
The same RPUs should be observed for every RBS, since the normalization by the standard reference used for RPUs computation should eliminate the RBS contribute. Here different RPUs are observed, maybe due to nonlinear RBS behavior or to saturation phenomena occurring with this very strong promoter. The switch point and linear boundaries are quite constant in all the cases, showing that the linear region of this system is not affected by RBS changes.</p><br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/5/50/E17_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/5/50/E17_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/c/c4/E18_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c4/E18_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/7/78/E19_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/78/E19_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/5/55/E20_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/5/55/E20_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!-- ----------------- --><br />
<!-- -pTet description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='pTet'></a><h2>p<sub>Tet</sub> promoter</h2><br />
<br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> (wiki name: E21 ) p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> (wiki name: E22 ) p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) p<sub>Tet</sub>-RBS32-mRFP-TT<br />
</li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT <br />
</li></ul><br />
</ul><br />
</ol><br />
<br />
<div align="justify"><br />
<p>The protocols for the characterization of p<sub>Tet</sub> promoter are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#pTet_protocol'>p<sub>Tet</sub> measurement section</a>.</p><br />
<p>The data collected from the mRFP measurement systems were processed as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'> data analysis section</a>. The induction curves were obtained by fitting a Hill function as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>modelling section</a> and the estimated <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>parameters</a> for p<sub>Tet</sub> are reported in the pictures and in table below. </p><br />
<p>This promoter is widely studied and characterized usually using the strong RBS BBa_B0034. Here we have characterized its transcriptional strength as a function of aTc induction (ng/ml) for different RBSs. Three different induction curves were obtained and are reported in figure:</p></div><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<br />
<br />
<!-- <td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/9/93/E33_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/9/93/E33_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td> --><br />
<br />
<table width='100%'><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/9/99/E34_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/9/99/E34_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/e/e4/E35_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e4/E35_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<p align='justify'><br />
The estimated parameters of the Hill curves described in the figures are summarized in the table below:<br />
</p><br />
<br><br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub> [(AUr/min)/cell]</b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub> [-]</b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub> [-]</b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub> [nM]</b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">230.67 [3.7]</td><br />
<td class="row">0.028 [91.61]</td><br />
<td class="row">4.61 [23.73]</td><br />
<td class="row">8.75 [4.16]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">55.77 [12]</td><br />
<td class="row">1.53E-11 [>>100]</td><br />
<td class="row">4.98 [57.62]</td><br />
<td class="row">7.26 [14.98]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">120 [5.95]</td><br />
<td class="row">0.085 [40.6]</td><br />
<td class="row">24.85 [47.6]</td><br />
<td class="row">9 [5.43]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br><br><br />
<br />
<p align='justify'><br />
The measurement system p<sub>Tet</sub>-B0031-mRFP-TT couldn't be assayed because its fluorescence output is under the detectability threshold of our measurement instrument. For this reason, the parameters of the corresponding Hill curve couldn't be estimated and are reported as 'Not Determined' ND. <br />
</p><br />
<br />
<div align='justify'><br />
<p>&alpha; parameter (representing the maximum trascriptional rate in the studied range of induction) varies as expected with the RBS variation and also the &delta; and &eta; parameters are quite different among the RBS variations.</p><br />
<p>The k<sub>p<sub>Tet</sub></sub> parameter is quite constant among the RBS variations, thus suggesting that in this case the RBS variation doesn't substantially affect the switch point of the Hill curve, even if the amplitude and the maximum slope are not quite maintained (for the &eta; parameter, maybe fitting problems). <br />
<br />
</p><br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p></div><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [ng/ml]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [ng/ml]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>1.53</td><td class='row'>~0</td><td class='row'>7.95</td><td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>3.16</td><td class='row'>~0</td><td class='row'>6.7</td><td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>2.73</td><td class='row'>0.23</td><td class='row'>8.96</td><td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
From these parameters, it is evident that whilst the switch-point is almost maintained for all the RBSs, the linear boundaries are similar for RBS30 and RBS32 but for RBS34 are moved on the right of one order of magnitude. <br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!-- ----------------- --><br />
<!-- --LuxI description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='LuxI'></a><h2>LuxI gene - BBa_C0061</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002'>modelling section</a>) using the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>p<sub>Tet</sub>-RBSx-LuxI-TT</a> measurement systems. <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here: </p><br />
<br />
<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg" class="thumbimage" height="70%" width="68%"></a></div></div><br />
<br />
<br />
<br />
<p align='justify'><br />
Since the measurement systems are only assayed in the exponential growth phase, in the equation (3) N &lt; <N<sub>max</sub> is assumed. <br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known and summarized in the table below:</p><br />
<br />
<div align="center"><br />
<table align="center" class='data' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.004925</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
</div><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, k<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>p<sub>Tet</sub>-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
<br />
<div align="justify"><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div></div><br />
<br />
<br />
<div align="justify"><p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p></div><br />
<br />
<br />
<div align=center"><br />
<table align="center" class='data'><br />
<tr><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>k<sub>M,LuxI</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>3.56*10<sup>-9</sup></td><br />
<td class='row'>6.87*10<sup>3</sup></td><br />
<td class='row'> 87 </td><br />
<td class='row'>8.5</td><br />
<td class='row'>ND</td><br />
<td class='row'> 252 </td><br />
</tr></table><br />
</div><br />
<br> <br />
</p><br />
</p><br />
<br />
<p align='justify'><br />
The provided parameters k<sub>M</sub> and V<sub>max</sub> represent the enzymatic activity of LuxI, described by our model. They must not be confused with the operative parameters of the Michaelis-Menten relation. <br />
These synthetic parameters have a great importance, since they can be used in more complicated models in order to predict the behavior of complex circuits.<br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!-- ----------------- --><br />
<!-- --AiiA description-- --><br />
<!-- ----------------- --><br />
<br />
<br />
<a name='AiiA'></a><h2>AiiA gene - BBa_C0060</h2><br />
<br />
<p align='justify'><br />
The activity of AiiA enzyme has been evaluated by testing the measurement systems <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a>. <br />
Similar to LuxI, a system of differential equations (referring to <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>) has been derived. <br />
</p><br />
<br />
<div style='text-align:justify'><br />
<div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg" class="thumbimage" height="70%" width="68%"></a><br />
</div></div><br />
<br />
<p align='justify'><br />
The parameters k<sub>cat</sub>, k<sub>M,AiiA</sub> and &alpha;<sub>RBSx</sub> would have been estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>p<sub>Tet</sub>-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
Unfortunately, their estimation revealed difficult.<br><br />
In the first experiments with the measurement system <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a> in LOW-COPY at pH=7 no degradation of HSL was observed. The collected data are shown in the figure below. HSL degradation is identical in the measurement system and in the negative control after 21 hours.<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<div align="justify">Further investigation on the enzyme were performed in more suitable conditions, to evaluate its intrinsic activity. AiiA activity was investigated in HIGH COPY plasmid in <em>E. coli</em> TOP10, in order to understand if the enzyme worked. In this case, a significant difference in degradation between p<sub>Tet</sub>-RBSx-AiiA-TT and the negative control was observed, also just after 7 hours. That was the proof of the good functioning (in HIGH COPY) of the enzyme.</div><br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<div align="justify"><br />
Since the enzyme activity had been assessed, some suitable working conditions were investigated for AiiA. Since our studies on HSL stability for different pHs has shown that its stability improves at pH=6.0, this experimental condition was used to test once again MGZ1 strain with the system expressing AiiA in low copy number plasmid. <br />
<br />
Unfortunately, we had some problems with the biosensor, BBa_T9002, that didn't work as usual, resulting in a calibration curve modified. So, k<sub>M,AiiA</sub> and k<sub>cat</sub> were impossible to be estimated from these data. We decided to estimate them from experimental data coming from Tecan tests preformed in <em>E.COLI</em> TOP10, where aiiA gene was cloned in a high copy numebr plasmid, pSB1A2, downstream p<sub>Tet</sub> with different RBSs; in this way we could have some semi-quantitative information about the order of magnitude of these parameters. Taking into account the different copy number, we tried to simulate our model behavior with reasonable values for AiiA parameters.</div><br />
<br><br />
<br />
</p><br />
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<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!-- ----------------- --><br />
<!-- ---pSB1C3-mRFP-- --><br />
<!-- ----------------- --><br />
<br />
<a name='K999'></a><h2>pSB1C3 plasmid with mRFP between S and P bearing p<sub>Tet</sub> (easy-to-clone)</h2><br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/e/ee/BBa_I13507.jpg" class="thumbimage" width="50%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<div align="justify">This vector was designed and realized in order to facilitate the cloning of coding sequences downstream of the strong promoter p<sub>Tet</sub>. This vector was assembled by ligating S-P excided mRFP coding sequence from BBa_J61002 and ligating it in BBa_R0040 cut with S and P. Thus, the resulting vector contains mRFP between S and P. p<sub>Tet</sub> can be easily excided (E-P) and moved in the desired vector (E-P) and then the desired coding sequence can be easily assembled by digesting S-P the vector and X-P the coding sequence, thus obtaining a final part that is standard10-compatible.</div><br><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<br />
<a name='Improvement'></a><h1>Existing parts: sequence debugging</h1><br />
<br />
<div align="justify"><br />
<br />
<ol><ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516021 "> BBa_K516021 </a> (wiki name: E10 ) RBS31-AiiA-TT Rebuilt existing part from BBa_I13914 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516022 "> BBa_K516022 </a> (wiki name: E11 ) RBS32-AiiA-TT Rebuilt existing part from BBa_I13912 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516030 "> BBa_K516030 </a> (wiki name: E5 ) RBS30-mRFP-TT Rebuilt existing part from BBa_S04180 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516032 "> BBa_K516032 </a> (wiki name: E7 ) RBS32-mRFP-TT Rebuilt existing part from BBa_ J133000 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) p<sub>Tet</sub>-RBS34-LuxI Rebuilt existing part from BBa_S03623 (DNA available, only 2008 kit, inconsistent) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) p<sub>Tet</sub>-RBS32-AiiA-TT Rebuilt existing part from BBa_ J22071 (2008 only, Bad sequencing) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) p<sub>Tet</sub>-RBS34-AiiA-TT Rebuilt existing part from BBa_K077047 (Part deleted) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) p<sub>Tet</sub>-RBS32-mRFP-TT Rebuilt existing part from BBa_I20252 (DNA planning) </li></ul></ol><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/File:UNIPV_HSL.PNGFile:UNIPV HSL.PNG2011-09-21T17:45:01Z<p>Nickpv: uploaded a new version of &quot;File:UNIPV HSL.PNG&quot;</p>
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<div></div>Nickpvhttp://2011.igem.org/File:UNIPV_HSL.PNGFile:UNIPV HSL.PNG2011-09-21T17:44:03Z<p>Nickpv: uploaded a new version of &quot;File:UNIPV HSL.PNG&quot;</p>
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<div></div>Nickpvhttp://2011.igem.org/File:UNIPV_HSL.PNGFile:UNIPV HSL.PNG2011-09-21T17:43:13Z<p>Nickpv: uploaded a new version of &quot;File:UNIPV HSL.PNG&quot;</p>
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<div></div>Nickpvhttp://2011.igem.org/File:UNIPV_HSL.pngFile:UNIPV HSL.png2011-09-21T17:38:00Z<p>Nickpv: </p>
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<div></div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Project/ResultsTeam:UNIPV-Pavia/Project/Results2011-09-21T17:05:53Z<p>Nickpv: </p>
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<div>{{main}}<br />
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<h2 class="art-postheader"><br />
Results<br />
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<div class="cleared"></div><br />
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#assembly"><span class="tocnumber">1</span> <span class="toctext">Part assembly</span></a> <br />
<li class="toclevel-1"><a href="#characterization"><span class="tocnumber">1</span> <span class="toctext">Characterization of basic modules</span></a> <br />
<ul><br />
<li class="toclevel-2"><a href="#promoters"><span class="tocnumber">2.1</span> <span class="toctext">Promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#enzymes"><span class="tocnumber">2.2</span> <span class="toctext">Characterization of the activity of the enzymes AiiA and LuxI</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.3</span> <span class="toctext">Characterization of RBS efficiency</span></a></li><br />
</ul><br />
<li class="toclevel-1"><a href="#growth"><span class="tocnumber">3</span> <span class="toctext">Identification of bacterial growth parameters</span></a></li><br />
<li class="toclevel-1"><a href="#HSL"><span class="tocnumber">4</span> <span class="toctext">Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</span></a></li><br />
<li class="toclevel-1"><a href="#t9002"><span class="tocnumber">5</span> <span class="toctext">Characterization of BBa_T9002 biosensor</span></a></li><br />
</ul></td></tr></table><br />
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<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C. For the cloning of the parts, <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#TOP10'><em>E. coli</em> TOP10</a> was used. <br />
</em><br />
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<a name='assembly'></a><h1>Parts assembly</h1><br />
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<a name='characterization'></a><h1>Characterization of basic modules</h1><br />
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<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------pTet and pLux-----------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<a name='promoters'></a><h2>Characterization of promoters pTet and pLux</h2><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!--------------------------------><br />
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<!--------AiiA and LuxI-----------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='enzymes'></a><h2>Characterization of enzymes AiiA and LuxI</h2><br />
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<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!--------------------------------><br />
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<!--------------RBS---------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
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<a name='rbs'></a><h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
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<br />
<a name='growth'></a><h2>Identification of bacterial growth parameters</h2><br />
<br />
<div class="listcircle"><br />
<p align='justify'><br />
<br />
The bacterial growth curve has been modelled as a logistic curve and is represented by the following equation:<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br><br />
N(0)=n<sub>0</sub><br />
<br><br><br />
<br />
where &mu; represents the growth rate of the cells (<em>E. coli</em> MGZ1 in M9 supplemented medium) and N<sub>max</sub> represents the maximum number of cells in the well. For a detailed description of the parameters, see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>modelling section</a>. For details on parameters identification, see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#N'>identification section.</a> <br />
The growth curves in all the performed experiments are measured in O.D.<sub>600</sub>. Since the <em>N</em> species in the model is expressed in <em>cell number</em>, a conversion factor has been estimated. The conversion factor <b>K<sub>O.D.toC.F.U.</sub></b> has been estimated as follows.<br />
<ol><ul><br />
<li>Two cultures C1 and C2 (MGZ1 cells) were grown in 1ml M9 medium till saturation (ON liquid culture, 37°C, 220 rpm).</li><br />
<li>Next morning, both C1 and C2 were diluted in M9 medium with a final volume of 1ml with the following dilution factors:<br />
<ul><li>1:1</li><li>1:10</li><li>1:100</li><li>1:1000</li><br />
</ul> in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm.<br />
</li><br />
<li>After 1 hour, O.D.<sub>600</sub> was measured using TECAN microplate reader (don't forget to measure a M9 sample for blanking!)<br><br />
<em>NB: from now on, cultures must be placed in ice to stop cell growth.</em></li><br />
<li>At the same time, proper dilution of the cultures were plated on LB agar plates.<br><br />
<em>NB: All the dilutions are performed moving 100 &mu;l of culture in previously ice-chilled 900 &mu;l fresch M9. 100 &mu;l of the final dilution are plated (It still represents a 1:10 dilution!)</em></li><br />
<li>Plates were grown overnight and next morning C.F.U. were counted.</li><br />
<li>C.F.U. values were corrected by the dilution factor and a linear regression (N vs O.D.<sub>600</sub>) was performed in order to evaluate the conversion factor <b>K<sub>O.D.toC.F.U.</sub></b>. </li><br />
<li><b>K<sub>O.D.toC.F.U.</sub></b> was used as conversion factor to multiply the O.D.<sub>600</sub> value of saturation in the growth curves (~0,5). </li><br />
<br />
</ul></ol><br />
<br />
<p>The results are summarized in the table and in the figure below: </p><br />
<br />
<center><br />
<table class='data'><tr><td class='row'><b> Culture </b></td><td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><td class='row'><b> C.F.U. </b></td><td class='row'><b> Dilution Factor (10^) </b></td><td class='row'><b> N </b></td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,367950002 </td><td class='row'> 0,758004416 </td><td class='row'> 990 </td><td class='row'> 5 </td><td class='row'> 990000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,044649998 </td><td class='row'> 0,091982322 </td><td class='row'> 141 </td><td class='row'> 6 </td><td class='row'> 1410000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,004799999 </td><td class='row'> 0,009888356 </td><td class='row'> 136 </td><td class='row'> 5 </td><td class='row'> 136000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,000549998 </td><td class='row'> 0,001133037 </td><td class='row'> 20 </td><td class='row'> 6 </td><td class='row'> 200000000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,54840003 </td><td class='row'> 1,129744917 </td><td class='row'> 165 </td><td class='row'> 4 </td><td class='row'> 16500000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,058200002 </td><td class='row'> 0,119896339 </td><td class='row'> 23 </td><td class='row'> 5 </td><td class='row'> 23000000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,008700002 </td><td class='row'> 0,017922652 </td><td class='row'> 251 </td><td class='row'> 3 </td><td class='row'> 2510000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,000100002 </td><td class='row'> 0,000206011 </td><td class='row'> 24 </td><td class='row'> 4 </td><td class='row'> 2400000 </td> </tr><br />
</table></center><br><br />
</p><br />
<br />
<center><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"><br />
</center><br />
<br />
<p>The estimation of &mu; parameter was performed by determining the slope of the logarithmic curve of O.D.<sub>600</sub> in exponential phase. Exponential phase was determined by visual inspection as the linear phase of the logarithmic curve of O.D.<sub>600</sub>. <br />
<br><br><br />
The estimated parameters are summarized in the table below:<br />
<br><br />
</p><br />
<center><br />
<table width='50%' class='data'><tr><br />
<td class='row'><b>N<sub>max</sub> [cell number]</b></td><br />
<td class='row'><b>&mu; [min<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><td class='row'>1*10<sup>9</sup></td><br />
<td class='row'>0.004925</td><br />
</tr><br />
</table><br />
</center><br />
<p>&nbsp;</p><br />
<p>The reported value of &mu; corresponds to a doubling time of 142 minutes.<br />
<br />
</p><br />
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<a name='HSL'></a><h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'><br />
In order to estimate the spontaneous degradation rate of HSL in M9 medium and in a culture of MGZ1 cells as a function of pH, two simple tests have been performed.<br><br />
Two different M9 media were prepared, one with the nominal pH (7.0) and one with pH=6.0.<br><br />
These media, now named respectively M9<sub>pH 7</sub> and M9<sub>pH 6</sub>, were added with a known concentration of HSL (100 nM) and then incubated at 37°C, 220 rpm (NB: the media were not infected with any culture but the standard growth conditions were reproduced). The amount of HSL present in the medium was assayed through the BBa_T9002 biosensor at 4 time points: <br><br />
<ul><br />
<li>t=0 h;</li><br />
<li>t=1 h;</li><br />
<li>t=2 h;</li><br />
<li>t=4 h;</li><br />
<li>t=8 h;</li><br />
</ul><br />
<p>The obtained time series of HSL amounts were processed to evaluate the time constant governing the dynamic of HSL degradation, supposing an exponential decay. The results are reported in the table below: </p><br />
<br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><br />
</td><br />
<td class='row'><br />
<b>t<sub>1/2</sub><sup>*</sup> [h]</b><br />
</td><td class='row'><br />
<b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b><br />
</td><br />
</tr><br />
<tr><td class='row'><b>M9<sub>pH 6</sub></b><br />
</td><br />
<td class='row'>32<br />
</td><br />
<td class='row'>0.022<br />
</td><br />
</tr><br />
<tr><td class='row'><b>M9<sub>pH 7</sub></b><br />
</td><br />
<td class='row'>8<br />
</td><br />
<td class='row'>0.087<br />
</td><br />
</tr><br />
</table><br />
</center><br />
<p><br />
The described experiment was repeated with a MGZ1 culture in order to evaluate the effect of culture on HSL stability. The estimated values are reported in the table below:</p><br />
<br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><br />
</td><br />
<td class='row'><br />
<b>t<sub>1/2</sub><sup>*</sup> [h]</b><br />
</td><td class='row'><br />
<b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b><br />
</td><br />
</tr><br />
<tr><td class='row'><b>Culture<sub>pH 6</sub></b><br />
</td><br />
<td class='row'>&#8734;<br />
</td><br />
<td class='row'>0<br />
</td><br />
</tr><br />
<tr><td class='row'><b>Culture<sub>pH 7</sub></b><br />
</td><br />
<td class='row'>19<br />
</td><br />
<td class='row'>0.037<br />
</td><br />
</tr><br />
</table><br />
</center><br />
<br />
<p>It is evident from the reported data that the spontaneous degradation of HSL is negligible when CTRL+E is implemented in MGZ1 cells grown in M9 medium with pH=6.0 and pH=7.0.<br><br />
Thus in the simulations we set &gamma;<sub>HSL</sub>=0; <br />
<br />
</p><br />
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<a name='t9002'></a><h2>Characterization of BBa_T9002 biosensor</h2><br />
As described in the <a href="https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002">modelling section</a>, BioBrick <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> is an HSL biosensor, which provides a non linear relationship between HSL input and S<sub>cell</sub> output. More precisely, the characteristic sigmoidal curve requires synthetic parameters for its accurate identification. These are the minimum and maximum values, the swtich point (i.e., the curve inflection point), and the upper and lower boundaries of linearity. This biosensor revealed greatly reliable, providing measurement repeatability and minimal experimental noise. Referring to its activation formula, the calibration curve is shown below.<br><br><br />
<br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage" width="47%" height="50%"></a></div></div><br />
<br><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage" width="100%"></a></div></div><br />
<br />
<br />
<center><br />
<table width='50%' class='data'><tr><br />
<td class='row'><b>Minimum [S<sub>cell</sub>]</b></td><br />
<td class='row'><b>Maximum [S<sub>cell</sub>]</b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Lower boundary of linearity [nM]</b></td><br />
<td class='row'><b>Upper boundary of linearity [nM]</b></td><br />
</tr><br />
<br />
<tr><br />
<td class='row'>17.31</td><br />
<td class='row'>739.4</td><br />
<td class='row'>1.39</td><br />
<td class='row'>0.38</td><br />
<td class='row'>5.07</td><br />
<br />
</tr><br />
</table><br />
</center><br />
<br><br><br />
In order to determine the threshold sensitivity of T9002 biosensor, experiments were performed with several HSL inductions minimally interspaced in the region of low detectability. Hypothesizing that the inducer is 1:20 diluted (as for all of our tests), the minimum detectable HSL concentration is 3 nM.<br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Parts/CharacterizedTeam:UNIPV-Pavia/Parts/Characterized2011-09-21T16:51:00Z<p>Nickpv: </p>
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<div>{{main}}<br />
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Characterized Parts<br />
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#new"><span class="tocnumber">1</span> <span class="toctext">New Parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#J101"><span class="tocnumber">1.1</span> <span class="toctext">J23101x series with different RBSs</span></a></li><br />
<li class="toclevel-2"><a href="#pTetLuxI"><span class="tocnumber">1.2</span> <span class="toctext">LuxI Expression cassette driven by aTc</span></a></li><br />
<li class="toclevel-2"><a href="#pTetAiiA"><span class="tocnumber">1.3</span> <span class="toctext">AiiA Expression cassette driven by aTc</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Existing"><span class="tocnumber">2</span> <span class="toctext">Characterization of existing parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#note"><span class="tocnumber">2.1</span> <span class="toctext">Notes on promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.2</span> <span class="toctext">RBSs from the community collection</span></a></li><br />
<li class="toclevel-2"><a href="#pLux"><span class="tocnumber">2.3</span> <span class="toctext">p<sub>Lux</sub> - a 3OC<sub>6</sub>-HSL-in PoPs-out device</span></a></li><br />
<li class="toclevel-2"><a href="#pTet"><span class="tocnumber">2.4</span> <span class="toctext">p<sub>Tet</sub> - BBa_R0040</span></a></li><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.5</span> <span class="toctext">LuxI - BBa_C0061</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.6</span> <span class="toctext">AiiA - BBa_C0060</span></a></li><br />
<li class="toclevel-2"><a href="#K999"><span class="tocnumber">2.7</span> <span class="toctext">pSB1C3 vector with mRFP between S and P for easy cloning</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Improvement"><span class="tocnumber">3</span> <span class="toctext">Sequence debugging</span></a></li><br />
</ul></td></tr></table><br />
</div><br><br />
<br />
<br />
<br />
<br><br><br />
<br />
<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part: pSB4C5">pSB4C5</a>. <br />
</em><br />
<br><br />
<br><br />
<br />
<br />
<div class="listcircle"><br />
<a name='new'></a><h1>New parts</h1><br />
<a name='J101'></a><h2>J23101x series</h2><br />
<br />
<ol type='1'><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> (wiki name: J101-E5 ) J101-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> (wiki name: J101-31 ) J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> (wiki name: J101-E7 ) J101-RBS32-mRFP-TT </li><br />
</ol><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- J23101x description --><br />
<!-- ----------------- --><br />
<p align='justify'><br />
<div align="justify"><p>BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in '<a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>data analysis</a>' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene.</p><br />
<p>The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P. This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts.</p><br />
<p>Salis et al. [Nat Biotec, 2009] stated that <em> 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels'</em> and again <em>'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.'</em></p><br />
<br />
<p>For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS. In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.</p><br />
<br />
<p><br><em><b>NB</b>: in the RPU computation, the J23101-RBS34-mRFP-TT (<A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_J23101">BBa_J23101</a>) in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency.</em></p></div><br />
<br />
<table align='center'><tr><td width='50%' ><br />
<table class='data'><br />
<tr><br />
<td class='row'><b>J23101 promoter with RBS</b><br />
</td><br />
<td class='row'><b>S<sub>cell</sub></b><br />
</td><br />
<td class='row'><b>R.P.U.s</b><br />
</td><br />
</tr><br />
<tr><td class='row'>RBS30<br />
</td><br />
<td class='row'>122 [13.23]</td><br />
<td class='row'>2.45 [0.27]</td><br />
</tr><br />
<tr><td class='row'>RBS31<br />
</td><br />
<td class='row'>2 [0.25]</td><br />
<td class='row'>0.04 [0.01]</td><br />
</tr><br />
<tr><td class='row'>RBS32<br />
</td><br />
<td class='row'>20 [1.7]</td><br />
<td class='row'>0.40 [0.03]</td><br />
</tr><br />
<tr><td class='row'>RBS34<br />
</td><br />
<td class='row'>50 [1.87]</td><br />
<td class='row'>1 [0.02]</td><br />
</tr><br />
</table></td><br />
<div align="center">Data are provided as average [Standard error].</div><br><br />
<td width='50%' ><br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="image"><img alt="RPUs of J23101 promoter with different RBSs" src="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="thumbimage" width="80%"></a></div></div><br />
</td><br />
</tr></table><br />
<br><br><br />
<div align="justify">On the other hand, if the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.</div><br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- pTetLuxI description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetLuxI'></a><h2>LuxI expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> (wiki name: E13 ) pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> (wiki name: E14 ) pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> (wiki name: E15 ) pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI </li></ul></ol><br />
<br />
<br><br><br />
<p align='justify'><em>Though these parts don't have a transcriptional terminator, they have been characterized in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a>, that contains the BBa_B0054 terminator. This choice is motivated by the need to reproduce the exact experimental context of the final circuit, as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Solution#Circuit_design'>solution section</a>. <br />
</em></p><br />
<br><br><br />
<p align='justify'><br />
LuxI has been characterized in terms of enzymatic activity under the regulation of p<sub>Tet</sub> promoter. <br />
</p><br />
<p align='justify'><br />
K<sub>M,LuxI</sub> and V<sub>max</sub> parameters representing its activity have been estimated and the promoter strength (represented by a synthetic parameter &alpha;<sub>pTet</sub> for every pTet-RBS combination) at full induction (100 ng/ml) has been estimated too with a simultaneous fitting of the available data. <br />
<br />
</p><br />
<br />
<br />
<p align='justify'><br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002'>modelling section</a>). <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system describing the behavior of this measurement circuit is reported here: </p><br />
<br />
<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg" class="thumbimage" height="70%" width="68%"></a></div></div><br />
<br />
<br />
<br />
<p align='justify'><br />
Since the measurement systems are only assayed in the exponential growth phase, in the equation (3) N &lt; N<sub>max</sub> is assumed. <br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known and summarized in the table below:</p><br />
<br />
<div align="center"><br />
<table align="center" class='data' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.004925</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
</div><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, k<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>pTet-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
<br />
<div align="justify"><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div></div><br />
<br />
<br />
<div align="justify"><p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p></div><br />
<br />
<br />
<div align="center"><br />
<table align="center" class='data'><br />
<tr><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>k<sub>M,LuxI</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>3.56*10<sup>-9</sup></td><br />
<td class='row'>6.87*10<sup>3</sup></td><br />
<td class='row'>87</td><br />
<td class='row'>8.5</td><br />
<td class='row'>ND</td><br />
<td class='row'>252</td><br />
</tr></table><br />
</div><br />
<br> <br />
</p><br />
</p><br />
<br />
<br />
</p><br />
<br />
<br />
<p align='justify'><br />
The collected data have been used to identify the parameters of our model. Despite the data-poor context, the model predictions fit the experimental data, thus demonstrating that the equation that models the HSL synthesis by LuxI is a good approximation of real processes. <br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- pTetAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetAiiA'></a><h2>AiiA expression cassette driven by aTc-inducible p<sub>Tet</sub> promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> (wiki name: E24 ) p<sub>Tet</sub>-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> (wiki name: E25 ) p<sub>Tet</sub>-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) p<sub>Tet</sub>-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) p<sub>Tet</sub>-RBS34-AiiA-TT </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity.<br />
<br />
Similar to LuxI, a system of differential equations (referring to <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>) has been derived. <br />
</p><br />
<br />
<div style='text-align:justify'><br />
<div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg" class="thumbimage" height="70%" width="68%"></a><br />
</div></div><br />
<br />
<p align='justify'><br />
The parameters k<sub>cat</sub>, k<sub>M,AiiA</sub> and &alpha;<sub>RBSx</sub> would have been estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>p<sub>Tet</sub>-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
Unfortunately, their estimation revealed difficult.<br><br />
In the first experiments with the measurement system <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a> in LOW-COPY at pH=7 no degradation of HSL was observed. The collected data are shown in the figure below. HSL degradation is identical in the measurement system and in the negative control after 21 hours.<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<div align="justify">Further investigation on the enzyme were performed in more suitable conditions, to evaluate its intrinsic activity. AiiA activity was investigated in HIGH COPY plasmid in <em>E. coli</em> TOP10, in order to understand if the enzyme worked. In this case, a significant difference in degradation between p<sub>Tet</sub>-RBSx-AiiA-TT and the negative control was observed, also just after 7 hours. That was the proof of the good functioning (in HIGH COPY) of the enzyme.</div><br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<div align="justify"><br />
Experiments on these parts gave us the opportunity to characterize only the activity of the enzyme in <em>E. COLI</em> TOP10 in high copy number plasmid, providing only some information about the order of magnitude of the model parameters, which has been designed to work in <em>E. COLI</em> MGZ1 in low copy number plasmid.<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<a name='Existing'></a><h1>Existing parts</h1><br />
<br />
<a name='note'></a><h2>Notes for promoter characterization</h2><br />
<p align='justify'><br />
<p>Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection.</p><br />
<p>The assembled RBSs are:</p><br />
<br><br />
<div align='center'><br />
<table class='data'><br />
<tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr><br />
<tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr><br />
<tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr><br />
<tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr><br />
<tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr><br />
</table></div><br />
<br><br />
<div align="justify"><p>For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.</p><br />
<p>The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. <br />
RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP).</p><br />
<p>For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.</p><br />
</p><br />
<p>Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least squares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs:</p><p></p><br />
<p><ol><ul><li><b><br />
RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity</p><br />
</li><p><li><b><br />
RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity</p><br />
</li><p><li><br />
<b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and it is representative of the position of linear region</p><br />
</li><br />
<p><li><br />
<b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</div></li></p><br />
</ul></ol><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --RBS description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='rbs'></a><h2>RBSs</h2><br />
<p align='justify'><br />
<p><div align="justify">The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of protein produced. <br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element.</p><br />
<p>The evaluation of RBS efficiency can be performed in a very intuitive fashion:</p><br />
<ol><br />
<li>select the RBSs you want to study</li><br />
<li>assemble them in a Promoter - XX - Coding sequence circuit</li><br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/d/d1/Vettore_base.jpg" class="thumbimage" width="60%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<li>measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS (<a href='http://partsregistry.org/wiki/index.php/Part: BBa_B0034'>BBa_B0034</a>). </li></div></ol><br><br />
<br />
<br />
<div align="justify"><p>This simple measurement system allows the quantification of RBS efficiency depending on the experimental context (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments.</p><br />
<p>To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.</p><br />
<p>In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, p<sub>Tet</sub>, p<sub>Lux</sub>). The system output was measured and the RBS efficiency evaluated. The results are summarized in the table below:</div></p><br />
<br><br />
<div align="center"><table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>p<sub>Lux</sub></sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>p<sub>Tet</sub></sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>J23101</sub></b><sup>**</sup></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>0.40</td><td class='row'>1.6814</td><td class='row'>2.45</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.01</td><td class='row'>ND</td><td class='row'>0.04</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.19</td><td class='row'>0.4193</td><td class='row'>0.40</td><td class='row'>0,3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table></div><br />
<br />
<br><br />
<div align="justify">On the other hand, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (p<sub>Tet</sub>-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below:</div><br><br />
<br />
<div align="center"><table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>mRFP</sub></b><sup>**</sup></td><br />
<td class='row'><b>eff<sub>AiiA</sub></b><sup>***</sup></td><br />
<td class='row'><b>eff<sub>LuxI</sub><sup>****</sup></b></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>1.72</td><td class='row'>0.53</td><td class='row'>0.45</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.03</td><td class='row'>0.83</td><td class='row'>0.028</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.37</td><td class='row'>0.50</td><td class='row'>N.D.</td><td class='row'>0.3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table></div><br />
<br><br />
<p align='justify'><br />
<div align="justify"><p><sup>*</sup> The RBS efficiency for inducible devices expressing mRFP was estimated as the ratio of the AUCs (Area under the curve) of the induction curve of the system with the studied RBS and the B0034 reference: AUC<sub>P, RBSx</sub>/AUC<sub>P, B0034</sub></p><br />
<p><sup>**</sup> The RBS efficiency for constitutive promoters expressing mRFP was computed as the ratio between S<sub>cell<sub>P, RBSx</sub></sub>/S<sub>cell<sub>P, B0034</sub></sub></p><br />
<p><sup>***</sup> The RBS efficiency for p<sub>Tet</sub> promoter driving the expression of AiiA enzyme was computed as the ratio &alpha;<sub>p<sub>Tet</sub>, RBSx</sub>/&alpha;<sub>p<sub>Tet</sub>, B0034</sub> estimated for the measurement system p<sub>Tet</sub>-RBSx-AiiA-TT. &alpha;<sub>p<sub>Tet</sub></sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#AiiA'>here</a>. p<sub>Tet</sub> was tested at full induction (100 ng/ml).</p><br />
<p><sup>****</sup> The RBS efficiency for promoters driving the expression of LuxI was computed as the ratio &alpha;<sub>p<sub>Tet</sub>, RBSx</sub>/&alpha;<sub>p<sub>Tet</sub>, B0034</sub> estimated from the measurement systems p<sub>Tet</sub>-RBSx-LuxI. &alpha;<sub>p<sub>Tet</sub></sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#LuxI'>here</a>. p<sub>Tet</sub> was tested at full induction (100 ng/ml).</p></div><br />
<br />
<br><br />
</p><br />
The parts we used to characterize the RBSs are listed here:<br />
<ol><br />
<ul><li>mRFP expression with different promoters</li><br />
<ul><br />
<li>J23101</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> J101-RBS30-RFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> J101-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_J23101 "> BBa_J23101 </a> J101-RBS34-mRFP-TT </li><br />
</ul><br />
<li>p<sub>Tet</sub></li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> p<sub>Tet</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
<li>p<sub>Lux</sub></li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
</ul><br />
<li>p<sub>Tet</sub> driving the expression of different genes</li><br />
<ul><li>mRFP</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> p<sub>Tet</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
<li>AiiA</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> p<sub>Tet</sub>-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> p<sub>Tet</sub>-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> p<sub>Tet</sub>-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> p<sub>Tet</sub>-RBS34-AiiA-TT </li><br />
</ul><br />
<li>LuxI</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> p<sub>Tet</sub>-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> p<sub>Tet</sub>-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> p<sub>Tet</sub>-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> p<sub>Tet</sub>-RBS34-LuxI </li><br />
</ul></ul><br />
</ul></ol><br />
<br />
<br />
</p><br />
<p align='justify'><br />
The results reported in the table suggest that the RBS efficiency ranking is not always maintained. In particular, for the different promoters driving the expression of mRFP, the ranking of the declared efficiencies is maintained for p<sub>Lux</sub>, but not for p<sub>Tet</sub> and J23101. The RBS B0030 results to be the most efficient for both J23101 and p<sub>Tet</sub>, but not for p<sub>Lux</sub> (NB: this effect might be due to an effective non-modularity of RBS, but also to saturating phenomena occurring for this very strong promoter at full induction). RBS B0031 always shows a very low efficiency, while B0032 an intermediate efficiency between B0031 and the stronger RBSs B0030 and B0034. <br><br />
For what concerns the encoded gene variation, more significant differences can be observed. RBS B0030 has the higher efficiency only for mRFP, while the values for AiiA and LuxI are similar (~0.5). Unexpectedly, the weak RBS B0031 has a higher efficiency with AiiA gene (0.83), while with mRFP and LuxI. With B0032 no activity was observed for LuxI, while for AiiA and mRFP the results are quite consistent with the one reported above. <br />
</p><br />
<p align='justify'><br />
These results are encouraging: though the partial non-modularity of RBS with the encoded gene is confirmed, the hypothesis of modularity with the promoter is to some extent confirmed. Three classes of efficiencies were identified:<br />
<ul><br />
<li>low efficiency RBS (B0031)</li><br />
<li>medium efficiency RBS (B0032)</li><br />
<li>high efficiency RBSs (B0030, B0034)</li><br />
</ul> <br />
</p><br />
<br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --pLux description- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<a name='pLux'></a><h2>p<sub>Lux</sub> promoter</h2><br />
<p align='justify'><br />
<br />
<br />
</p><br />
<ol><ul><br />
<br />
<br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> (wiki name: E17 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> (wiki name: E18 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> (wiki name: E19 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> (wiki name: E20 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS34-mRFP-TT </li></ul><br />
</ol><br />
<br />
<p align='justify'><br />
The estimated parameters for the Hill functions are summarized in the table below. For more details on parameter estimation, see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>model section</a>. <br />
</p><br />
<br />
<table class='data' width='100%'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Lux</sub></sub> [(AUr/min)/cell]</b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Lux</sub></sub> [-]</b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Lux</sub></sub> [-]</b></td><br />
<td class="row"><b>k<sub>p<sub>Lux</sub></sub> [ng/ml]</b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">438 [10]</td><br />
<td class="row">0.05 [>100]</td><br />
<td class="row">2 [47]</td><br />
<td class="row">1.88 [27]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">9.8 [7]</td><br />
<td class="row">0.11 [57]</td><br />
<td class="row">1.2 [29]</td><br />
<td class="row">1.5 [26]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">206 [3]</td><br />
<td class="row">0 [>>100]</td><br />
<td class="row">1.36 [10]</td><br />
<td class="row">1.87 [9]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">1105 [6]</td><br />
<td class="row">0.02 [>100]</td><br />
<td class="row">1.33 [19]</td><br />
<td class="row">2.34 [18]</td><br />
</tr><br />
</table><br />
<div align="center">Data are provided as average [CV%].</div><br><br />
<br />
<br />
<div align="justify"><br />
<p>From this table, it is evident that, whilst &alpha;<sub>p<sub>Lux</sub></sub> assumes significantly different values for different RBSs, &eta;<sub>pLux</sub> and k<sub>pLux</sub> assume very similar values. This result shows that RBS variation modulates the amplitude of Hill function, not affecting the shape of the curve. The four induction curves result to be the same Hill function modulated in amplitude by a parameter, that is the estimated RBS efficiency for this measurement system.</p><br />
<p>These results are quite encouraging, because suggest that, given the non-modular behavior of RBS dpending on the encoded gene, the RBS has a modular behaviour respect to the promoter.</p><br />
<p>The operative parameters are summarized in the table below:</p></div><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>4.28</td><td class='row'>0.20</td><td class='row'>1.08</td><td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>4.93</td><td class='row'>0.55</td><td class='row'>0.25</td><td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>9.49</td><td class='row'>0.02</td><td class='row'>0.47</td><td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>21.53</td><td class='row'>0.51</td><td class='row'>0.53</td><td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<p align='justify'>These operative parameters provide useful information on the behavior of this 3OC6-HSL inducible device. RPU<sub>max</sub> assumes very different values in terms of RPUs.<br />
This can't be explained by RBS modulation, since RPUs have been evaluated by normalizing S<sub>cell</sub> of p<sub>Lux</sub>-RBSx for the one of J23101-RBSx. It is evident that some nonlinear effect on maximum strength, maybe due to saturation phenomena on protein expression, occur. <br />
The same RPUs should be observed for every RBS, since the normalization by the standard reference used for RPUs computation should eliminate the RBS contribute. Here different RPUs are observed, maybe due to nonlinear RBS behavior or to saturation phenomena occurring with this very strong promoter. The switch point and linear boundaries are quite constant in all the cases, showing that the linear region of this system is not affected by RBS changes.</p><br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/5/50/E17_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/5/50/E17_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/c/c4/E18_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c4/E18_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/7/78/E19_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/78/E19_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/5/55/E20_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/5/55/E20_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- -pTet description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='pTet'></a><h2>p<sub>Tet</sub> promoter</h2><br />
<br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> (wiki name: E21 ) p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> (wiki name: E22 ) p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) p<sub>Tet</sub>-RBS32-mRFP-TT<br />
</li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT <br />
</li></ul><br />
</ul><br />
</ol><br />
<br />
<div align="justify"><br />
<p>The protocols for the characterization of p<sub>Tet</sub> promoter are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#pTet_protocol'>p<sub>Tet</sub> measurement section</a>.</p><br />
<p>The data collected from the mRFP measurement systems were processed as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'> data analysis section</a>. The induction curves were obtained by fitting a Hill function as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>modelling section</a> and the estimated <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>parameters</a> for p<sub>Tet</sub> are reported in the pictures and in table below. </p><br />
<p>This promoter is widely studied and characterized usually using the strong RBS BBa_B0034. Here we have characterized its transcriptional strength as a function of aTc induction (ng/ul) for different RBSs. Three different induction curves were obtained and are reported in figure:</p></div><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<br />
<br />
<!-- <td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/9/93/E33_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/9/93/E33_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td> --><br />
<br />
<table width='100%'><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/9/99/E34_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/9/99/E34_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/e/e4/E35_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e4/E35_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<p align='justify'><br />
The estimated parameters of the Hill curves described in the figures are summarized in the table below:<br />
</p><br />
<br><br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub> [(AUr/min)/cell]</b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub> [-]</b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub> [-]</b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub> [nM]</b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">230.67 [3.7]</td><br />
<td class="row">0.028 [91.61]</td><br />
<td class="row">4.61 [23.73]</td><br />
<td class="row">8.75 [4.16]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">55.77 [12]</td><br />
<td class="row">1.53E-11 [>>100]</td><br />
<td class="row">4.98 [57.62]</td><br />
<td class="row">7.26 [14.98]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">120 [5.95]</td><br />
<td class="row">0.085 [40.6]</td><br />
<td class="row">24.85 [47.6]</td><br />
<td class="row">9 [5.43]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br><br><br />
<br />
<p align='justify'><br />
The measurement system p<sub>Tet</sub>-B0031-mRFP-TT couldn't be assayed because its fluorescence output is under the detectability threshold of our measurement instrument. For this reason, the parameters of the corresponding Hill curve couldn't be estimated and are reported as 'Not Determined' ND. <br />
</p><br />
<br />
<div align='justify'><br />
<p>&alpha; parameter (representing the maximum trascriptional rate in the studied range of induction) varies as expected with the RBS variation and also the &delta; and &eta; parameters are quite different among the RBS variations.</p><br />
<p>The k<sub>p<sub>Tet</sub></sub> parameter is quite constant among the RBS variations, thus suggesting that in this case the RBS variation doesn't substantially affect the switch point of the Hill curve, even if the amplitude and the maximum slope are not quite maintained (for the &eta; parameter, maybe fitting problems). <br />
<br />
</p><br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p></div><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [ng/ml]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [ng/ml]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>1.53</td><td class='row'>~0</td><td class='row'>7.95</td><td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>3.16</td><td class='row'>~0</td><td class='row'>6.7</td><td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>2.73</td><td class='row'>0.23</td><td class='row'>8.96</td><td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
From these parameters, it is evident that whilst the switch-point is almost maintained for all the RBSs, the linear boundaries are similar for RBS30 and RBS32 but for RBS34 are moved on the right of one order of magnitude. <br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!-- ----------------- --><br />
<!-- --LuxI description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='LuxI'></a><h2>LuxI gene - BBa_C0061</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002'>modelling section</a>) using the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>p<sub>Tet</sub>-RBSx-LuxI-TT</a> measurement systems. <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here: </p><br />
<br />
<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg" class="thumbimage" height="70%" width="68%"></a></div></div><br />
<br />
<br />
<br />
<p align='justify'><br />
Since the measurement systems are only assayed in the exponential growth phase, in the equation (3) N &lt; <N<sub>max</sub> is assumed. <br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known and summarized in the table below:</p><br />
<br />
<div align="center"><br />
<table align="center" class='data' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.004925</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
</div><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, k<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>p<sub>Tet</sub>-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
<br />
<div align="justify"><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div></div><br />
<br />
<br />
<div align="justify"><p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p></div><br />
<br />
<br />
<div align=center"><br />
<table align="center" class='data'><br />
<tr><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>k<sub>M,LuxI</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>3.56*10<sup>-9</sup></td><br />
<td class='row'>6.87*10<sup>3</sup></td><br />
<td class='row'> 87 </td><br />
<td class='row'>8.5</td><br />
<td class='row'>ND</td><br />
<td class='row'> 252 </td><br />
</tr></table><br />
</div><br />
<br> <br />
</p><br />
</p><br />
<br />
<p align='justify'><br />
The provided parameters k<sub>M</sub> and V<sub>max</sub> represent the enzymatic activity of LuxI, described by our model. They must not be confused with the operative parameters of the Michaelis-Menten relation. <br />
These synthetic parameters have a great importance, since they can be used in more complicated models in order to predict the behavior of complex circuits.<br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
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<!-- ----------------- --><br />
<!-- --AiiA description-- --><br />
<!-- ----------------- --><br />
<br />
<br />
<a name='AiiA'></a><h2>AiiA gene - BBa_C0060</h2><br />
<br />
<p align='justify'><br />
The activity of AiiA enzyme has been evaluated by testing the measurement systems <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a>. <br />
Similar to LuxI, a system of differential equations (referring to <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>) has been derived. <br />
</p><br />
<br />
<div style='text-align:justify'><br />
<div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg" class="thumbimage" height="70%" width="68%"></a><br />
</div></div><br />
<br />
<p align='justify'><br />
The parameters k<sub>cat</sub>, k<sub>M,AiiA</sub> and &alpha;<sub>RBSx</sub> would have been estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>p<sub>Tet</sub>-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
Unfortunately, their estimation revealed difficult.<br><br />
In the first experiments with the measurement system <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a> in LOW-COPY at pH=7 no degradation of HSL was observed. The collected data are shown in the figure below. HSL degradation is identical in the measurement system and in the negative control after 21 hours.<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<div align="justify">Further investigation on the enzyme were performed in more suitable conditions, to evaluate its intrinsic activity. AiiA activity was investigated in HIGH COPY plasmid in <em>E. coli</em> TOP10, in order to understand if the enzyme worked. In this case, a significant difference in degradation between p<sub>Tet</sub>-RBSx-AiiA-TT and the negative control was observed, also just after 7 hours. That was the proof of the good functioning (in HIGH COPY) of the enzyme.</div><br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<div align="justify"><br />
Since the enzyme activity had been assessed, some suitable working conditions were investigated for AiiA. Since our studies on HSL stability for different pHs has shown that its stability improves at pH=6.0, this experimental condition was used to test once again MGZ1 strain with the system expressing AiiA in low copy number plasmid. <br />
<br />
Unfortunately, we had some problems with the biosensor, BBa_T9002, that didn't work as usual, resulting in a calibration curve modified. So, k<sub>M,AiiA</sub> and k<sub>cat</sub> were impossible to be estimated from these data. We decided to estimate them from experimental data coming from Tecan tests preformed in <em>E.COLI</em> TOP10, where aiiA gene was cloned in a high copy numebr plasmid, pSB1A2, downstream p<sub>Tet</sub> with different RBSs; in this way we could have some semi-quantitative information about the order of magnitude of these parameters. Taking into account the different copy number, we tried to simulate our model behavior with reasonable values for AiiA parameters.</div><br />
<br><br />
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</p><br />
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<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!-- ----------------- --><br />
<!-- ---pSB1C3-mRFP-- --><br />
<!-- ----------------- --><br />
<br />
<a name='K999'></a><h2>pSB1C3 plasmid with mRFP between S and P bearing p<sub>Tet</sub> (easy-to-clone)</h2><br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/e/ee/BBa_I13507.jpg" class="thumbimage" width="50%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<div align="justify">This vector was designed and realized in order to facilitate the cloning of coding sequences downstream of the strong promoter p<sub>Tet</sub>. This vector was assembled by ligating S-P excided mRFP coding sequence from BBa_J61002 and ligating it in BBa_R0040 cut with S and P. Thus, the resulting vector contains mRFP between S and P. p<sub>Tet</sub> can be easily excided (E-P) and moved in the desired vector (E-P) and then the desired coding sequence can be easily assembled by digesting S-P the vector and X-P the coding sequence, thus obtaining a final part that is standard10-compatible.</div><br><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<a name='Improvement'></a><h1>Existing parts: sequence debugging</h1><br />
<br />
<div align="justify"><br />
<br />
<ol><ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516021 "> BBa_K516021 </a> (wiki name: E10 ) RBS31-AiiA-TT Rebuilt existing part from BBa_I13914 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516022 "> BBa_K516022 </a> (wiki name: E11 ) RBS32-AiiA-TT Rebuilt existing part from BBa_I13912 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516030 "> BBa_K516030 </a> (wiki name: E5 ) RBS30-mRFP-TT Rebuilt existing part from BBa_S04180 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516032 "> BBa_K516032 </a> (wiki name: E7 ) RBS32-mRFP-TT Rebuilt existing part from BBa_ J133000 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) p<sub>Tet</sub>-RBS34-LuxI Rebuilt existing part from BBa_S03623 (DNA available, only 2008 kit, inconsistent) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) p<sub>Tet</sub>-RBS32-AiiA-TT Rebuilt existing part from BBa_ J22071 (2008 only, Bad sequencing) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) p<sub>Tet</sub>-RBS34-AiiA-TT Rebuilt existing part from BBa_K077047 (Part deleted) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) p<sub>Tet</sub>-RBS32-mRFP-TT Rebuilt existing part from BBa_I20252 (DNA planning) </li></ul></ol><br />
</div><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Parts/CharacterizedTeam:UNIPV-Pavia/Parts/Characterized2011-09-21T16:43:19Z<p>Nickpv: </p>
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<div>{{main}}<br />
<html><br />
<a name='indice'></a><br />
<h2 class="art-postheader"><br />
Characterized Parts<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br><br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#new"><span class="tocnumber">1</span> <span class="toctext">New Parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#J101"><span class="tocnumber">1.1</span> <span class="toctext">J23101x series with different RBSs</span></a></li><br />
<li class="toclevel-2"><a href="#pTetLuxI"><span class="tocnumber">1.2</span> <span class="toctext">LuxI Expression cassette driven by aTc</span></a></li><br />
<li class="toclevel-2"><a href="#pTetAiiA"><span class="tocnumber">1.3</span> <span class="toctext">AiiA Expression cassette driven by aTc</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Existing"><span class="tocnumber">2</span> <span class="toctext">Characterization of existing parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#note"><span class="tocnumber">2.1</span> <span class="toctext">Notes on promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.2</span> <span class="toctext">RBSs from the community collection</span></a></li><br />
<li class="toclevel-2"><a href="#pLux"><span class="tocnumber">2.3</span> <span class="toctext">p<sub>Lux</sub> - a 3OC<sub>6</sub>-HSL-in PoPs-out device</span></a></li><br />
<li class="toclevel-2"><a href="#pTet"><span class="tocnumber">2.4</span> <span class="toctext">p<sub>Tet</sub> - BBa_R0040</span></a></li><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.5</span> <span class="toctext">LuxI - BBa_C0061</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.6</span> <span class="toctext">AiiA - BBa_C0060</span></a></li><br />
<li class="toclevel-2"><a href="#K999"><span class="tocnumber">2.7</span> <span class="toctext">pSB1C3 vector with mRFP between S and P for easy cloning</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Improvement"><span class="tocnumber">3</span> <span class="toctext">Sequence debugging</span></a></li><br />
</ul></td></tr></table><br />
</div><br><br />
<br />
<br />
<br />
<br><br><br />
<br />
<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part: pSB4C5">pSB4C5</a>. <br />
</em><br />
<br><br />
<br><br />
<br />
<br />
<div class="listcircle"><br />
<a name='new'></a><h1>New parts</h1><br />
<a name='J101'></a><h2>J23101x series</h2><br />
<br />
<ol type='1'><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> (wiki name: J101-E5 ) J101-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> (wiki name: J101-31 ) J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> (wiki name: J101-E7 ) J101-RBS32-mRFP-TT </li><br />
</ol><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- J23101x description --><br />
<!-- ----------------- --><br />
<p align='justify'><br />
<div align="justify"><p>BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in '<a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>data analysis</a>' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene.</p><br />
<p>The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P. This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts.</p><br />
<p>Salis et al. [Nat Biotec, 2009] stated that <em> 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels'</em> and again <em>'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.'</em></p><br />
<br />
<p>For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS. In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.</p><br />
<br />
<p><br><em><b>NB</b>: in the RPU computation, the J23101-RBS34-mRFP-TT (<A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_J23101">BBa_J23101</a>) in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency.</em></p></div><br />
<br />
<table align='center'><tr><td width='50%' ><br />
<table class='data'><br />
<tr><br />
<td class='row'><b>J23101 promoter with RBS</b><br />
</td><br />
<td class='row'><b>S<sub>cell</sub></b><br />
</td><br />
<td class='row'><b>R.P.U.s</b><br />
</td><br />
</tr><br />
<tr><td class='row'>RBS30<br />
</td><br />
<td class='row'>122 [13.23]</td><br />
<td class='row'>2.45 [0.27]</td><br />
</tr><br />
<tr><td class='row'>RBS31<br />
</td><br />
<td class='row'>2 [0.25]</td><br />
<td class='row'>0.04 [0.01]</td><br />
</tr><br />
<tr><td class='row'>RBS32<br />
</td><br />
<td class='row'>20 [1.7]</td><br />
<td class='row'>0.40 [0.03]</td><br />
</tr><br />
<tr><td class='row'>RBS34<br />
</td><br />
<td class='row'>50 [1.87]</td><br />
<td class='row'>1 [0.02]</td><br />
</tr><br />
</table></td><br />
<div align="center">Data are provided as average [Standard error].</div><br><br />
<td width='50%' ><br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="image"><img alt="RPUs of J23101 promoter with different RBSs" src="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="thumbimage" width="80%"></a></div></div><br />
</td><br />
</tr></table><br />
<br><br><br />
<div align="justify">On the other hand, if the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.</div><br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- pTetLuxI description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetLuxI'></a><h2>LuxI expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> (wiki name: E13 ) pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> (wiki name: E14 ) pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> (wiki name: E15 ) pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI </li></ul></ol><br />
<br />
<br><br><br />
<p align='justify'><em>Though these parts don't have a transcriptional terminator, they have been characterized in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a>, that contains the BBa_B0054 terminator. This choice is motivated by the need to reproduce the exact experimental context of the final circuit, as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Solution#Circuit_design'>solution section</a>. <br />
</em></p><br />
<br><br><br />
<p align='justify'><br />
LuxI has been characterized in terms of enzymatic activity under the regulation of p<sub>Tet</sub> promoter. <br />
</p><br />
<p align='justify'><br />
K<sub>M,LuxI</sub> and V<sub>max</sub> parameters representing its activity have been estimated and the promoter strength (represented by a synthetic parameter &alpha;<sub>pTet</sub> for every pTet-RBS combination) at full induction (100 ng/ml) has been estimated too with a simultaneous fitting of the available data. <br />
<br />
</p><br />
<br />
<br />
<p align='justify'><br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002'>modelling section</a>). <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system describing the behavior of this measurement circuit is reported here: </p><br />
<br />
<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg" class="thumbimage" height="70%" width="68%"></a></div></div><br />
<br />
<br />
<br />
<p align='justify'><br />
Since the measurement systems are only assayed in the exponential growth phase, in the equation (3) N &lt; N<sub>max</sub> is assumed. <br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known and summarized in the table below:</p><br />
<br />
<div align="center"><br />
<table align="center" class='data' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.004925</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
</div><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, k<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>pTet-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
<br />
<div align="justify"><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div></div><br />
<br />
<br />
<div align="justify"><p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p></div><br />
<br />
<br />
<div align="center"><br />
<table align="center" class='data'><br />
<tr><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>k<sub>M,LuxI</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>3.56*10<sup>-9</sup></td><br />
<td class='row'>6.87*10<sup>3</sup></td><br />
<td class='row'>87</td><br />
<td class='row'>8.5</td><br />
<td class='row'>ND</td><br />
<td class='row'>252</td><br />
</tr></table><br />
</div><br />
<br> <br />
</p><br />
</p><br />
<br />
<br />
</p><br />
<br />
<br />
<p align='justify'><br />
The collected data have been used to identify the parameters of our model. Despite the data-poor context, the model predictions fit the experimental data, thus demonstrating that the equation that models the HSL synthesis by LuxI is a good approximation of real processes. <br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- pTetAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetAiiA'></a><h2>AiiA expression cassette driven by aTc-inducible p<sub>Tet</sub> promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> (wiki name: E24 ) p<sub>Tet</sub>-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> (wiki name: E25 ) p<sub>Tet</sub>-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) p<sub>Tet</sub>-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) p<sub>Tet</sub>-RBS34-AiiA-TT </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity.<br />
<br />
Similar to LuxI, a system of differential equations (referring to <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>) has been derived. <br />
</p><br />
<br />
<div style='text-align:justify'><br />
<div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg" class="thumbimage" height="70%" width="68%"></a><br />
</div></div><br />
<br />
<p align='justify'><br />
The parameters k<sub>cat</sub>, k<sub>M,AiiA</sub> and &alpha;<sub>RBSx</sub> would have been estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>p<sub>Tet</sub>-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
Unfortunately, their estimation revealed difficult.<br><br />
In the first experiments with the measurement system <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a> in LOW-COPY at pH=7 no degradation of HSL was observed. The collected data are shown in the figure below. HSL degradation is identical in the measurement system and in the negative control after 21 hours.<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<div align="justify">Further investigation on the enzyme were performed in more suitable conditions, to evaluate its intrinsic activity. AiiA activity was investigated in HIGH COPY plasmid in <em>E. coli</em> TOP10, in order to understand if the enzyme worked. In this case, a significant difference in degradation between p<sub>Tet</sub>-RBSx-AiiA-TT and the negative control was observed, also just after 7 hours. That was the proof of the good functioning (in HIGH COPY) of the enzyme.</div><br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<div align="justify"><br />
Experiments on these parts gave us the opportunity to characterize only the activity of the enzyme in <em>E. COLI</em> TOP10 in high copy number plasmid, providing only some information about the order of magnitude of the model parameters, which has been designed to work in <em>E. COLI</em> MGZ1 in low copy number plasmid.<br />
<br />
<br />
<a name='Existing'></a><h1>Existing parts</h1><br />
<br />
<a name='note'></a><h2>Notes for promoter characterization</h2><br />
<p align='justify'><br />
<p>Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection.</p><br />
<p>The assembled RBSs are:</p><br />
<br><br />
<div align='center'><br />
<table class='data'><br />
<tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr><br />
<tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr><br />
<tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr><br />
<tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr><br />
<tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr><br />
</table></div><br />
<br><br />
<div align="justify"><p>For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.</p><br />
<p>The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. <br />
RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP).</p><br />
<p>For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.</p><br />
</p><br />
<p>Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least squares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs:</p><p></p><br />
<p><ol><ul><li><b><br />
RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity</p><br />
</li><p><li><b><br />
RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity</p><br />
</li><p><li><br />
<b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and it is representative of the position of linear region</p><br />
</li><br />
<p><li><br />
<b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</div></li></p><br />
</ul></ol><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --RBS description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='rbs'></a><h2>RBSs</h2><br />
<p align='justify'><br />
<p><div align="justify">The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of protein produced. <br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element.</p><br />
<p>The evaluation of RBS efficiency can be performed in a very intuitive fashion:</p><br />
<ol><br />
<li>select the RBSs you want to study</li><br />
<li>assemble them in a Promoter - XX - Coding sequence circuit</li><br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/d/d1/Vettore_base.jpg" class="thumbimage" width="60%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<li>measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS (<a href='http://partsregistry.org/wiki/index.php/Part: BBa_B0034'>BBa_B0034</a>). </li></div></ol><br><br />
<br />
<br />
<div align="justify"><p>This simple measurement system allows the quantification of RBS efficiency depending on the experimental context (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments.</p><br />
<p>To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.</p><br />
<p>In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, p<sub>Tet</sub>, p<sub>Lux</sub>). The system output was measured and the RBS efficiency evaluated. The results are summarized in the table below:</div></p><br />
<br><br />
<div align="center"><table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>p<sub>Lux</sub></sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>p<sub>Tet</sub></sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>J23101</sub></b><sup>**</sup></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>0.40</td><td class='row'>1.6814</td><td class='row'>2.45</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.01</td><td class='row'>ND</td><td class='row'>0.04</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.19</td><td class='row'>0.4193</td><td class='row'>0.40</td><td class='row'>0,3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table></div><br />
<br />
<br><br />
<div align="justify">On the other hand, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (p<sub>Tet</sub>-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below:</div><br><br />
<br />
<div align="center"><table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>mRFP</sub></b><sup>**</sup></td><br />
<td class='row'><b>eff<sub>AiiA</sub></b><sup>***</sup></td><br />
<td class='row'><b>eff<sub>LuxI</sub><sup>****</sup></b></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>1.72</td><td class='row'>0.53</td><td class='row'>0.45</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.03</td><td class='row'>0.83</td><td class='row'>0.028</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.37</td><td class='row'>0.50</td><td class='row'>N.D.</td><td class='row'>0.3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table></div><br />
<br><br />
<p align='justify'><br />
<div align="justify"><p><sup>*</sup> The RBS efficiency for inducible devices expressing mRFP was estimated as the ratio of the AUCs (Area under the curve) of the induction curve of the system with the studied RBS and the B0034 reference: AUC<sub>P, RBSx</sub>/AUC<sub>P, B0034</sub></p><br />
<p><sup>**</sup> The RBS efficiency for constitutive promoters expressing mRFP was computed as the ratio between S<sub>cell<sub>P, RBSx</sub></sub>/S<sub>cell<sub>P, B0034</sub></sub></p><br />
<p><sup>***</sup> The RBS efficiency for p<sub>Tet</sub> promoter driving the expression of AiiA enzyme was computed as the ratio &alpha;<sub>p<sub>Tet</sub>, RBSx</sub>/&alpha;<sub>p<sub>Tet</sub>, B0034</sub> estimated for the measurement system p<sub>Tet</sub>-RBSx-AiiA-TT. &alpha;<sub>p<sub>Tet</sub></sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#AiiA'>here</a>. p<sub>Tet</sub> was tested at full induction (100 ng/ml).</p><br />
<p><sup>****</sup> The RBS efficiency for promoters driving the expression of LuxI was computed as the ratio &alpha;<sub>p<sub>Tet</sub>, RBSx</sub>/&alpha;<sub>p<sub>Tet</sub>, B0034</sub> estimated from the measurement systems p<sub>Tet</sub>-RBSx-LuxI. &alpha;<sub>p<sub>Tet</sub></sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#LuxI'>here</a>. p<sub>Tet</sub> was tested at full induction (100 ng/ml).</p></div><br />
<br />
<br><br />
</p><br />
The parts we used to characterize the RBSs are listed here:<br />
<ol><br />
<ul><li>mRFP expression with different promoters</li><br />
<ul><br />
<li>J23101</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> J101-RBS30-RFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> J101-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_J23101 "> BBa_J23101 </a> J101-RBS34-mRFP-TT </li><br />
</ul><br />
<li>p<sub>Tet</sub></li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> p<sub>Tet</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
<li>p<sub>Lux</sub></li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
</ul><br />
<li>p<sub>Tet</sub> driving the expression of different genes</li><br />
<ul><li>mRFP</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> p<sub>Tet</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
<li>AiiA</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> p<sub>Tet</sub>-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> p<sub>Tet</sub>-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> p<sub>Tet</sub>-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> p<sub>Tet</sub>-RBS34-AiiA-TT </li><br />
</ul><br />
<li>LuxI</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> p<sub>Tet</sub>-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> p<sub>Tet</sub>-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> p<sub>Tet</sub>-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> p<sub>Tet</sub>-RBS34-LuxI </li><br />
</ul></ul><br />
</ul></ol><br />
<br />
<br />
</p><br />
<p align='justify'><br />
The results reported in the table suggest that the RBS efficiency ranking is not always maintained. In particular, for the different promoters driving the expression of mRFP, the ranking of the declared efficiencies is maintained for p<sub>Lux</sub>, but not for p<sub>Tet</sub> and J23101. The RBS B0030 results to be the most efficient for both J23101 and p<sub>Tet</sub>, but not for p<sub>Lux</sub> (NB: this effect might be due to an effective non-modularity of RBS, but also to saturating phenomena occurring for this very strong promoter at full induction). RBS B0031 always shows a very low efficiency, while B0032 an intermediate efficiency between B0031 and the stronger RBSs B0030 and B0034. <br><br />
For what concerns the encoded gene variation, more significant differences can be observed. RBS B0030 has the higher efficiency only for mRFP, while the values for AiiA and LuxI are similar (~0.5). Unexpectedly, the weak RBS B0031 has a higher efficiency with AiiA gene (0.83), while with mRFP and LuxI. With B0032 no activity was observed for LuxI, while for AiiA and mRFP the results are quite consistent with the one reported above. <br />
</p><br />
<p align='justify'><br />
These results are encouraging: though the partial non-modularity of RBS with the encoded gene is confirmed, the hypothesis of modularity with the promoter is to some extent confirmed. Three classes of efficiencies were identified:<br />
<ul><br />
<li>low efficiency RBS (B0031)</li><br />
<li>medium efficiency RBS (B0032)</li><br />
<li>high efficiency RBSs (B0030, B0034)</li><br />
</ul> <br />
</p><br />
<br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --pLux description- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<a name='pLux'></a><h2>p<sub>Lux</sub> promoter</h2><br />
<p align='justify'><br />
<br />
<br />
</p><br />
<ol><ul><br />
<br />
<br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> (wiki name: E17 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> (wiki name: E18 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> (wiki name: E19 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> (wiki name: E20 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS34-mRFP-TT </li></ul><br />
</ol><br />
<br />
<p align='justify'><br />
The estimated parameters for the Hill functions are summarized in the table below. For more details on parameter estimation, see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>model section</a>. <br />
</p><br />
<br />
<table class='data' width='100%'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Lux</sub></sub> [(AUr/min)/cell]</b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Lux</sub></sub> [-]</b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Lux</sub></sub> [-]</b></td><br />
<td class="row"><b>k<sub>p<sub>Lux</sub></sub> [ng/ml]</b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">438 [10]</td><br />
<td class="row">0.05 [>100]</td><br />
<td class="row">2 [47]</td><br />
<td class="row">1.88 [27]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">9.8 [7]</td><br />
<td class="row">0.11 [57]</td><br />
<td class="row">1.2 [29]</td><br />
<td class="row">1.5 [26]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">206 [3]</td><br />
<td class="row">0 [>>100]</td><br />
<td class="row">1.36 [10]</td><br />
<td class="row">1.87 [9]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">1105 [6]</td><br />
<td class="row">0.02 [>100]</td><br />
<td class="row">1.33 [19]</td><br />
<td class="row">2.34 [18]</td><br />
</tr><br />
</table><br />
<div align="center">Data are provided as average [CV%].</div><br><br />
<br />
<br />
<div align="justify"><br />
<p>From this table, it is evident that, whilst &alpha;<sub>p<sub>Lux</sub></sub> assumes significantly different values for different RBSs, &eta;<sub>pLux</sub> and k<sub>pLux</sub> assume very similar values. This result shows that RBS variation modulates the amplitude of Hill function, not affecting the shape of the curve. The four induction curves result to be the same Hill function modulated in amplitude by a parameter, that is the estimated RBS efficiency for this measurement system.</p><br />
<p>These results are quite encouraging, because suggest that, given the non-modular behavior of RBS dpending on the encoded gene, the RBS has a modular behaviour respect to the promoter.</p><br />
<p>The operative parameters are summarized in the table below:</p></div><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>4.28</td><td class='row'>0.20</td><td class='row'>1.08</td><td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>4.93</td><td class='row'>0.55</td><td class='row'>0.25</td><td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>9.49</td><td class='row'>0.02</td><td class='row'>0.47</td><td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>21.53</td><td class='row'>0.51</td><td class='row'>0.53</td><td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<p align='justify'>These operative parameters provide useful information on the behavior of this 3OC6-HSL inducible device. RPU<sub>max</sub> assumes very different values in terms of RPUs.<br />
This can't be explained by RBS modulation, since RPUs have been evaluated by normalizing S<sub>cell</sub> of p<sub>Lux</sub>-RBSx for the one of J23101-RBSx. It is evident that some nonlinear effect on maximum strength, maybe due to saturation phenomena on protein expression, occur. <br />
The same RPUs should be observed for every RBS, since the normalization by the standard reference used for RPUs computation should eliminate the RBS contribute. Here different RPUs are observed, maybe due to nonlinear RBS behavior or to saturation phenomena occurring with this very strong promoter. The switch point and linear boundaries are quite constant in all the cases, showing that the linear region of this system is not affected by RBS changes.</p><br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/5/50/E17_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/5/50/E17_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/c/c4/E18_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c4/E18_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/7/78/E19_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/78/E19_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/5/55/E20_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/5/55/E20_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- -pTet description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='pTet'></a><h2>p<sub>Tet</sub> promoter</h2><br />
<br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> (wiki name: E21 ) p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> (wiki name: E22 ) p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) p<sub>Tet</sub>-RBS32-mRFP-TT<br />
</li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT <br />
</li></ul><br />
</ul><br />
</ol><br />
<br />
<div align="justify"><br />
<p>The protocols for the characterization of p<sub>Tet</sub> promoter are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#pTet_protocol'>p<sub>Tet</sub> measurement section</a>.</p><br />
<p>The data collected from the mRFP measurement systems were processed as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'> data analysis section</a>. The induction curves were obtained by fitting a Hill function as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>modelling section</a> and the estimated <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>parameters</a> for p<sub>Tet</sub> are reported in the pictures and in table below. </p><br />
<p>This promoter is widely studied and characterized usually using the strong RBS BBa_B0034. Here we have characterized its transcriptional strength as a function of aTc induction (ng/ul) for different RBSs. Three different induction curves were obtained and are reported in figure:</p></div><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<br />
<br />
<!-- <td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/9/93/E33_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/9/93/E33_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td> --><br />
<br />
<table width='100%'><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/9/99/E34_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/9/99/E34_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/e/e4/E35_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e4/E35_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<p align='justify'><br />
The estimated parameters of the Hill curves described in the figures are summarized in the table below:<br />
</p><br />
<br><br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub> [(AUr/min)/cell]</b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub> [-]</b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub> [-]</b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub> [nM]</b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">230.67 [3.7]</td><br />
<td class="row">0.028 [91.61]</td><br />
<td class="row">4.61 [23.73]</td><br />
<td class="row">8.75 [4.16]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">55.77 [12]</td><br />
<td class="row">1.53E-11 [>>100]</td><br />
<td class="row">4.98 [57.62]</td><br />
<td class="row">7.26 [14.98]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">120 [5.95]</td><br />
<td class="row">0.085 [40.6]</td><br />
<td class="row">24.85 [47.6]</td><br />
<td class="row">9 [5.43]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br><br><br />
<br />
<p align='justify'><br />
The measurement system p<sub>Tet</sub>-B0031-mRFP-TT couldn't be assayed because its fluorescence output is under the detectability threshold of our measurement instrument. For this reason, the parameters of the corresponding Hill curve couldn't be estimated and are reported as 'Not Determined' ND. <br />
</p><br />
<br />
<div align='justify'><br />
<p>&alpha; parameter (representing the maximum trascriptional rate in the studied range of induction) varies as expected with the RBS variation and also the &delta; and &eta; parameters are quite different among the RBS variations.</p><br />
<p>The k<sub>p<sub>Tet</sub></sub> parameter is quite constant among the RBS variations, thus suggesting that in this case the RBS variation doesn't substantially affect the switch point of the Hill curve, even if the amplitude and the maximum slope are not quite maintained (for the &eta; parameter, maybe fitting problems). <br />
<br />
</p><br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p></div><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [ng/ml]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [ng/ml]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>1.53</td><td class='row'>~0</td><td class='row'>7.95</td><td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>3.16</td><td class='row'>~0</td><td class='row'>6.7</td><td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>2.73</td><td class='row'>0.23</td><td class='row'>8.96</td><td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
From these parameters, it is evident that whilst the switch-point is almost maintained for all the RBSs, the linear boundaries are similar for RBS30 and RBS32 but for RBS34 are moved on the right of one order of magnitude. <br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<br />
<br />
<!-- ----------------- --><br />
<!-- --LuxI description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='LuxI'></a><h2>LuxI gene - BBa_C0061</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002'>modelling section</a>) using the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>p<sub>Tet</sub>-RBSx-LuxI-TT</a> measurement systems. <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here: </p><br />
<br />
<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg" class="thumbimage" height="70%" width="68%"></a></div></div><br />
<br />
<br />
<br />
<p align='justify'><br />
Since the measurement systems are only assayed in the exponential growth phase, in the equation (3) N &lt; <N<sub>max</sub> is assumed. <br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known and summarized in the table below:</p><br />
<br />
<div align="center"><br />
<table align="center" class='data' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.004925</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
</div><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, k<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>p<sub>Tet</sub>-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
<br />
<div align="justify"><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div></div><br />
<br />
<br />
<div align="justify"><p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p></div><br />
<br />
<br />
<div align=center"><br />
<table align="center" class='data'><br />
<tr><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>k<sub>M,LuxI</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>3.56*10<sup>-9</sup></td><br />
<td class='row'>6.87*10<sup>3</sup></td><br />
<td class='row'> 87 </td><br />
<td class='row'>8.5</td><br />
<td class='row'>ND</td><br />
<td class='row'> 252 </td><br />
</tr></table><br />
</div><br />
<br> <br />
</p><br />
</p><br />
<br />
<p align='justify'><br />
The provided parameters k<sub>M</sub> and V<sub>max</sub> represent the enzymatic activity of LuxI, described by our model. They must not be confused with the operative parameters of the Michaelis-Menten relation. <br />
These synthetic parameters have a great importance, since they can be used in more complicated models in order to predict the behavior of complex circuits.<br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
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<br />
<!-- ----------------- --><br />
<!-- --AiiA description-- --><br />
<!-- ----------------- --><br />
<br />
<br />
<a name='AiiA'></a><h2>AiiA gene - BBa_C0060</h2><br />
<br />
<p align='justify'><br />
The activity of AiiA enzyme has been evaluated by testing the measurement systems <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a>. <br />
Similar to LuxI, a system of differential equations (referring to <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>) has been derived. <br />
</p><br />
<br />
<div style='text-align:justify'><br />
<div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg" class="thumbimage" height="70%" width="68%"></a><br />
</div></div><br />
<br />
<p align='justify'><br />
The parameters k<sub>cat</sub>, k<sub>M,AiiA</sub> and &alpha;<sub>RBSx</sub> would have been estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>p<sub>Tet</sub>-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
Unfortunately, their estimation revealed difficult.<br><br />
In the first experiments with the measurement system <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a> in LOW-COPY at pH=7 no degradation of HSL was observed. The collected data are shown in the figure below. HSL degradation is identical in the measurement system and in the negative control after 21 hours.<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<div align="justify">Further investigation on the enzyme were performed in more suitable conditions, to evaluate its intrinsic activity. AiiA activity was investigated in HIGH COPY plasmid in <em>E. coli</em> TOP10, in order to understand if the enzyme worked. In this case, a significant difference in degradation between p<sub>Tet</sub>-RBSx-AiiA-TT and the negative control was observed, also just after 7 hours. That was the proof of the good functioning (in HIGH COPY) of the enzyme.</div><br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<div align="justify"><br />
Since the enzyme activity had been assessed, some suitable working conditions were investigated for AiiA. Since our studies on HSL stability for different pHs has shown that its stability improves at pH=6.0, this experimental condition was used to test once again MGZ1 strain with the system expressing AiiA in low copy number plasmid. <br />
<br />
Unfortunately, we had some problems with the biosensor, BBa_T9002, that didn't work as usual, resulting in a calibration curve modified. So, k<sub>M,AiiA</sub> and k<sub>cat</sub> were impossible to be estimated from these data. We decided to estimate them from experimental data coming from Tecan tests preformed in <em>E.COLI</em> TOP10, where aiiA gene was cloned in a high copy numebr plasmid, pSB1A2, downstream p<sub>Tet</sub> with different RBSs; in this way we could have some semi-quantitative information about the order of magnitude of these parameters. Taking into account the different copy number, we tried to simulate our model behavior with reasonable values for AiiA parameters.</div><br />
<br><br />
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</p><br />
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<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!-- ----------------- --><br />
<!-- ---pSB1C3-mRFP-- --><br />
<!-- ----------------- --><br />
<br />
<a name='K999'></a><h2>pSB1C3 plasmid with mRFP between S and P bearing p<sub>Tet</sub> (easy-to-clone)</h2><br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/e/ee/BBa_I13507.jpg" class="thumbimage" width="50%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<div align="justify">This vector was designed and realized in order to facilitate the cloning of coding sequences downstream of the strong promoter p<sub>Tet</sub>. This vector was assembled by ligating S-P excided mRFP coding sequence from BBa_J61002 and ligating it in BBa_R0040 cut with S and P. Thus, the resulting vector contains mRFP between S and P. p<sub>Tet</sub> can be easily excided (E-P) and moved in the desired vector (E-P) and then the desired coding sequence can be easily assembled by digesting S-P the vector and X-P the coding sequence, thus obtaining a final part that is standard10-compatible.</div><br><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<a name='Improvement'></a><h1>Existing parts: sequence debugging</h1><br />
<br />
<div align="justify"><br />
<br />
<ol><ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516021 "> BBa_K516021 </a> (wiki name: E10 ) RBS31-AiiA-TT Rebuilt existing part from BBa_I13914 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516022 "> BBa_K516022 </a> (wiki name: E11 ) RBS32-AiiA-TT Rebuilt existing part from BBa_I13912 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516030 "> BBa_K516030 </a> (wiki name: E5 ) RBS30-mRFP-TT Rebuilt existing part from BBa_S04180 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516032 "> BBa_K516032 </a> (wiki name: E7 ) RBS32-mRFP-TT Rebuilt existing part from BBa_ J133000 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) p<sub>Tet</sub>-RBS34-LuxI Rebuilt existing part from BBa_S03623 (DNA available, only 2008 kit, inconsistent) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) p<sub>Tet</sub>-RBS32-AiiA-TT Rebuilt existing part from BBa_ J22071 (2008 only, Bad sequencing) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) p<sub>Tet</sub>-RBS34-AiiA-TT Rebuilt existing part from BBa_K077047 (Part deleted) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) p<sub>Tet</sub>-RBS32-mRFP-TT Rebuilt existing part from BBa_I20252 (DNA planning) </li></ul></ol><br />
</div><br />
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</html><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Parts/CharacterizedTeam:UNIPV-Pavia/Parts/Characterized2011-09-21T16:34:55Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<html><br />
<a name='indice'></a><br />
<h2 class="art-postheader"><br />
Characterized Parts<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br><br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#new"><span class="tocnumber">1</span> <span class="toctext">New Parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#J101"><span class="tocnumber">1.1</span> <span class="toctext">J23101x series with different RBSs</span></a></li><br />
<li class="toclevel-2"><a href="#pTetLuxI"><span class="tocnumber">1.2</span> <span class="toctext">LuxI Expression cassette driven by aTc</span></a></li><br />
<li class="toclevel-2"><a href="#pTetAiiA"><span class="tocnumber">1.3</span> <span class="toctext">AiiA Expression cassette driven by aTc</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Existing"><span class="tocnumber">2</span> <span class="toctext">Characterization of existing parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#note"><span class="tocnumber">2.1</span> <span class="toctext">Notes on promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.2</span> <span class="toctext">RBSs from the community collection</span></a></li><br />
<li class="toclevel-2"><a href="#pLux"><span class="tocnumber">2.3</span> <span class="toctext">p<sub>Lux</sub> - a 3OC<sub>6</sub>-HSL-in PoPs-out device</span></a></li><br />
<li class="toclevel-2"><a href="#pTet"><span class="tocnumber">2.4</span> <span class="toctext">p<sub>Tet</sub> - BBa_R0040</span></a></li><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.5</span> <span class="toctext">LuxI - BBa_C0061</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.6</span> <span class="toctext">AiiA - BBa_C0060</span></a></li><br />
<li class="toclevel-2"><a href="#K999"><span class="tocnumber">2.7</span> <span class="toctext">pSB1C3 vector with mRFP between S and P for easy cloning</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Improvement"><span class="tocnumber">3</span> <span class="toctext">Sequence debugging</span></a></li><br />
</ul></td></tr></table><br />
</div><br><br />
<br />
<br />
<br />
<br><br><br />
<br />
<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part: pSB4C5">pSB4C5</a>. <br />
</em><br />
<br><br />
<br><br />
<br />
<br />
<div class="listcircle"><br />
<a name='new'></a><h1>New parts</h1><br />
<a name='J101'></a><h2>J23101x series</h2><br />
<br />
<ol type='1'><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> (wiki name: J101-E5 ) J101-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> (wiki name: J101-31 ) J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> (wiki name: J101-E7 ) J101-RBS32-mRFP-TT </li><br />
</ol><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- J23101x description --><br />
<!-- ----------------- --><br />
<p align='justify'><br />
<div align="justify"><p>BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in '<a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>data analysis</a>' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene.</p><br />
<p>The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P. This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts.</p><br />
<p>Salis et al. [Nat Biotec, 2009] stated that <em> 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels'</em> and again <em>'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.'</em></p><br />
<br />
<p>For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS. In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.</p><br />
<br />
<p><br><em><b>NB</b>: in the RPU computation, the J23101-RBS34-mRFP-TT (<A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_J23101">BBa_J23101</a>) in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency.</em></p></div><br />
<br />
<table align='center'><tr><td width='50%' ><br />
<table class='data'><br />
<tr><br />
<td class='row'><b>J23101 promoter with RBS</b><br />
</td><br />
<td class='row'><b>S<sub>cell</sub></b><br />
</td><br />
<td class='row'><b>R.P.U.s</b><br />
</td><br />
</tr><br />
<tr><td class='row'>RBS30<br />
</td><br />
<td class='row'>122 [13.23]</td><br />
<td class='row'>2.45 [0.27]</td><br />
</tr><br />
<tr><td class='row'>RBS31<br />
</td><br />
<td class='row'>2 [0.25]</td><br />
<td class='row'>0.04 [0.01]</td><br />
</tr><br />
<tr><td class='row'>RBS32<br />
</td><br />
<td class='row'>20 [1.7]</td><br />
<td class='row'>0.40 [0.03]</td><br />
</tr><br />
<tr><td class='row'>RBS34<br />
</td><br />
<td class='row'>50 [1.87]</td><br />
<td class='row'>1 [0.02]</td><br />
</tr><br />
</table></td><br />
<div align="center">Data are provided as average [Standard error].</div><br><br />
<td width='50%' ><br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="image"><img alt="RPUs of J23101 promoter with different RBSs" src="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="thumbimage" width="80%"></a></div></div><br />
</td><br />
</tr></table><br />
<br><br><br />
<div align="justify">On the other hand, if the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.</div><br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- pTetLuxI description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetLuxI'></a><h2>LuxI expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> (wiki name: E13 ) pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> (wiki name: E14 ) pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> (wiki name: E15 ) pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI </li></ul></ol><br />
<br />
<br><br><br />
<p align='justify'><em>Though these parts don't have a transcriptional terminator, they have been characterized in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a>, that contains the BBa_B0054 terminator. This choice is motivated by the need to reproduce the exact experimental context of the final circuit, as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Solution#Circuit_design'>solution section</a>. <br />
</em></p><br />
<br><br><br />
<p align='justify'><br />
LuxI has been characterized in terms of enzymatic activity under the regulation of p<sub>Tet</sub> promoter. <br />
</p><br />
<p align='justify'><br />
K<sub>M,LuxI</sub> and V<sub>max</sub> parameters representing its activity have been estimated and the promoter strength (represented by a synthetic parameter &alpha;<sub>pTet</sub> for every pTet-RBS combination) at full induction (100 ng/ml) has been estimated too with a simultaneous fitting of the available data. <br />
<br />
</p><br />
<br />
<br />
<p align='justify'><br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002'>modelling section</a>). <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system describing the behavior of this measurement circuit is reported here: </p><br />
<br />
<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg" class="thumbimage" height="70%" width="68%"></a></div></div><br />
<br />
<br />
<br />
<p align='justify'><br />
Since the measurement systems are only assayed in the exponential growth phase, in the equation (3) N &lt; N<sub>max</sub> is assumed. <br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known and summarized in the table below:</p><br />
<br />
<div align="center"><br />
<table align="center" class='data' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.004925</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
</div><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, k<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>pTet-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
<br />
<div align="justify"><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div></div><br />
<br />
<br />
<div align="justify"><p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p></div><br />
<br />
<br />
<div align="center"><br />
<table align="center" class='data'><br />
<tr><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>k<sub>M,LuxI</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>3.56*10<sup>-9</sup></td><br />
<td class='row'>6.87*10<sup>3</sup></td><br />
<td class='row'>87</td><br />
<td class='row'>8.5</td><br />
<td class='row'>ND</td><br />
<td class='row'>252</td><br />
</tr></table><br />
</div><br />
<br> <br />
</p><br />
</p><br />
<br />
<br />
</p><br />
<br />
<br />
<p align='justify'><br />
The collected data have been used to identify the parameters of our model. Despite the data-poor context, the model predictions fit the experimental data, thus demonstrating that the equation that models the HSL synthesis by LuxI is a good approximation of real processes. <br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- pLuxAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetAiiA'></a><h2>AiiA expression cassette driven by aTc-inducible p<sub>Tet</sub> promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> (wiki name: E24 ) p<sub>Tet</sub>-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> (wiki name: E25 ) p<sub>Tet</sub>-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) p<sub>Tet</sub>-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) p<sub>Tet</sub>-RBS34-AiiA-TT </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity.<br />
<br />
Similar to LuxI, a system of differential equations (referring to <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>) has been derived. <br />
</p><br />
<br />
<div style='text-align:justify'><br />
<div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg" class="thumbimage" height="70%" width="68%"></a><br />
</div></div><br />
<br />
<p align='justify'><br />
The parameters k<sub>cat</sub>, k<sub>M,AiiA</sub> and &alpha;<sub>RBSx</sub> would have been estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>p<sub>Tet</sub>-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
Unfortunately, their estimation revealed difficult.<br><br />
In the first experiments with the measurement system <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a> in LOW-COPY at pH=7 no degradation of HSL was observed. The collected data are shown in the figure below. HSL degradation is identical in the measurement system and in the negative control after 21 hours.<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<div align="justify">Further investigation on the enzyme were performed in more suitable conditions, to evaluate its intrinsic activity. AiiA activity was investigated in HIGH COPY plasmid in <em>E. coli</em> TOP10, in order to understand if the enzyme worked. In this case, a significant difference in degradation between p<sub>Tet</sub>-RBSx-AiiA-TT and the negative control was observed, also just after 7 hours. That was the proof of the good functioning (in HIGH COPY) of the enzyme.</div><br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<div align="justify"><br />
Experiments on these parts gave us the opportunity to characterize only the activity of the enzyme in <em>E. COLI</em> TOP10 in high copy number plasmid, providing only some information about the order of magnitude of the model parameters, which has been designed to work in <em>E. COLI</em> MGZ1 in low copy number plasmid. More suitable tests will be performed in order to decide which parts will be used to obtain a desired behavior.<br />
<br />
<br />
<a name='Existing'></a><h1>Existing parts</h1><br />
<br />
<a name='note'></a><h2>Notes for promoter characterization</h2><br />
<p align='justify'><br />
<p>Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection.</p><br />
<p>The assembled RBSs are:</p><br />
<br><br />
<div align='center'><br />
<table class='data'><br />
<tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr><br />
<tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr><br />
<tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr><br />
<tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr><br />
<tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr><br />
</table></div><br />
<br><br />
<div align="justify"><p>For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.</p><br />
<p>The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. <br />
RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP).</p><br />
<p>For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.</p><br />
</p><br />
<p>Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least squares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs:</p><p></p><br />
<p><ol><ul><li><b><br />
RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity</p><br />
</li><p><li><b><br />
RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity</p><br />
</li><p><li><br />
<b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and it is representative of the position of linear region</p><br />
</li><br />
<p><li><br />
<b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</div></li></p><br />
</ul></ol><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --RBS description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='rbs'></a><h2>RBSs</h2><br />
<p align='justify'><br />
<p><div align="justify">The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of protein produced. <br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element.</p><br />
<p>The evaluation of RBS efficiency can be performed in a very intuitive fashion:</p><br />
<ol><br />
<li>select the RBSs you want to study</li><br />
<li>assemble them in a Promoter - XX - Coding sequence circuit</li><br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/d/d1/Vettore_base.jpg" class="thumbimage" width="60%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<li>measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS (<a href='http://partsregistry.org/wiki/index.php/Part: BBa_B0034'>BBa_B0034</a>). </li></div></ol><br><br />
<br />
<br />
<div align="justify"><p>This simple measurement system allows the quantification of RBS efficiency depending on the experimental context (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments.</p><br />
<p>To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.</p><br />
<p>In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, p<sub>Tet</sub>, p<sub>Lux</sub>). The system output was measured and the RBS efficiency evaluated. The results are summarized in the table below:</div></p><br />
<br><br />
<div align="center"><table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>p<sub>Lux</sub></sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>p<sub>Tet</sub></sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>J23101</sub></b><sup>**</sup></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>0.40</td><td class='row'>1.6814</td><td class='row'>2.45</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.01</td><td class='row'>ND</td><td class='row'>0.04</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.19</td><td class='row'>0.4193</td><td class='row'>0.40</td><td class='row'>0,3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table></div><br />
<br />
<br><br />
<div align="justify">On the other hand, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (p<sub>Tet</sub>-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below:</div><br><br />
<br />
<div align="center"><table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>mRFP</sub></b><sup>**</sup></td><br />
<td class='row'><b>eff<sub>AiiA</sub></b><sup>***</sup></td><br />
<td class='row'><b>eff<sub>LuxI</sub><sup>****</sup></b></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>1.72</td><td class='row'>0.53</td><td class='row'>0.45</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.03</td><td class='row'>0.83</td><td class='row'>0.028</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.37</td><td class='row'>0.50</td><td class='row'>N.D.</td><td class='row'>0.3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table></div><br />
<br><br />
<p align='justify'><br />
<div align="justify"><p><sup>*</sup> The RBS efficiency for inducible devices expressing mRFP was estimated as the ratio of the AUCs (Area under the curve) of the induction curve of the system with the studied RBS and the B0034 reference: AUC<sub>P, RBSx</sub>/AUC<sub>P, B0034</sub></p><br />
<p><sup>**</sup> The RBS efficiency for constitutive promoters expressing mRFP was computed as the ratio between S<sub>cell<sub>P, RBSx</sub></sub>/S<sub>cell<sub>P, B0034</sub></sub></p><br />
<p><sup>***</sup> The RBS efficiency for p<sub>Tet</sub> promoter driving the expression of AiiA enzyme was computed as the ratio &alpha;<sub>p<sub>Tet</sub>, RBSx</sub>/&alpha;<sub>p<sub>Tet</sub>, B0034</sub> estimated for the measurement system p<sub>Tet</sub>-RBSx-AiiA-TT. &alpha;<sub>p<sub>Tet</sub></sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#AiiA'>here</a>. p<sub>Tet</sub> was tested at full induction (100 ng/ml).</p><br />
<p><sup>****</sup> The RBS efficiency for promoters driving the expression of LuxI was computed as the ratio &alpha;<sub>p<sub>Tet</sub>, RBSx</sub>/&alpha;<sub>p<sub>Tet</sub>, B0034</sub> estimated from the measurement systems p<sub>Tet</sub>-RBSx-LuxI. &alpha;<sub>p<sub>Tet</sub></sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#LuxI'>here</a>. p<sub>Tet</sub> was tested at full induction (100 ng/ml).</p></div><br />
<br />
<br><br />
</p><br />
The parts we used to characterize the RBSs are listed here:<br />
<ol><br />
<ul><li>mRFP expression with different promoters</li><br />
<ul><br />
<li>J23101</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> J101-RBS30-RFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> J101-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_J23101 "> BBa_J23101 </a> J101-RBS34-mRFP-TT </li><br />
</ul><br />
<li>p<sub>Tet</sub></li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> p<sub>Tet</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
<li>p<sub>Lux</sub></li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
</ul><br />
<li>p<sub>Tet</sub> driving the expression of different genes</li><br />
<ul><li>mRFP</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> p<sub>Tet</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
<li>AiiA</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> p<sub>Tet</sub>-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> p<sub>Tet</sub>-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> p<sub>Tet</sub>-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> p<sub>Tet</sub>-RBS34-AiiA-TT </li><br />
</ul><br />
<li>LuxI</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> p<sub>Tet</sub>-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> p<sub>Tet</sub>-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> p<sub>Tet</sub>-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> p<sub>Tet</sub>-RBS34-LuxI </li><br />
</ul></ul><br />
</ul></ol><br />
<br />
<br />
</p><br />
<p align='justify'><br />
The results reported in the table suggest that the RBS efficiency ranking is not always maintained. In particular, for the different promoters driving the expression of mRFP, the ranking of the declared efficiencies is maintained for p<sub>Lux</sub>, but not for p<sub>Tet</sub> and J23101. The RBS B0030 results to be the most efficient for both J23101 and p<sub>Tet</sub>, but not for p<sub>Lux</sub> (NB: this effect might be due to an effective non-modularity of RBS, but also to saturating phenomena occurring for this very strong promoter at full induction). RBS B0031 always shows a very low efficiency, while B0032 an intermediate efficiency between B0031 and the stronger RBSs B0030 and B0034. <br><br />
For what concerns the encoded gene variation, more significant differences can be observed. RBS B0030 has the higher efficiency only for mRFP, while the values for AiiA and LuxI are similar (~0.5). Unexpectedly, the weak RBS B0031 has a higher efficiency with AiiA gene (0.83), while with mRFP and LuxI. With B0032 no activity was observed for LuxI, while for AiiA and mRFP the results are quite consistent with the one reported above. <br />
</p><br />
<p align='justify'><br />
These results are encouraging: though the partial non-modularity of RBS with the encoded gene is confirmed, the hypothesis of modularity with the promoter is to some extent confirmed. Three classes of efficiencies were identified:<br />
<ul><br />
<li>low efficiency RBS (B0031)</li><br />
<li>medium efficiency RBS (B0032)</li><br />
<li>high efficiency RBSs (B0030, B0034)</li><br />
</ul> <br />
</p><br />
<br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --pLux description- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<a name='pLux'></a><h2>p<sub>Lux</sub> promoter</h2><br />
<p align='justify'><br />
<br />
<br />
</p><br />
<ol><ul><br />
<br />
<br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> (wiki name: E17 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> (wiki name: E18 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> (wiki name: E19 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> (wiki name: E20 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS34-mRFP-TT </li></ul><br />
</ol><br />
<br />
<p align='justify'><br />
The estimated parameters for the Hill functions are summarized in the table below. For more details on parameter estimation, see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>model section</a>. <br />
</p><br />
<br />
<table class='data' width='100%'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Lux</sub></sub> [(AUr/min)/cell]</b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Lux</sub></sub> [-]</b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Lux</sub></sub> [-]</b></td><br />
<td class="row"><b>k<sub>p<sub>Lux</sub></sub> [ng/ml]</b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">438 [10]</td><br />
<td class="row">0.05 [>100]</td><br />
<td class="row">2 [47]</td><br />
<td class="row">1.88 [27]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">9.8 [7]</td><br />
<td class="row">0.11 [57]</td><br />
<td class="row">1.2 [29]</td><br />
<td class="row">1.5 [26]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">206 [3]</td><br />
<td class="row">0 [>>100]</td><br />
<td class="row">1.36 [10]</td><br />
<td class="row">1.87 [9]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">1105 [6]</td><br />
<td class="row">0.02 [>100]</td><br />
<td class="row">1.33 [19]</td><br />
<td class="row">2.34 [18]</td><br />
</tr><br />
</table><br />
<div align="center">Data are provided as average [CV%].</div><br><br />
<br />
<br />
<div align="justify"><br />
<p>From this table, it is evident that, whilst &alpha;<sub>p<sub>Lux</sub></sub> assumes significantly different values for different RBSs, &eta;<sub>pLux</sub> and k<sub>pLux</sub> assume very similar values. This result shows that RBS variation modulates the amplitude of Hill function, not affecting the shape of the curve. The four induction curves result to be the same Hill function modulated in amplitude by a parameter, that is the estimated RBS efficiency for this measurement system.</p><br />
<p>These results are quite encouraging, because suggest that, given the non-modular behavior of RBS dpending on the encoded gene, the RBS has a modular behaviour respect to the promoter.</p><br />
<p>The operative parameters are summarized in the table below:</p></div><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>4.28</td><td class='row'>0.20</td><td class='row'>1.08</td><td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>4.93</td><td class='row'>0.55</td><td class='row'>0.25</td><td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>9.49</td><td class='row'>0.02</td><td class='row'>0.47</td><td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>21.53</td><td class='row'>0.51</td><td class='row'>0.53</td><td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<p align='justify'>These operative parameters provide useful information on the behavior of this 3OC6-HSL inducible device. RPU<sub>max</sub> assumes very different values in terms of RPUs.<br />
This can't be explained by RBS modulation, since RPUs have been evaluated by normalizing S<sub>cell</sub> of p<sub>Lux</sub>-RBSx for the one of J23101-RBSx. It is evident that some nonlinear effect on maximum strength, maybe due to saturation phenomena on protein expression, occur. <br />
The same RPUs should be observed for every RBS, since the normalization by the standard reference used for RPUs computation should eliminate the RBS contribute. Here different RPUs are observed, maybe due to nonlinear RBS behavior or to saturation phenomena occurring with this very strong promoter. The switch point and linear boundaries are quite constant in all the cases, showing that the linear region of this system is not affected by RBS changes.</p><br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/5/50/E17_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/5/50/E17_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/c/c4/E18_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c4/E18_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/7/78/E19_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/78/E19_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/5/55/E20_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/5/55/E20_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- -pTet description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='pTet'></a><h2>p<sub>Tet</sub> promoter</h2><br />
<br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> (wiki name: E21 ) p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> (wiki name: E22 ) p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) p<sub>Tet</sub>-RBS32-mRFP-TT<br />
</li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT <br />
</li></ul><br />
</ul><br />
</ol><br />
<br />
<div align="justify"><br />
<p>The protocols for the characterization of p<sub>Tet</sub> promoter are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#pTet_protocol'>p<sub>Tet</sub> measurement section</a>.</p><br />
<p>The data collected from the mRFP measurement systems were processed as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'> data analysis section</a>. The induction curves were obtained by fitting a Hill function as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>modelling section</a> and the estimated <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>parameters</a> for p<sub>Tet</sub> are reported in the pictures and in table below. </p><br />
<p>This promoter is widely studied and characterized usually using the strong RBS BBa_B0034. Here we have characterized its transcriptional strength as a function of aTc induction (ng/ul) for different RBSs. Three different induction curves were obtained and are reported in figure:</p></div><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<br />
<br />
<!-- <td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/9/93/E33_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/9/93/E33_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td> --><br />
<br />
<table width='100%'><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/9/99/E34_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/9/99/E34_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/e/e4/E35_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e4/E35_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<p align='justify'><br />
The estimated parameters of the Hill curves described in the figures are summarized in the table below:<br />
</p><br />
<br><br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub> [(AUr/min)/cell]</b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub> [-]</b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub> [-]</b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub> [nM]</b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">230.67 [3.7]</td><br />
<td class="row">0.028 [91.61]</td><br />
<td class="row">4.61 [23.73]</td><br />
<td class="row">8.75 [4.16]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">55.77 [12]</td><br />
<td class="row">1.53E-11 [>>100]</td><br />
<td class="row">4.98 [57.62]</td><br />
<td class="row">7.26 [14.98]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">120 [5.95]</td><br />
<td class="row">0.085 [40.6]</td><br />
<td class="row">24.85 [47.6]</td><br />
<td class="row">9 [5.43]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br><br><br />
<br />
<p align='justify'><br />
The measurement system p<sub>Tet</sub>-B0031-mRFP-TT couldn't be assayed because its fluorescence output is under the detectability threshold of our measurement instrument. For this reason, the parameters of the corresponding Hill curve couldn't be estimated and are reported as 'Not Determined' ND. <br />
</p><br />
<br />
<div align='justify'><br />
<p>&alpha; parameter (representing the maximum trascriptional rate in the studied range of induction) varies as expected with the RBS variation and also the &delta; and &eta; parameters are quite different among the RBS variations.</p><br />
<p>The k<sub>p<sub>Tet</sub></sub> parameter is quite constant among the RBS variations, thus suggesting that in this case the RBS variation doesn't substantially affect the switch point of the Hill curve, even if the amplitude and the maximum slope are not quite maintained (for the &eta; parameter, maybe fitting problems). <br />
<br />
</p><br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p></div><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [ng/ml]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [ng/ml]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>1.53</td><td class='row'>~0</td><td class='row'>7.95</td><td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>3.16</td><td class='row'>~0</td><td class='row'>6.7</td><td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>2.73</td><td class='row'>0.23</td><td class='row'>8.96</td><td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
From these parameters, it is evident that whilst the switch-point is almost maintained for all the RBSs, the linear boundaries are similar for RBS30 and RBS32 but for RBS34 are moved on the right of one order of magnitude. <br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<br />
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<br />
<br />
<!-- ----------------- --><br />
<!-- --LuxI description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='LuxI'></a><h2>LuxI gene - BBa_C0061</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002'>modelling section</a>) using the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>p<sub>Tet</sub>-RBSx-LuxI-TT</a> measurement systems. <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here: </p><br />
<br />
<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg" class="thumbimage" height="70%" width="68%"></a></div></div><br />
<br />
<br />
<br />
<p align='justify'><br />
Since the measurement systems are only assayed in the exponential growth phase, in the equation (3) N &lt; <N<sub>max</sub> is assumed. <br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known and summarized in the table below:</p><br />
<br />
<div align="center"><br />
<table align="center" class='data' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.004925</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
</div><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, k<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>p<sub>Tet</sub>-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
<br />
<div align="justify"><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div></div><br />
<br />
<br />
<div align="justify"><p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p></div><br />
<br />
<br />
<div align=center"><br />
<table align="center" class='data'><br />
<tr><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>k<sub>M,LuxI</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>3.56*10<sup>-9</sup></td><br />
<td class='row'>6.87*10<sup>3</sup></td><br />
<td class='row'> 87 </td><br />
<td class='row'>8.5</td><br />
<td class='row'>ND</td><br />
<td class='row'> 252 </td><br />
</tr></table><br />
</div><br />
<br> <br />
</p><br />
</p><br />
<br />
<p align='justify'><br />
The provided parameters k<sub>M</sub> and V<sub>max</sub> represent the enzymatic activity of LuxI, described by our model. They must not be confused with the operative parameters of the Michaelis-Menten relation. <br />
These synthetic parameters have a great importance, since they can be used in more complicated models in order to predict the behavior of complex circuits.<br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
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<br />
<br />
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<br />
<br />
<!-- ----------------- --><br />
<!-- --AiiA description-- --><br />
<!-- ----------------- --><br />
<br />
<br />
<a name='AiiA'></a><h2>AiiA gene - BBa_C0060</h2><br />
<br />
<p align='justify'><br />
The activity of AiiA enzyme has been evaluated by testing the measurement systems <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a>. <br />
Similar to LuxI, a system of differential equations (referring to <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>) has been derived. <br />
</p><br />
<br />
<div style='text-align:justify'><br />
<div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg" class="thumbimage" height="70%" width="68%"></a><br />
</div></div><br />
<br />
<p align='justify'><br />
The parameters k<sub>cat</sub>, k<sub>M,AiiA</sub> and &alpha;<sub>RBSx</sub> would have been estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>p<sub>Tet</sub>-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
Unfortunately, their estimation revealed difficult.<br><br />
In the first experiments with the measurement system <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a> in LOW-COPY at pH=7 no degradation of HSL was observed. The collected data are shown in the figure below. HSL degradation is identical in the measurement system and in the negative control after 21 hours.<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<div align="justify">Further investigation on the enzyme were performed in more suitable conditions, to evaluate its intrinsic activity. AiiA activity was investigated in HIGH COPY plasmid in <em>E. coli</em> TOP10, in order to understand if the enzyme worked. In this case, a significant difference in degradation between p<sub>Tet</sub>-RBSx-AiiA-TT and the negative control was observed, also just after 7 hours. That was the proof of the good functioning (in HIGH COPY) of the enzyme.</div><br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<div align="justify"><br />
Since the enzyme activity had been assessed, some suitable working conditions were investigated for AiiA. Since our studies on HSL stability for different pHs has shown that its stability improves at pH=6.0, this experimental condition was used to test once again MGZ1 strain with the system expressing AiiA in low copy number plasmid. <br />
<br />
Unfortunately, we had some problems with the biosensor, BBa_T9002, that didn't work as usual, resulting in a calibration curve modified. So, k<sub>M,AiiA</sub> and k<sub>cat</sub> were impossible to be estimated from these data. We decided to estimate them from experimental data coming from Tecan tests preformed in <em>E.COLI</em> TOP10, where aiiA gene was cloned in a high copy numebr plasmid, pSB1A2, downstream p<sub>Tet</sub> with different RBSs; in this way we could have some semi-quantitative information about the order of magnitude of these parameters. Taking into account the different copy number, we tried to simulate our model behavior with reasonable values for AiiA parameters.</div><br />
<br><br />
<br />
</p><br />
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<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!-- ----------------- --><br />
<!-- ---pSB1C3-mRFP-- --><br />
<!-- ----------------- --><br />
<br />
<a name='K999'></a><h2>pSB1C3 plasmid with mRFP between S and P bearing p<sub>Tet</sub> (easy-to-clone)</h2><br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/e/ee/BBa_I13507.jpg" class="thumbimage" width="50%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<div align="justify">This vector was designed and realized in order to facilitate the cloning of coding sequences downstream of the strong promoter p<sub>Tet</sub>. This vector was assembled by ligating S-P excided mRFP coding sequence from BBa_J61002 and ligating it in BBa_R0040 cut with S and P. Thus, the resulting vector contains mRFP between S and P. p<sub>Tet</sub> can be easily excided (E-P) and moved in the desired vector (E-P) and then the desired coding sequence can be easily assembled by digesting S-P the vector and X-P the coding sequence, thus obtaining a final part that is standard10-compatible.</div><br><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<a name='Improvement'></a><h1>Existing parts: sequence debugging</h1><br />
<br />
<div align="justify"><br />
<br />
<ol><ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516021 "> BBa_K516021 </a> (wiki name: E10 ) RBS31-AiiA-TT Rebuilt existing part from BBa_I13914 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516022 "> BBa_K516022 </a> (wiki name: E11 ) RBS32-AiiA-TT Rebuilt existing part from BBa_I13912 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516030 "> BBa_K516030 </a> (wiki name: E5 ) RBS30-mRFP-TT Rebuilt existing part from BBa_S04180 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516032 "> BBa_K516032 </a> (wiki name: E7 ) RBS32-mRFP-TT Rebuilt existing part from BBa_ J133000 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) p<sub>Tet</sub>-RBS34-LuxI Rebuilt existing part from BBa_S03623 (DNA available, only 2008 kit, inconsistent) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) p<sub>Tet</sub>-RBS32-AiiA-TT Rebuilt existing part from BBa_ J22071 (2008 only, Bad sequencing) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) p<sub>Tet</sub>-RBS34-AiiA-TT Rebuilt existing part from BBa_K077047 (Part deleted) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) p<sub>Tet</sub>-RBS32-mRFP-TT Rebuilt existing part from BBa_I20252 (DNA planning) </li></ul></ol><br />
</div><br />
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</html><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Parts/CharacterizedTeam:UNIPV-Pavia/Parts/Characterized2011-09-21T15:28:42Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<html><br />
<a name='indice'></a><br />
<h2 class="art-postheader"><br />
Characterized Parts<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br><br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#new"><span class="tocnumber">1</span> <span class="toctext">New Parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#J101"><span class="tocnumber">1.1</span> <span class="toctext">J23101x series with different RBSs</span></a></li><br />
<li class="toclevel-2"><a href="#pTetLuxI"><span class="tocnumber">1.2</span> <span class="toctext">LuxI Expression cassette driven by aTc</span></a></li><br />
<li class="toclevel-2"><a href="#pTetAiiA"><span class="tocnumber">1.3</span> <span class="toctext">AiiA Expression cassette driven by aTc</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Existing"><span class="tocnumber">2</span> <span class="toctext">Characterization of existing parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#note"><span class="tocnumber">2.1</span> <span class="toctext">Notes on promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.2</span> <span class="toctext">RBSs from the community collection</span></a></li><br />
<li class="toclevel-2"><a href="#pLux"><span class="tocnumber">2.3</span> <span class="toctext">p<sub>Lux</sub> - a 3OC<sub>6</sub>-HSL-in PoPs-out device</span></a></li><br />
<li class="toclevel-2"><a href="#pTet"><span class="tocnumber">2.4</span> <span class="toctext">p<sub>Tet</sub> - BBa_R0040</span></a></li><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.5</span> <span class="toctext">LuxI - BBa_C0061</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.6</span> <span class="toctext">AiiA - BBa_C0060</span></a></li><br />
<li class="toclevel-2"><a href="#K999"><span class="tocnumber">2.7</span> <span class="toctext">pSB1C3 vector with mRFP between S and P for easy cloning</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Improvement"><span class="tocnumber">3</span> <span class="toctext">Sequence debugging</span></a></li><br />
</ul></td></tr></table><br />
</div><br><br />
<br />
<br />
<br />
<br><br><br />
<br />
<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part: pSB4C5">pSB4C5</a>. <br />
</em><br />
<br><br />
<br><br />
<br />
<br />
<div class="listcircle"><br />
<a name='new'></a><h1>New parts</h1><br />
<a name='J101'></a><h2>J23101x series</h2><br />
<br />
<ol type='1'><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> (wiki name: J101-E5 ) J101-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> (wiki name: J101-31 ) J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> (wiki name: J101-E7 ) J101-RBS32-mRFP-TT </li><br />
</ol><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- J23101x description --><br />
<!-- ----------------- --><br />
<p align='justify'><br />
<div align="justify"><p>BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in '<a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>data analysis</a>' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene.</p><br />
<p>The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P. This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts.</p><br />
<p>Salis et al. [Nat Biotec, 2009] stated that <em> 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels'</em> and again <em>'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.'</em></p><br />
<br />
<p>For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS. In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.</p><br />
<br />
<p><br><em><b>NB</b>: in the RPU computation, the J23101-RBS34-mRFP-TT (<A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_J23101">BBa_J23101</a>) in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency.</em></p></div><br />
<br />
<table align='center'><tr><td width='50%' ><br />
<table class='data'><br />
<tr><br />
<td class='row'><b>J23101 promoter with RBS</b><br />
</td><br />
<td class='row'><b>S<sub>cell</sub></b><br />
</td><br />
<td class='row'><b>R.P.U.s</b><br />
</td><br />
</tr><br />
<tr><td class='row'>RBS30<br />
</td><br />
<td class='row'>122 [13.23]</td><br />
<td class='row'>2.45 [0.27]</td><br />
</tr><br />
<tr><td class='row'>RBS31<br />
</td><br />
<td class='row'>2 [0.25]</td><br />
<td class='row'>0.04 [0.01]</td><br />
</tr><br />
<tr><td class='row'>RBS32<br />
</td><br />
<td class='row'>20 [1.7]</td><br />
<td class='row'>0.40 [0.03]</td><br />
</tr><br />
<tr><td class='row'>RBS34<br />
</td><br />
<td class='row'>50 [1.87]</td><br />
<td class='row'>1 [0.02]</td><br />
</tr><br />
</table></td><br />
<div align="center">Data are provided as average [Standard error].</div><br><br />
<td width='50%' ><br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="image"><img alt="RPUs of J23101 promoter with different RBSs" src="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="thumbimage" width="80%"></a></div></div><br />
</td><br />
</tr></table><br />
<br><br><br />
<div align="justify">On the other hand, if the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.</div><br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- pTetLuxI description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetLuxI'></a><h2>LuxI expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> (wiki name: E13 ) pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> (wiki name: E14 ) pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> (wiki name: E15 ) pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI </li></ul></ol><br />
<br />
<br><br><br />
<p align='justify'><em>Though these parts don't have a transcriptional terminator, they have been characterized in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a>, that contains the BBa_B0054 terminator. This choice is motivated by the need to reproduce the exact experimental context of the final circuit, as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Solution#Circuit_design'>solution section</a>. <br />
</em></p><br />
<br><br><br />
<p align='justify'><br />
LuxI has been characterized in terms of enzymatic activity under the regulation of p<sub>Tet</sub> promoter. <br />
</p><br />
<p align='justify'><br />
K<sub>M,LuxI</sub> and V<sub>max</sub> parameters representing its activity have been estimated and the promoter strength (represented by a synthetic parameter &alpha;<sub>pTet</sub> for every pTet-RBS combination) at full induction (100 ng/ml) has been estimated too with a simultaneous fitting of the available data. <br />
<br />
</p><br />
<br />
<br />
<p align='justify'><br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002'>modelling section</a>). <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system describing the behavior of this measurement circuit is reported here: </p><br />
<br />
<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg" class="thumbimage" height="70%" width="68%"></a></div></div><br />
<br />
<br />
<br />
<p align='justify'><br />
Since the measurement systems are only assayed in the exponential growth phase, in the equation (3) N &lt; N<sub>max</sub> is assumed. <br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known and summarized in the table below:</p><br />
<br />
<div align="center"><br />
<table align="center" class='data' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.004925</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
</div><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, k<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>pTet-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
<br />
<div align="justify"><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div></div><br />
<br />
<br />
<div align="justify"><p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p></div><br />
<br />
<br />
<div align="center"><br />
<table align="center" class='data'><br />
<tr><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>k<sub>M,LuxI</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>3.56*10<sup>-9</sup></td><br />
<td class='row'>6.87*10<sup>3</sup></td><br />
<td class='row'>87</td><br />
<td class='row'>8.5</td><br />
<td class='row'>ND</td><br />
<td class='row'>252</td><br />
</tr></table><br />
</div><br />
<br> <br />
</p><br />
</p><br />
<br />
<br />
</p><br />
<br />
<br />
<p align='justify'><br />
The collected data have been used to identify the parameters of our model. Despite the data-poor context, the model predictions fit the experimental data, thus demonstrating that the equation that models the HSL synthesis by LuxI is a good approximation of real processes. <br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- pLuxAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetAiiA'></a><h2>AiiA expression cassette driven by aTc-inducible p<sub>Tet</sub> promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> (wiki name: E24 ) p<sub>Tet</sub>-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> (wiki name: E25 ) p<sub>Tet</sub>-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) p<sub>Tet</sub>-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) p<sub>Tet</sub>-RBS34-AiiA-TT </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<br />
<a name='Existing'></a><h1>Existing parts</h1><br />
<br />
<a name='note'></a><h2>Notes for promoter characterization</h2><br />
<p align='justify'><br />
<p>Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection.</p><br />
<p>The assembled RBSs are:</p><br />
<br><br />
<div align='center'><br />
<table class='data'><br />
<tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr><br />
<tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr><br />
<tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr><br />
<tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr><br />
<tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr><br />
</table></div><br />
<br><br />
<div align="justify"><p>For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.</p><br />
<p>The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. <br />
RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP).</p><br />
<p>For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.</p><br />
</p><br />
<p>Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least squares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs:</p><p></p><br />
<p><ol><ul><li><b><br />
RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity</p><br />
</li><p><li><b><br />
RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity</p><br />
</li><p><li><br />
<b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and it is representative of the position of linear region</p><br />
</li><br />
<p><li><br />
<b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</div></li></p><br />
</ul></ol><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --RBS description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='rbs'></a><h2>RBSs</h2><br />
<p align='justify'><br />
<p><div align="justify">The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of protein produced. <br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element.</p><br />
<p>The evaluation of RBS efficiency can be performed in a very intuitive fashion:</p><br />
<ol><br />
<li>select the RBSs you want to study</li><br />
<li>assemble them in a Promoter - XX - Coding sequence circuit</li><br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/d/d1/Vettore_base.jpg" class="thumbimage" width="60%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<li>measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS (<a href='http://partsregistry.org/wiki/index.php/Part: BBa_B0034'>BBa_B0034</a>). </li></div></ol><br><br />
<br />
<br />
<div align="justify"><p>This simple measurement system allows the quantification of RBS efficiency depending on the experimental context (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments.</p><br />
<p>To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.</p><br />
<p>In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, p<sub>Tet</sub>, p<sub>Lux</sub>). The system output was measured and the RBS efficiency evaluated. The results are summarized in the table below:</div></p><br />
<br><br />
<div align="center"><table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>p<sub>Lux</sub></sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>p<sub>Tet</sub></sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>J23101</sub></b><sup>**</sup></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>0.40</td><td class='row'>1.6814</td><td class='row'>2.45</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.01</td><td class='row'>ND</td><td class='row'>0.04</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.19</td><td class='row'>0.4193</td><td class='row'>0.40</td><td class='row'>0,3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table></div><br />
<br />
<br><br />
<div align="justify">On the other hand, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (p<sub>Tet</sub>-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below:</div><br><br />
<br />
<div align="center"><table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>mRFP</sub></b><sup>**</sup></td><br />
<td class='row'><b>eff<sub>AiiA</sub></b><sup>***</sup></td><br />
<td class='row'><b>eff<sub>LuxI</sub><sup>****</sup></b></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>1.72</td><td class='row'>0.53</td><td class='row'>0.45</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.03</td><td class='row'>0.83</td><td class='row'>0.028</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.37</td><td class='row'>0.50</td><td class='row'>N.D.</td><td class='row'>0.3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table></div><br />
<br><br />
<p align='justify'><br />
<div align="justify"><p><sup>*</sup> The RBS efficiency for inducible devices expressing mRFP was estimated as the ratio of the AUCs (Area under the curve) of the induction curve of the system with the studied RBS and the B0034 reference: AUC<sub>P, RBSx</sub>/AUC<sub>P, B0034</sub></p><br />
<p><sup>**</sup> The RBS efficiency for constitutive promoters expressing mRFP was computed as the ratio between S<sub>cell<sub>P, RBSx</sub></sub>/S<sub>cell<sub>P, B0034</sub></sub></p><br />
<p><sup>***</sup> The RBS efficiency for p<sub>Tet</sub> promoter driving the expression of AiiA enzyme was computed as the ratio &alpha;<sub>p<sub>Tet</sub>, RBSx</sub>/&alpha;<sub>p<sub>Tet</sub>, B0034</sub> estimated for the measurement system p<sub>Tet</sub>-RBSx-AiiA-TT. &alpha;<sub>p<sub>Tet</sub></sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#AiiA'>here</a>. p<sub>Tet</sub> was tested at full induction (100 ng/ml).</p><br />
<p><sup>****</sup> The RBS efficiency for promoters driving the expression of LuxI was computed as the ratio &alpha;<sub>p<sub>Tet</sub>, RBSx</sub>/&alpha;<sub>p<sub>Tet</sub>, B0034</sub> estimated from the measurement systems p<sub>Tet</sub>-RBSx-LuxI. &alpha;<sub>p<sub>Tet</sub></sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#LuxI'>here</a>. p<sub>Tet</sub> was tested at full induction (100 ng/ml).</p></div><br />
<br />
<br><br />
</p><br />
The parts we used to characterize the RBSs are listed here:<br />
<ol><br />
<ul><li>mRFP expression with different promoters</li><br />
<ul><br />
<li>J23101</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> J101-RBS30-RFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> J101-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_J23101 "> BBa_J23101 </a> J101-RBS34-mRFP-TT </li><br />
</ul><br />
<li>p<sub>Tet</sub></li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> p<sub>Tet</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
<li>p<sub>Lux</sub></li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
</ul><br />
<li>p<sub>Tet</sub> driving the expression of different genes</li><br />
<ul><li>mRFP</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> p<sub>Tet</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
<li>AiiA</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> p<sub>Tet</sub>-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> p<sub>Tet</sub>-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> p<sub>Tet</sub>-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> p<sub>Tet</sub>-RBS34-AiiA-TT </li><br />
</ul><br />
<li>LuxI</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> p<sub>Tet</sub>-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> p<sub>Tet</sub>-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> p<sub>Tet</sub>-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> p<sub>Tet</sub>-RBS34-LuxI </li><br />
</ul></ul><br />
</ul></ol><br />
<br />
<br />
</p><br />
<p align='justify'><br />
The results reported in the table suggest that the RBS efficiency ranking is not always maintained. In particular, for the different promoters driving the expression of mRFP, the ranking of the declared efficiencies is maintained for p<sub>Lux</sub>, but not for p<sub>Tet</sub> and J23101. The RBS B0030 results to be the most efficient for both J23101 and p<sub>Tet</sub>, but not for p<sub>Lux</sub> (NB: this effect might be due to an effective non-modularity of RBS, but also to saturating phenomena occurring for this very strong promoter at full induction). RBS B0031 always shows a very low efficiency, while B0032 an intermediate efficiency between B0031 and the stronger RBSs B0030 and B0034. <br><br />
For what concerns the encoded gene variation, more significant differences can be observed. RBS B0030 has the higher efficiency only for mRFP, while the values for AiiA and LuxI are similar (~0.5). Unexpectedly, the weak RBS B0031 has a higher efficiency with AiiA gene (0.83), while with mRFP and LuxI. With B0032 no activity was observed for LuxI, while for AiiA and mRFP the results are quite consistent with the one reported above. <br />
</p><br />
<p align='justify'><br />
These results are encouraging: though the partial non-modularity of RBS with the encoded gene is confirmed, the hypothesis of modularity with the promoter is to some extent confirmed. Three classes of efficiencies were identified:<br />
<ul><br />
<li>low efficiency RBS (B0031)</li><br />
<li>medium efficiency RBS (B0032)</li><br />
<li>high efficiency RBSs (B0030, B0034)</li><br />
</ul> <br />
</p><br />
<br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --pLux description- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<a name='pLux'></a><h2>p<sub>Lux</sub> promoter</h2><br />
<p align='justify'><br />
<br />
<br />
</p><br />
<ol><ul><br />
<br />
<br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> (wiki name: E17 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> (wiki name: E18 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> (wiki name: E19 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> (wiki name: E20 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS34-mRFP-TT </li></ul><br />
</ol><br />
<br />
<p align='justify'><br />
The estimated parameters for the Hill functions are summarized in the table below. For more details on parameter estimation, see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>model section</a>. <br />
</p><br />
<br />
<table class='data' width='100%'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Lux</sub></sub> [(AUr/min)/cell]</b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Lux</sub></sub> [-]</b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Lux</sub></sub> [-]</b></td><br />
<td class="row"><b>k<sub>p<sub>Lux</sub></sub> [ng/ml]</b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">438 [10]</td><br />
<td class="row">0.05 [>100]</td><br />
<td class="row">2 [47]</td><br />
<td class="row">1.88 [27]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">9.8 [7]</td><br />
<td class="row">0.11 [57]</td><br />
<td class="row">1.2 [29]</td><br />
<td class="row">1.5 [26]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">206 [3]</td><br />
<td class="row">0 [>>100]</td><br />
<td class="row">1.36 [10]</td><br />
<td class="row">1.87 [9]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">1105 [6]</td><br />
<td class="row">0.02 [>100]</td><br />
<td class="row">1.33 [19]</td><br />
<td class="row">2.34 [18]</td><br />
</tr><br />
</table><br />
<div align="center">Data are provided as average [CV%].</div><br><br />
<br />
<br />
<div align="justify"><br />
<p>From this table, it is evident that, whilst &alpha;<sub>p<sub>Lux</sub></sub> assumes significantly different values for different RBSs, &eta;<sub>pLux</sub> and k<sub>pLux</sub> assume very similar values. This result shows that RBS variation modulates the amplitude of Hill function, not affecting the shape of the curve. The four induction curves result to be the same Hill function modulated in amplitude by a parameter, that is the estimated RBS efficiency for this measurement system.</p><br />
<p>These results are quite encouraging, because suggest that, given the non-modular behavior of RBS dpending on the encoded gene, the RBS has a modular behaviour respect to the promoter.</p><br />
<p>The operative parameters are summarized in the table below:</p></div><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>4.28</td><td class='row'>0.20</td><td class='row'>1.08</td><td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>4.93</td><td class='row'>0.55</td><td class='row'>0.25</td><td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>9.49</td><td class='row'>0.02</td><td class='row'>0.47</td><td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>21.53</td><td class='row'>0.51</td><td class='row'>0.53</td><td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<p align='justify'>These operative parameters provide useful information on the behavior of this 3OC6-HSL inducible device. RPU<sub>max</sub> assumes very different values in terms of RPUs.<br />
This can't be explained by RBS modulation, since RPUs have been evaluated by normalizing S<sub>cell</sub> of p<sub>Lux</sub>-RBSx for the one of J23101-RBSx. It is evident that some nonlinear effect on maximum strength, maybe due to saturation phenomena on protein expression, occur. <br />
The same RPUs should be observed for every RBS, since the normalization by the standard reference used for RPUs computation should eliminate the RBS contribute. Here different RPUs are observed, maybe due to nonlinear RBS behavior or to saturation phenomena occurring with this very strong promoter. The switch point and linear boundaries are quite constant in all the cases, showing that the linear region of this system is not affected by RBS changes.</p><br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/5/50/E17_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/5/50/E17_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/c/c4/E18_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c4/E18_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/7/78/E19_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/78/E19_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/5/55/E20_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/5/55/E20_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- -pTet description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='pTet'></a><h2>p<sub>Tet</sub> promoter</h2><br />
<br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> (wiki name: E21 ) p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> (wiki name: E22 ) p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) p<sub>Tet</sub>-RBS32-mRFP-TT<br />
</li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT <br />
</li></ul><br />
</ul><br />
</ol><br />
<br />
<div align="justify"><br />
<p>The protocols for the characterization of p<sub>Tet</sub> promoter are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#pTet_protocol'>p<sub>Tet</sub> measurement section</a>.</p><br />
<p>The data collected from the mRFP measurement systems were processed as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'> data analysis section</a>. The induction curves were obtained by fitting a Hill function as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>modelling section</a> and the estimated <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>parameters</a> for p<sub>Tet</sub> are reported in the pictures and in table below. </p><br />
<p>This promoter is widely studied and characterized usually using the strong RBS BBa_B0034. Here we have characterized its transcriptional strength as a function of aTc induction (ng/ul) for different RBSs. Three different induction curves were obtained and are reported in figure:</p></div><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<br />
<br />
<!-- <td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/9/93/E33_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/9/93/E33_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td> --><br />
<br />
<table width='100%'><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/9/99/E34_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/9/99/E34_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/e/e4/E35_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e4/E35_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<p align='justify'><br />
The estimated parameters of the Hill curves described in the figures are summarized in the table below:<br />
</p><br />
<br><br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub> [(AUr/min)/cell]</b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub> [-]</b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub> [-]</b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub> [nM]</b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">230.67 [3.7]</td><br />
<td class="row">0.028 [91.61]</td><br />
<td class="row">4.61 [23.73]</td><br />
<td class="row">8.75 [4.16]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">55.77 [12]</td><br />
<td class="row">1.53E-11 [>>100]</td><br />
<td class="row">4.98 [57.62]</td><br />
<td class="row">7.26 [14.98]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">120 [5.95]</td><br />
<td class="row">0.085 [40.6]</td><br />
<td class="row">24.85 [47.6]</td><br />
<td class="row">9 [5.43]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br><br><br />
<br />
<p align='justify'><br />
The measurement system p<sub>Tet</sub>-B0031-mRFP-TT couldn't be assayed because its fluorescence output is under the detectability threshold of our measurement instrument. For this reason, the parameters of the corresponding Hill curve couldn't be estimated and are reported as 'Not Determined' ND. <br />
</p><br />
<br />
<div align='justify'><br />
<p>&alpha; parameter (representing the maximum trascriptional rate in the studied range of induction) varies as expected with the RBS variation and also the &delta; and &eta; parameters are quite different among the RBS variations.</p><br />
<p>The k<sub>p<sub>Tet</sub></sub> parameter is quite constant among the RBS variations, thus suggesting that in this case the RBS variation doesn't substantially affect the switch point of the Hill curve, even if the amplitude and the maximum slope are not quite maintained (for the &eta; parameter, maybe fitting problems). <br />
<br />
</p><br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p></div><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [ng/ml]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [ng/ml]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>1.53</td><td class='row'>~0</td><td class='row'>7.95</td><td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>3.16</td><td class='row'>~0</td><td class='row'>6.7</td><td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>2.73</td><td class='row'>0.23</td><td class='row'>8.96</td><td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
From these parameters, it is evident that whilst the switch-point is almost maintained for all the RBSs, the linear boundaries are similar for RBS30 and RBS32 but for RBS34 are moved on the right of one order of magnitude. <br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --LuxI description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='LuxI'></a><h2>LuxI gene - BBa_C0061</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002'>modelling section</a>) using the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>p<sub>Tet</sub>-RBSx-LuxI-TT</a> measurement systems. <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here: </p><br />
<br />
<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg" class="thumbimage" height="70%" width="68%"></a></div></div><br />
<br />
<br />
<br />
<p align='justify'><br />
Since the measurement systems are only assayed in the exponential growth phase, in the equation (3) N &lt; <N<sub>max</sub> is assumed. <br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known and summarized in the table below:</p><br />
<br />
<div align="center"><br />
<table align="center" class='data' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.004925</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
</div><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, k<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>p<sub>Tet</sub>-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
<br />
<div align="justify"><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div></div><br />
<br />
<br />
<div align="justify"><p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p></div><br />
<br />
<br />
<div align=center"><br />
<table align="center" class='data'><br />
<tr><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>k<sub>M,LuxI</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>3.56*10<sup>-9</sup></td><br />
<td class='row'>6.87*10<sup>3</sup></td><br />
<td class='row'> 87 </td><br />
<td class='row'>8.5</td><br />
<td class='row'>ND</td><br />
<td class='row'> 252 </td><br />
</tr></table><br />
</div><br />
<br> <br />
</p><br />
</p><br />
<br />
<p align='justify'><br />
The provided parameters k<sub>M</sub> and V<sub>max</sub> represent the enzymatic activity of LuxI, described by our model. They must not be confused with the operative parameters of the Michaelis-Menten relation. <br />
These synthetic parameters have a great importance, since they can be used in more complicated models in order to predict the behavior of complex circuits.<br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --AiiA description-- --><br />
<!-- ----------------- --><br />
<br />
<br />
<a name='AiiA'></a><h2>AiiA gene - BBa_C0060</h2><br />
<br />
<p align='justify'><br />
The activity of AiiA enzyme has been evaluated by testing the measurement systems <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a>. <br />
Similar to LuxI, a system of differential equations (referring to <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>) has been derived. <br />
</p><br />
<br />
<div style='text-align:justify'><br />
<div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg" class="thumbimage" height="70%" width="68%"></a><br />
</div></div><br />
<br />
<p align='justify'><br />
The parameters k<sub>cat</sub>, k<sub>M,AiiA</sub> and &alpha;<sub>RBSx</sub> would have been estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>p<sub>Tet</sub>-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
Unfortunately, their estimation revealed difficult.<br><br />
In the first experiments with the measurement system <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a> in LOW-COPY at pH=7 no degradation of HSL was observed. The collected data are shown in the figure below. HSL degradation is identical in the measurement system and in the negative control after 21 hours.<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<div align="justify">Further investigation on the enzyme were performed in more suitable conditions, to evaluate its intrinsic activity. AiiA activity was investigated in HIGH COPY plasmid in <em>E. coli</em> TOP10, in order to understand if the enzyme worked. In this case, a significant difference in degradation between p<sub>Tet</sub>-RBSx-AiiA-TT and the negative control was observed, also just after 7 hours. That was the proof of the good functioning (in HIGH COPY) of the enzyme.</div><br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/6c/Aiia_HC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/e/ec/Aiia_HC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/b/b2/Aiia_HC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/d/d7/Aiia_HC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<div align="justify"><br />
Since the enzyme activity had been assessed, some suitable working conditions were investigated for AiiA. Since our studies on HSL stability for different pHs has shown that its stability improves at pH=6.0, this experimental condition was used to test once again MGZ1 strain with the system expressing AiiA in low copy number plasmid. <br />
<br />
Unfortunately, we had some problems with the biosensor, BBa_T9002, that didn't work as usual, resulting in a calibration curve modified. So, k<sub>M,AiiA</sub> and k<sub>cat</sub> were impossible to be estimated from these data. We decided to estimate them from experimental data coming from Tecan tests preformed in <em>E.COLI</em> TOP10, where aiiA gene was cloned in a high copy numebr plasmid, pSB1A2, downstream p<sub>Tet</sub> with different RBSs; in this way we could have some semi-quantitative information about the order of magnitude of these parameters. Taking into account the different copy number, we tried to simulate our model behavior with reasonable values for AiiA parameters.</div><br />
<br><br />
<br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
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<br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- ---pSB1C3-mRFP-- --><br />
<!-- ----------------- --><br />
<br />
<a name='K999'></a><h2>pSB1C3 plasmid with mRFP between S and P bearing p<sub>Tet</sub> (easy-to-clone)</h2><br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/e/ee/BBa_I13507.jpg" class="thumbimage" width="50%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<div align="justify">This vector was designed and realized in order to facilitate the cloning of coding sequences downstream of the strong promoter p<sub>Tet</sub>. This vector was assembled by ligating S-P excided mRFP coding sequence from BBa_J61002 and ligating it in BBa_R0040 cut with S and P. Thus, the resulting vector contains mRFP between S and P. p<sub>Tet</sub> can be easily excided (E-P) and moved in the desired vector (E-P) and then the desired coding sequence can be easily assembled by digesting S-P the vector and X-P the coding sequence, thus obtaining a final part that is standard10-compatible.</div><br><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<a name='Improvement'></a><h1>Existing parts: sequence debugging</h1><br />
<br />
<div align="justify"><br />
<br />
<ol><ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516021 "> BBa_K516021 </a> (wiki name: E10 ) RBS31-AiiA-TT Rebuilt existing part from BBa_I13914 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516022 "> BBa_K516022 </a> (wiki name: E11 ) RBS32-AiiA-TT Rebuilt existing part from BBa_I13912 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516030 "> BBa_K516030 </a> (wiki name: E5 ) RBS30-mRFP-TT Rebuilt existing part from BBa_S04180 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516032 "> BBa_K516032 </a> (wiki name: E7 ) RBS32-mRFP-TT Rebuilt existing part from BBa_ J133000 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) p<sub>Tet</sub>-RBS34-LuxI Rebuilt existing part from BBa_S03623 (DNA available, only 2008 kit, inconsistent) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) p<sub>Tet</sub>-RBS32-AiiA-TT Rebuilt existing part from BBa_ J22071 (2008 only, Bad sequencing) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) p<sub>Tet</sub>-RBS34-AiiA-TT Rebuilt existing part from BBa_K077047 (Part deleted) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) p<sub>Tet</sub>-RBS32-mRFP-TT Rebuilt existing part from BBa_I20252 (DNA planning) </li></ul></ol><br />
</div><br />
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<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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</div><br />
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</html><br />
<br />
{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Parts/CharacterizedTeam:UNIPV-Pavia/Parts/Characterized2011-09-21T11:58:09Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<html><br />
<a name='indice'></a><br />
<h2 class="art-postheader"><br />
Characterized Parts<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br><br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#new"><span class="tocnumber">1</span> <span class="toctext">New Parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#J101"><span class="tocnumber">1.1</span> <span class="toctext">J23101x series with different RBSs</span></a></li><br />
<li class="toclevel-2"><a href="#pTetLuxI"><span class="tocnumber">1.2</span> <span class="toctext">LuxI Expression cassette driven by aTc</span></a></li><br />
<li class="toclevel-2"><a href="#pTetAiiA"><span class="tocnumber">1.3</span> <span class="toctext">AiiA Expression cassette driven by aTc</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Existing"><span class="tocnumber">2</span> <span class="toctext">Characterization of existing parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#note"><span class="tocnumber">2.1</span> <span class="toctext">Notes on promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.2</span> <span class="toctext">RBSs from the community collection</span></a></li><br />
<li class="toclevel-2"><a href="#pLux"><span class="tocnumber">2.3</span> <span class="toctext">p<sub>Lux</sub> - a 3OC<sub>6</sub>-HSL-in PoPs-out device</span></a></li><br />
<li class="toclevel-2"><a href="#pTet"><span class="tocnumber">2.4</span> <span class="toctext">p<sub>Tet</sub> - BBa_R0040</span></a></li><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.5</span> <span class="toctext">LuxI - BBa_C0061</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.6</span> <span class="toctext">AiiA - BBa_C0060</span></a></li><br />
<li class="toclevel-2"><a href="#K999"><span class="tocnumber">2.7</span> <span class="toctext">pSB1C3 vector with mRFP between S and P for easy cloning</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Improvement"><span class="tocnumber">3</span> <span class="toctext">Sequence debugging</span></a></li><br />
</ul></td></tr></table><br />
</div><br><br />
<br />
<br />
<br />
<br><br><br />
<br />
<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part: pSB4C5">pSB4C5</a>. <br />
</em><br />
<br><br />
<br><br />
<br />
<br />
<div class="listcircle"><br />
<a name='new'></a><h1>New parts</h1><br />
<a name='J101'></a><h2>J23101x series</h2><br />
<br />
<ol type='1'><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> (wiki name: J101-E5 ) J101-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> (wiki name: J101-31 ) J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> (wiki name: J101-E7 ) J101-RBS32-mRFP-TT </li><br />
</ol><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- J23101x description --><br />
<!-- ----------------- --><br />
<p align='justify'><br />
<div align="justify"><p>BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in '<a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>data analysis</a>' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene.</p><br />
<p>The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P. This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts.</p><br />
<p>Salis et al. [Nat Biotec, 2009] stated that <em> 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels'</em> and again <em>'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.'</em></p><br />
<br />
<p>For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS. In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.</p><br />
<br />
<p><br><em><b>NB</b>: in the RPU computation, the J23101-RBS34-mRFP-TT (<A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_J23101">BBa_J23101</a>) in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a> construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency.</em></p></div><br />
<br />
<table align='center'><tr><td width='50%' ><br />
<table class='data'><br />
<tr><br />
<td class='row'><b>J23101 promoter with RBS</b><br />
</td><br />
<td class='row'><b>S<sub>cell</sub></b><br />
</td><br />
<td class='row'><b>R.P.U.s</b><br />
</td><br />
</tr><br />
<tr><td class='row'>RBS30<br />
</td><br />
<td class='row'>122 [13.23]</td><br />
<td class='row'>2.45 [0.27]</td><br />
</tr><br />
<tr><td class='row'>RBS31<br />
</td><br />
<td class='row'>2 [0.25]</td><br />
<td class='row'>0.04 [0.01]</td><br />
</tr><br />
<tr><td class='row'>RBS32<br />
</td><br />
<td class='row'>20 [1.7]</td><br />
<td class='row'>0.40 [0.03]</td><br />
</tr><br />
<tr><td class='row'>RBS34<br />
</td><br />
<td class='row'>50 [1.87]</td><br />
<td class='row'>1 [0.02]</td><br />
</tr><br />
</table></td><br />
<div align="center">Data are provided as average [Standard error].</div><br><br />
<td width='50%' ><br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="image"><img alt="RPUs of J23101 promoter with different RBSs" src="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="thumbimage" width="80%"></a></div></div><br />
</td><br />
</tr></table><br />
<br><br><br />
<div align="justify">On the other hand, if the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.</div><br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!-- ----------------- --><br />
<!-- pTetLuxI description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetLuxI'></a><h2>LuxI expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> (wiki name: E13 ) pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> (wiki name: E14 ) pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> (wiki name: E15 ) pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI </li></ul></ol><br />
<br />
<br><br><br />
<p align='justify'><em>Though these parts don't have a transcriptional terminator, they have been characterized in low copy plasmid <A HREF="http://partsregistry.org/wiki/index.php/Part:pSB4C5">pSB4C5</a>, that contains the BBa_B0054 terminator. This choice is motivated by the need to reproduce the exact experimental context of the final circuit, as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Solution#Circuit_design'>solution section</a>. <br />
</em></p><br />
<br><br><br />
<p align='justify'><br />
LuxI has been characterized in terms of enzymatic activity under the regulation of p<sub>Tet</sub> promoter. <br />
</p><br />
<p align='justify'><br />
K<sub>M,LuxI</sub> and V<sub>max</sub> parameters representing its activity have been estimated and the promoter strength (represented by a synthetic parameter &alpha;<sub>pTet</sub> for every pTet-RBS combination) at full induction (100 ng/ml) has been estimated too with a simultaneous fitting of the available data. <br />
<br />
</p><br />
<br />
<br />
<p align='justify'><br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002'>modelling section</a>). <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system describing the behavior of this measurement circuit is reported here: </p><br />
<br />
<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg" class="thumbimage" height="70%" width="68%"></a></div></div><br />
<br />
<br />
<br />
<p align='justify'><br />
Since the measurement systems are only assayed in the exponential growth phase, in the equation (3) N &lt; N<sub>max</sub> is assumed. <br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known and summarized in the table below:</p><br />
<br />
<div align="center"><br />
<table align="center" class='data' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.004925</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
</div><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, k<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>pTet-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
<br />
<div align="justify"><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div></div><br />
<br />
<br />
<div align="justify"><p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p></div><br />
<br />
<br />
<div align="center"><br />
<table align="center" class='data'><br />
<tr><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>k<sub>M,LuxI</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>3.56*10<sup>-9</sup></td><br />
<td class='row'>6.87*10<sup>3</sup></td><br />
<td class='row'>87</td><br />
<td class='row'>8.5</td><br />
<td class='row'>ND</td><br />
<td class='row'>252</td><br />
</tr></table><br />
</div><br />
<br> <br />
</p><br />
</p><br />
<br />
<br />
</p><br />
<br />
<br />
<p align='justify'><br />
The collected data have been used to identify the parameters of our model. Despite the data-poor context, the model predictions fit the experimental data, thus demonstrating that the equation that models the HSL synthesis by LuxI is a good approximation of real processes. <br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- pLuxAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetAiiA'></a><h2>AiiA expression cassette driven by aTc-inducible p<sub>Tet</sub> promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> (wiki name: E24 ) p<sub>Tet</sub>-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> (wiki name: E25 ) p<sub>Tet</sub>-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) p<sub>Tet</sub>-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) p<sub>Tet</sub>-RBS34-AiiA-TT </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<br />
<a name='Existing'></a><h1>Existing parts</h1><br />
<br />
<a name='note'></a><h2>Notes for promoter characterization</h2><br />
<p align='justify'><br />
<p>Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection.</p><br />
<p>The assembled RBSs are:</p><br />
<br><br />
<div align='center'><br />
<table class='data'><br />
<tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr><br />
<tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr><br />
<tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr><br />
<tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr><br />
<tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr><br />
</table></div><br />
<br><br />
<div align="justify"><p>For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.</p><br />
<p>The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. <br />
RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP).</p><br />
<p>For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.</p><br />
</p><br />
<p>Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least squares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs:</p><p></p><br />
<p><ol><ul><li><b><br />
RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity</p><br />
</li><p><li><b><br />
RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity</p><br />
</li><p><li><br />
<b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and it is representative of the position of linear region</p><br />
</li><br />
<p><li><br />
<b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</div></li></p><br />
</ul></ol><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --RBS description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='rbs'></a><h2>RBSs</h2><br />
<p align='justify'><br />
<p><div align="justify">The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of protein produced. <br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element.</p><br />
<p>The evaluation of RBS efficiency can be performed in a very intuitive fashion:</p><br />
<ol><br />
<li>select the RBSs you want to study</li><br />
<li>assemble them in a Promoter - XX - Coding sequence circuit</li><br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/d/d1/Vettore_base.jpg" class="thumbimage" width="60%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<li>measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS (<a href='http://partsregistry.org/wiki/index.php/Part: BBa_B0034'>BBa_B0034</a>). </li></div></ol><br><br />
<br />
<br />
<div align="justify"><p>This simple measurement system allows the quantification of RBS efficiency depending on the experimental context (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments.</p><br />
<p>To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.</p><br />
<p>In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, p<sub>Tet</sub>, p<sub>Lux</sub>). The system output was measured and the RBS efficiency evaluated. The results are summarized in the table below:</div></p><br />
<br><br />
<div align="center"><table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>p<sub>Lux</sub></sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>p<sub>Tet</sub></sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>J23101</sub></b><sup>**</sup></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>0.40</td><td class='row'>1.6814</td><td class='row'>2.45</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.01</td><td class='row'>ND</td><td class='row'>0.04</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.19</td><td class='row'>0.4193</td><td class='row'>0.40</td><td class='row'>0,3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table></div><br />
<br />
<br><br />
<div align="justify">On the other hand, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (p<sub>Tet</sub>-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below:</div><br><br />
<br />
<div align="center"><table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>mRFP</sub></b><sup>**</sup></td><br />
<td class='row'><b>eff<sub>AiiA</sub></b><sup>***</sup></td><br />
<td class='row'><b>eff<sub>LuxI</sub><sup>****</sup></b></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>1.72</td><td class='row'>0.53</td><td class='row'>0.45</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.03</td><td class='row'>0.83</td><td class='row'>0.028</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.37</td><td class='row'>0.50</td><td class='row'>N.D.</td><td class='row'>0.3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table></div><br />
<br><br />
<p align='justify'><br />
<div align="justify"><p><sup>*</sup> The RBS efficiency for inducible devices expressing mRFP was estimated as the ratio of the AUCs (Area under the curve) of the induction curve of the system with the studied RBS and the B0034 reference: AUC<sub>P, RBSx</sub>/AUC<sub>P, B0034</sub></p><br />
<p><sup>**</sup> The RBS efficiency for constitutive promoters expressing mRFP was computed as the ratio between S<sub>cell<sub>P, RBSx</sub></sub>/S<sub>cell<sub>P, B0034</sub></sub></p><br />
<p><sup>***</sup> The RBS efficiency for p<sub>Tet</sub> promoter driving the expression of AiiA enzyme was computed as the ratio &alpha;<sub>p<sub>Tet</sub>, RBSx</sub>/&alpha;<sub>p<sub>Tet</sub>, B0034</sub> estimated for the measurement system p<sub>Tet</sub>-RBSx-AiiA-TT. &alpha;<sub>p<sub>Tet</sub></sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#AiiA'>here</a>. p<sub>Tet</sub> was tested at full induction (100 ng/ml).</p><br />
<p><sup>****</sup> The RBS efficiency for promoters driving the expression of LuxI was computed as the ratio &alpha;<sub>p<sub>Tet</sub>, RBSx</sub>/&alpha;<sub>p<sub>Tet</sub>, B0034</sub> estimated from the measurement systems p<sub>Tet</sub>-RBSx-LuxI. &alpha;<sub>p<sub>Tet</sub></sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#LuxI'>here</a>. p<sub>Tet</sub> was tested at full induction (100 ng/ml).</p></div><br />
<br />
<br><br />
</p><br />
The parts we used to characterize the RBSs are listed here:<br />
<ol><br />
<ul><li>mRFP expression with different promoters</li><br />
<ul><br />
<li>J23101</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> J101-RBS30-RFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> J101-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_J23101 "> BBa_J23101 </a> J101-RBS34-mRFP-TT </li><br />
</ul><br />
<li>p<sub>Tet</sub></li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> p<sub>Tet</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
<li>p<sub>Lux</sub></li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
</ul><br />
<li>p<sub>Tet</sub> driving the expression of different genes</li><br />
<ul><li>mRFP</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> p<sub>Tet</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT </li><br />
</ul><br />
<li>AiiA</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> p<sub>Tet</sub>-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> p<sub>Tet</sub>-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> p<sub>Tet</sub>-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> p<sub>Tet</sub>-RBS34-AiiA-TT </li><br />
</ul><br />
<li>LuxI</li><br />
<ul class="disc"><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> p<sub>Tet</sub>-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> p<sub>Tet</sub>-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> p<sub>Tet</sub>-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> p<sub>Tet</sub>-RBS34-LuxI </li><br />
</ul></ul><br />
</ul></ol><br />
<br />
<br />
</p><br />
<p align='justify'><br />
The results reported in the table suggest that the RBS efficiency ranking is not always maintained. In particular, for the different promoters driving the expression of mRFP, the ranking of the declared efficiencies is maintained for p<sub>Lux</sub>, but not for p<sub>Tet</sub> and J23101. The RBS B0030 results to be the most efficient for both J23101 and p<sub>Tet</sub>, but not for p<sub>Lux</sub> (NB: this effect might be due to an effective non-modularity of RBS, but also to saturating phenomena occurring for this very strong promoter at full induction). RBS B0031 always shows a very low efficiency, while B0032 an intermediate efficiency between B0031 and the stronger RBSs B0030 and B0034. <br><br />
For what concerns the encoded gene variation, more significant differences can be observed. RBS B0030 has the higher efficiency only for mRFP, while the values for AiiA and LuxI are similar (~0.5). Unexpectedly, the weak RBS B0031 has a higher efficiency with AiiA gene (0.83), while with mRFP and LuxI. With B0032 no activity was observed for LuxI, while for AiiA and mRFP the results are quite consistent with the one reported above. <br />
</p><br />
<p align='justify'><br />
These results are encouraging: though the partial non-modularity of RBS with the encoded gene is confirmed, the hypothesis of modularity with the promoter is to some extent confirmed. Three classes of efficiencies were identified:<br />
<ul><br />
<li>low efficiency RBS (B0031)</li><br />
<li>medium efficiency RBS (B0032)</li><br />
<li>high efficiency RBSs (B0030, B0034)</li><br />
</ul> <br />
</p><br />
<br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --pLux description- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<a name='pLux'></a><h2>p<sub>Lux</sub> promoter</h2><br />
<p align='justify'><br />
<br />
<br />
</p><br />
<ol><ul><br />
<br />
<br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> (wiki name: E17 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> (wiki name: E18 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> (wiki name: E19 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> (wiki name: E20 ) pLambda-RBS30-LuxR-T-p<sub>Lux</sub>-RBS34-mRFP-TT </li></ul><br />
</ol><br />
<br />
<p align='justify'><br />
The estimated parameters for the Hill functions are summarized in the table below. For more details on parameter estimation, see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>model section</a>. <br />
</p><br />
<br />
<table class='data' width='100%'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Lux</sub></sub> [(AUr/min)/cell]</b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Lux</sub></sub> [-]</b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Lux</sub></sub> [-]</b></td><br />
<td class="row"><b>k<sub>p<sub>Lux</sub></sub> [ng/ml]</b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">438 [10]</td><br />
<td class="row">0.05 [>100]</td><br />
<td class="row">2 [47]</td><br />
<td class="row">1.88 [27]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">9.8 [7]</td><br />
<td class="row">0.11 [57]</td><br />
<td class="row">1.2 [29]</td><br />
<td class="row">1.5 [26]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">206 [3]</td><br />
<td class="row">0 [>>100]</td><br />
<td class="row">1.36 [10]</td><br />
<td class="row">1.87 [9]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">1105 [6]</td><br />
<td class="row">0.02 [>100]</td><br />
<td class="row">1.33 [19]</td><br />
<td class="row">2.34 [18]</td><br />
</tr><br />
</table><br />
<div align="center">Data are provided as average [CV%].</div><br><br />
<br />
<br />
<div align="justify"><br />
<p>From this table, it is evident that, whilst &alpha;<sub>p<sub>Lux</sub></sub> assumes significantly different values for different RBSs, &eta;<sub>pLux</sub> and k<sub>pLux</sub> assume very similar values. This result shows that RBS variation modulates the amplitude of Hill function, not affecting the shape of the curve. The four induction curves result to be the same Hill function modulated in amplitude by a parameter, that is the estimated RBS efficiency for this measurement system.</p><br />
<p>These results are quite encouraging, because suggest that, given the non-modular behavior of RBS dpending on the encoded gene, the RBS has a modular behaviour respect to the promoter.</p><br />
<p>The operative parameters are summarized in the table below:</p></div><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>4.28</td><td class='row'>0.20</td><td class='row'>1.08</td><td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>4.93</td><td class='row'>0.55</td><td class='row'>0.25</td><td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>9.49</td><td class='row'>0.02</td><td class='row'>0.47</td><td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>21.53</td><td class='row'>0.51</td><td class='row'>0.53</td><td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<p align='justify'>These operative parameters provide useful information on the behavior of this 3OC6-HSL inducible device. RPU<sub>max</sub> assumes very different values in terms of RPUs.<br />
This can't be explained by RBS modulation, since RPUs have been evaluated by normalizing S<sub>cell</sub> of p<sub>Lux</sub>-RBSx for the one of J23101-RBSx. It is evident that some nonlinear effect on maximum strength, maybe due to saturation phenomena on protein expression, occur. <br />
The same RPUs should be observed for every RBS, since the normalization by the standard reference used for RPUs computation should eliminate the RBS contribute. Here different RPUs are observed, maybe due to nonlinear RBS behavior or to saturation phenomena occurring with this very strong promoter. The switch point and linear boundaries are quite constant in all the cases, showing that the linear region of this system is not affected by RBS changes.</p><br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/5/50/E17_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/5/50/E17_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/c/c4/E18_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c4/E18_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/7/78/E19_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/78/E19_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/5/55/E20_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/5/55/E20_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- -pTet description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='pTet'></a><h2>p<sub>Tet</sub> promoter</h2><br />
<br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> (wiki name: E21 ) p<sub>Tet</sub>-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> (wiki name: E22 ) p<sub>Tet</sub>-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) p<sub>Tet</sub>-RBS32-mRFP-TT<br />
</li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> p<sub>Tet</sub>-RBS34-mRFP-TT <br />
</li></ul><br />
</ul><br />
</ol><br />
<br />
<div align="justify"><br />
<p>The protocols for the characterization of p<sub>Tet</sub> promoter are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#pTet_protocol'>p<sub>Tet</sub> measurement section</a>.</p><br />
<p>The data collected from the mRFP measurement systems were processed as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'> data analysis section</a>. The induction curves were obtained by fitting a Hill function as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>modelling section</a> and the estimated <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>parameters</a> for p<sub>Tet</sub> are reported in the pictures and in table below. </p><br />
<p>This promoter is widely studied and characterized usually using the strong RBS BBa_B0034. Here we have characterized its transcriptional strength as a function of aTc induction (ng/ul) for different RBSs. Three different induction curves were obtained and are reported in figure:</p></div><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<br />
<br />
<!-- <td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/9/93/E33_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/9/93/E33_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td> --><br />
<br />
<table width='100%'><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/9/99/E34_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/9/99/E34_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="https://static.igem.org/mediawiki/2011/e/e4/E35_RPU_80.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e4/E35_RPU_80.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<p align='justify'><br />
The estimated parameters of the Hill curves described in the figures are summarized in the table below:<br />
</p><br />
<br><br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub> [(AUr/min)/cell]</b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub> [-]</b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub> [-]</b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub> [nM]</b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">230.67 [3.7]</td><br />
<td class="row">0.028 [91.61]</td><br />
<td class="row">4.61 [23.73]</td><br />
<td class="row">8.75 [4.16]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">55.77 [12]</td><br />
<td class="row">1.53E-11 [>>100]</td><br />
<td class="row">4.98 [57.62]</td><br />
<td class="row">7.26 [14.98]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">120 [5.95]</td><br />
<td class="row">0.085 [40.6]</td><br />
<td class="row">24.85 [47.6]</td><br />
<td class="row">9 [5.43]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br><br><br />
<br />
<p align='justify'><br />
The measurement system p<sub>Tet</sub>-B0031-mRFP-TT couldn't be assayed because its fluorescence output is under the detectability threshold of our measurement instrument. For this reason, the parameters of the corresponding Hill curve couldn't be estimated and are reported as 'Not Determined' ND. <br />
</p><br />
<br />
<div align='justify'><br />
<p>&alpha; parameter (representing the maximum trascriptional rate in the studied range of induction) varies as expected with the RBS variation and also the &delta; and &eta; parameters are quite different among the RBS variations.</p><br />
<p>The k<sub>p<sub>Tet</sub></sub> parameter is quite constant among the RBS variations, thus suggesting that in this case the RBS variation doesn't substantially affect the switch point of the Hill curve, even if the amplitude and the maximum slope are not quite maintained (for the &eta; parameter, maybe fitting problems). <br />
<br />
</p><br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p></div><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [ng/ml]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [ng/ml]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>1.53</td><td class='row'>~0</td><td class='row'>7.95</td><td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>3.16</td><td class='row'>~0</td><td class='row'>6.7</td><td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>2.73</td><td class='row'>0.23</td><td class='row'>8.96</td><td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
From these parameters, it is evident that whilst the switch-point is almost maintained for all the RBSs, the linear boundaries are similar for RBS30 and RBS32 but for RBS34 are moved on the right of one order of magnitude. <br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<br />
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<br />
<!-- ----------------- --><br />
<!-- --LuxI description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='LuxI'></a><h2>LuxI gene - BBa_C0061</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002'>modelling section</a>) using the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>p<sub>Tet</sub>-RBSx-LuxI-TT</a> measurement systems. <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here: </p><br />
<br />
<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/a/a6/Pc_luxi.jpg" class="thumbimage" height="70%" width="68%"></a></div></div><br />
<br />
<br />
<br />
<p align='justify'><br />
Since the measurement systems are only assayed in the exponential growth phase, in the equation (3) N &lt; <N<sub>max</sub> is assumed. <br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known and summarized in the table below:</p><br />
<br />
<div align="center"><br />
<table align="center" class='data' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.004925</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
</div><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, k<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>p<sub>Tet</sub>-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
<br />
<div align="justify"><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div></div><br />
<br />
<br />
<div align="justify"><p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p></div><br />
<br />
<br />
<div align=center"><br />
<table align="center" class='data'><br />
<tr><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>k<sub>M,LuxI</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>3.56*10<sup>-9</sup></td><br />
<td class='row'>6.87*10<sup>3</sup></td><br />
<td class='row'> 87 </td><br />
<td class='row'>8.5</td><br />
<td class='row'>ND</td><br />
<td class='row'> 252 </td><br />
</tr></table><br />
</div><br />
<br> <br />
</p><br />
</p><br />
<br />
<p align='justify'><br />
The provided parameters k<sub>M</sub> and V<sub>max</sub> represent the enzymatic activity of LuxI, described by our model. They must not be confused with the operative parameters of the Michaelis-Menten relation. <br />
These synthetic parameters have a great importance, since they can be used in more complicated models in order to predict the behavior of complex circuits.<br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --AiiA description-- --><br />
<!-- ----------------- --><br />
<br />
<br />
<a name='AiiA'></a><h2>AiiA gene - BBa_C0060</h2><br />
<br />
<p align='justify'><br />
The activity of AiiA enzyme has been evaluated by testing the measurement systems <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a>. <br />
Similar to LuxI, a system of differential equations (referring to <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>) has been derived. <br />
</p><br />
<br />
<div style='text-align:justify'><br />
<div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg" class="thumbimage" height="70%" width="68%"></a><br />
</div></div><br />
<br />
<p align='justify'><br />
The parameters k<sub>cat</sub>, k<sub>M,AiiA</sub> and &alpha;<sub>RBSx</sub> would have been estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>p<sub>Tet</sub>-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
Unfortunately, their estimation revealed impossible.<br><br />
Firstly several tests were performed, considering the subpart <a href="https://static.igem.org/mediawiki/2011/1/11/Pc_aiia.jpg">p<sub>Tet</sub>-RBSx-AiiA-TT</a> in LOW-COPY at pH=7. An example of them is shown in the figure below: HSL is degraded in time as well as the negative control, which shouldn't degrade it even after 21 hours.<br />
<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
<a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<div align="justify">So, we decided to investigate the behaviour in HIGH COPY in another <em>E.COLI</em> strain, TOP10, in order to understand if the enzyme AiiA would have been worked well. In this case, a significant difference in degradation between p<sub>Tet</sub>-RBSx-AiiA-TT and the negative control was observed, also just after 7 hours. That was the proof of the good functioning (in HIGH COPY) of the enzyme.</div><br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><br />
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<div align="justify">Then, we decided to come back to MGZ1 LOW COPY, this time at pH=6. <br />
<br />
Unfortunately, we had some problems with the biosensor, BBa_T9002, that didn't work as usual, resulting in a calibration curve modified. So, k<sub>M,AiiA</sub> and k<sub>cat</sub> were impossible to be estimated from these data. We decided to estimate them from<br />
experimental data coming from Tecan tests preformed in <em>E.COLI</em> TOP10, where aiiA gene was cloned in a high copy numebr plasmid, pSB1A2, downstream p<sub>Tet</sub> with different RBSs; in this way we could have some semi-quantitative information about the order of<br />
magnitude of these parameters. Taking into account the different copy number, we tried to simulate our model behavior with reasonable values for AiiA parameters.</div><br />
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<a name='K999'></a><h2>pSB1C3 plasmid with mRFP between S and P bearing p<sub>Tet</sub> (easy-to-clone)</h2><br />
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<div align="justify">This vector was designed and realized in order to facilitate the cloning of coding sequences downstream of the strong promoter p<sub>Tet</sub>. This vector was assembled by ligating S-P excided mRFP coding sequence from BBa_J61002 and ligating it in BBa_R0040 cut with S and P. Thus, the resulting vector contains mRFP between S and P. p<sub>Tet</sub> can be easily excided (E-P) and moved in the desired vector (E-P) and then the desired coding sequence can be easily assembled by digesting S-P the vector and X-P the coding sequence, thus obtaining a final part that is standard10-compatible.</div><br><br />
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<a name='Improvement'></a><h1>Existing parts: sequence debugging</h1><br />
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<ol><ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516021 "> BBa_K516021 </a> (wiki name: E10 ) RBS31-AiiA-TT Rebuilt existing part from BBa_I13914 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516022 "> BBa_K516022 </a> (wiki name: E11 ) RBS32-AiiA-TT Rebuilt existing part from BBa_I13912 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516030 "> BBa_K516030 </a> (wiki name: E5 ) RBS30-mRFP-TT Rebuilt existing part from BBa_S04180 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516032 "> BBa_K516032 </a> (wiki name: E7 ) RBS32-mRFP-TT Rebuilt existing part from BBa_ J133000 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) p<sub>Tet</sub>-RBS34-LuxI Rebuilt existing part from BBa_S03623 (DNA available, only 2008 kit, inconsistent) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) p<sub>Tet</sub>-RBS32-AiiA-TT Rebuilt existing part from BBa_ J22071 (2008 only, Bad sequencing) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) p<sub>Tet</sub>-RBS34-AiiA-TT Rebuilt existing part from BBa_K077047 (Part deleted) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) p<sub>Tet</sub>-RBS32-mRFP-TT Rebuilt existing part from BBa_I20252 (DNA planning) </li></ul></ol><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Team/What_we_did...Team:UNIPV-Pavia/Team/What we did...2011-09-20T23:10:51Z<p>Nickpv: </p>
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<h2 class="art-postheader">What we did...</h2><br />
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<li><b>Edoardo Baldini</b> has been the wiki-master. He took care of the wiki design and layout very meticulously. Despite being an electronic engineer,<br />
he also learnt and carried out all the wet lab protocols, cloning parts with success and performing Tecan tests. During the brainstorming<br />
sessions of March and April he proposed a couple of interesting ideas. Finally he worked on our terrific sweaters!<br />
<br><br><br />
<li><b>Giuseppe Bertoni</b> has been fundamental during the brainstorming sessions, thanks to his enormous knowledge and passion in<br />
biology and biotechnology. He also worked on cloning BioBrick parts and on the design and performing of Tecan experiments. He also worked<br />
on the wiki, designing our fantastic logo.<br />
<br><br><br />
<li><b>Davide Bianchini</b> has been working hard on the model, deriving its equations and designing the Tecan tests for the parameter estimation.<br />
His work on the stability analysis was also very important; moreover he also helped in the wet lab, working on the cloning of BioBrick parts<br />
and their transferring in pSB1C3 shipping plasmid.<br />
<br><br><br />
<li><b>Niccolò Franceschi</b> has worked hard in the wet lab, both on the cloning and the testing of our BioBrick parts, verifying, together with Viola<br />
that all of our parts were correct. He also analyzed the scientific literature and actively worked on resuming the results of our tests.<br />
Least but not last, was fundamental in raising funds for our project.<br />
<br><br><br />
<li><b>Viola Ghio</b> worked hard in the wet lab. She cloned parts, performed screening on them and also verified that their sequences were correct.<br />
Moreover she worked on the transferring of selected BioBrick parts in pSB1C3 shipping vector. She actively took part in wiki editing and<br />
also took photographs for the Gallery and Team pages.<br />
<br><br><br />
<li><b>Tommaso Goggia</b>, one of the biomedical engineers of our group, worked very hard on all the activities, learning to perform all the wet lab<br />
protocols, designing and performing Tecan tests. Together with Davide he derived the model equations and carried out a precise data<br />
analysis for the quantitative characterization of our BioBrick parts and the identification of mathematical model parameters.<br />
<br />
<br><br><br />
<li><b>Emanuela Pasi</b> gave contribution in the wet lab, following all the Tecan tests and the BioBrick parts cloning. She was also involved in the<br />
analysis of the scientific literature connected with our project; freezer management was also one of her works. Finally she played an<br />
important role in the design of our poster and presentations.<br />
<br />
<br />
<br><br><br />
<li><b>Daniele Sartori</b> was the chemist specialist of the group, who worked on all the wet lab activities; he helped us understanding the behavior<br />
of HSL at different pHs. His support in cloning and transferring BioBrick parts in pSB1C3 was fundamental; he also designed the poster and the presentation<br />
together with Emanuela and decided which results to put in it.<br />
<br><br> <br />
<li><b>Nicolò Politi</b> planned and coordinated all the student and advisor activities in both wetlab and drylab and trained the students for the cloning and quantitative experiments. Instructor for the rest of the team about promoter measurement, modeling and data analysis. <br />
<br><br><br />
<li><b>Federica Sampietro</b> coordinated all the wet lab work. She played an important role in cloning all the parts and characterizing them, as she<br />
worked also on the data analysis and quantitative characterization. During the brainstorming meetings she analyzed all the ideas and collected<br />
a lot of information on the previous iGEM editions projects. She also took care of the freezer management together with Emanuela.<br />
<br><br><br />
<li><b>Susanna Zucca</b> coordinated the team during the brainstorming sessions, project discussions and during the submission and documentation of BioBrick parts to the Registry. Instructor for the rest of the team about physical and functional standardization in synthetic biology.<br />
<br><br><br />
<li><b>Maria Gabriella Cusella</b> is the leader of the wetlab in which the students worked during the summer. She organized the spaces, the instruments and the lab funds dedicated to the iGEM project. <br />
<br><br><br />
<li><b>Paolo Magni</b> coordinated all the project activities: meetings, project timeline, fundraising, contacts with media and institutions. Registry, wiki, poster and presentation supervisor (with the other advisors). He also performed the selection of the team students and he participated (with the other adivsors) to the selection and the definition of the project and to all the discussions about its problems and issues. <br />
<br><br><br />
<br />
<b>All team members contributed equally to brainstorming sessions, BioBrick parts selection and project documentation.</b><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Project/ModellingTeam:UNIPV-Pavia/Project/Modelling2011-09-20T22:53:38Z<p>Nickpv: </p>
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<h2 class="art-postheader">Modelling</h2><br />
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#Mathematical_modelling_page"><span class="tocnumber"></span> <span class="toctext">Mathematical modelling: introduction</span></a> <br />
<ul> <br><br />
<li class="toclevel-2"><a href="#The importance of the mathematical model"><span class="tocnumber">1</span> <span class="toctext">The importance of mathematical modelling</span></a></li><br />
<li class="toclevel-2"><a href="#Equations_for_gene_networks"><span class="tocnumber">2</span> <span class="toctext">Equations for gene networks</span></a></li><br />
<ul><br />
<li class="toclevel-3"><a href="#Hypothesis"><span class="tocnumber">2.1</span> <span class="toctext">Hypotheses</span></a></li> <br />
<li class="toclevel-3"><a href="#Equations_1_and_2"><span class="tocnumber">2.2</span> <span class="toctext">Equations (1) and (2)</span></a></li><br />
<li class="toclevel-3"><a href="#Equation_3"><span class="tocnumber">2.3</span> <span class="toctext">Equation (3)</span></a></li><br />
<li class="toclevel-3"><a href="#Equation_4"><span class="tocnumber">2.4</span> <span class="toctext">Equation (4)</span></a></li><br />
</ul> <br />
<br />
<li class="toclevel-2"><a href="#Table_of_parameters"><span class="tocnumber">3</span> <span class="toctext">Table of parameters</span></a></li><br />
<ul><br />
<li class="toclevel-3"><a href="#CV"><span class="tocnumber">3</span> <span class="toctext">Table of parameter CV</span></a></li><br />
</ul><br />
<br />
<li class="toclevel-2"><a href="#Parameter_estimation"><span class="tocnumber">4</span> <span class="toctext">Parameter estimation</span></a></li> <br />
<ul> <br />
<li class="toclevel-3"><a href="#Ptet_&_Plux"><span class="tocnumber">4.1</span> <span class="toctext">pTet & pLux</span></a></li> <br />
<li class="toclevel-3"><a href="#introduction_to_T9002"><span class="tocnumber">4.2</span> <span class="toctext">T9002 introduction</span></a></li><br />
<li class="toclevel-3"><a href="#Enzymes"><span class="tocnumber">4.3</span> <span class="toctext"> AiiA & LuxI</span></a></li><br />
<li class="toclevel-3"><a href="#N"><span class="tocnumber">4.4</span> <span class="toctext">N</span></a></li><br />
<li class="toclevel-3"><a href="#Degradation_rates"><span class="tocnumber">4.5</span> <span class="toctext">Degradation rates</span></a></li></ul><br />
<br />
<br />
<li class="toclevel-2"><a href="#Simulations"><span class="tocnumber">5</span> <span class="toctext">Simulations</span></a></li> <li class="toclevel-1"><a href="#Sensitivity_Analysis"><span class="tocnumber">6</span> <span class="toctext">Sensitivity Analysis of the steady state of enzyme expression in exponential phase</span></a></li><br />
<ul><br />
<li class="toclevel-2"><a href="#Steady state of enzyme expression"><span class="tocnumber">6.1</span> <span class="toctext">Steady state of enzyme expression</span></a></li><br />
<li class="toclevel-2"><a href="#Sensitivity analysis"><span class="tocnumber">6.2</span> <span class="toctext">Sensitivity analysis</span></a></li><br />
</ul><br />
<li class="toclevel-1"><a href="#References"><span class="tocnumber">7</span> <span class="toctext">References</span></a></li> <br />
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<a name="Mathematical_modelling_page"></a><h1><span class="mw-headline"> <b>Mathematical modelling: introduction</b> </span></h1> <br />
<div style='text-align:justify'><p>Mathematical modelling plays a central role in Synthetic Biology, due to its ability to serve as a crucial link between the concept and realization of a biological circuit: what we propose in this page is a mathematical modelling approach to the entire project, which has proven extremely useful before and after the "wet lab" activities.</p><br />
<br />
<p>Thus, immediately at the beginning, when there was little knowledge, a mathematical model based on a system of differential equations was derived and implemented using a set of reasonable values of model parameters, to validate the feasibility of the project. Once this became clear, starting from the characterization of each simple subpart created in the wet lab, some of the parameters of the mathematical model were estimated thanks to several ad-hoc experiments we performed within the iGEM project (others were derived from literature) and they were used to predict the final behaviour of the whole engineered closed-loop circuit. This approach is consistent with the typical one adopted for the analysis and synthesis of a biological circuit, as exemplified by <a href="#Pasotti"><i><b>Pasotti L</b> et al. 2011.</i></a></p><br />
<br />
<p>After a brief overview on the importance of the mathematical modelling approach, we deeply analyze the system of equations, underlining the role and function of the parameters involved.</p><br />
<p>Experimental procedures for parameter estimation are discussed and, finally, a different type of circuit is presented. <font color="red">Simulations were performed, using <em>ODEs</em> with MATLAB and used to explain the difference between a closed-loop control system model and an open one.</font></div></p><br />
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<a name="The importance of the mathematical model"></a><h2> <span class="mw-headline"> <b>The importance of mathematical modelling</b> </span></h2> <br />
<div style='text-align:justify'><p>Mathematical modelling reveals fundamental in the challenge of understanding and engineering complex biological systems. Indeed, these are characterized by a high degree of interconnection among the single constituent parts, requiring a comprehensive analysis of their behavior through mathematical formalisms and computational tools.</p><br />
<div>Synthetically, we can identify two major roles concerning mathematical models:</div><br />
<br />
<ul><br />
<br />
<br />
<br />
<p><li><b>Simulation</b>: mathematical models allow to analyse complex system dynamics and to reveal the relationships between the involved variables, starting from the knowledge of the single subparts behavior and from simple hypotheses of their interconnection. <a href="#Endler">(<i><b>Endler L</b> et al. 2009</i>)</a></li></p><br />
<br />
<p><li><b>Knowledge elicitation</b>: mathematical models summarize into a small set of parameters the results of several experiments (parameter identification), allowing a robust comparison among different experimental conditions and providing an efficient way to synthesize knowledge about biological processes. Then, through the simulation process, they make possible the re-usability of the knowledge coming from different experiments, engineering complex systems from the composition of its constituent subparts under appropriate experimental/environmental conditions <a href="#Braun">(<i><b>Braun D</b> et al. 2005</a>;<a href="#Canton"> <b>Canton B</b> et al 2008</a></i>)</font>.</li><br />
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<a name="Equations_for_gene_networks"></a><h2> <span class="mw-headline"> <b>Equations for gene networks</b> </span></h2> <br />
<p>Below is provided the system of equations of our mathematical model. </p><br />
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<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/43/Model_new.jpg"><br />
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<a name="Hypothesis"></a><h4> <span class="mw-headline"> <b>Hypotheses of the model</b> </span></h4><br />
<table class="data"><br />
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<td><br />
<div style='text-align:justify'><br />
<em><br />
<b>HP<sub>1</sub></b>: in equation (2) only HSL is considered as inducer, instead of the complex LuxR-HSL. <br />
This is motivated by the fact that our final device offers a constitutive LuxR production due to the upstream constitutive promoter P&lambda;. Assuming LuxR is abundant in the cytoplasm, we can understand this simplification of attributing pLux promoter induction only by HSL.<br />
<br><br />
<br><br />
<b>HP<sub>2</sub></b>: in system equation, LuxI and AiiA amounts are expressed per cell. For this reason, the whole equation (3), except for the <br />
term of intrinsic degradation of HSL, is multiplied by the number of cells N, due to the property of the lactone to diffuse freely inside/outside bacteria.<br />
<br><br />
<br><br />
<b>HP<sub>3</sub></b>: as regards promoters pTet and pLux, we assume their strengths (measured in PoPs), due to a given concentration of inducer (aTc and HSL for Ptet and Plux respectively), to be <br />
independent from the gene downstream.<br />
In other words, in our hypothesis, if the mRFP coding region is substituted with a region coding for another gene (in our case, AiiA or LuxI), we would obtain the same synthesis rate:<br />
this is the reason why the strength of the complex promoter-RBS is expressed in Arbitrary Units [AUr].<br />
<br><br />
<br><br />
<b>HP<sub>4</sub></b>: considering the exponential growth, the enzymes AiiA and LuxI concentration is supposed to be constant, because their production is equally compensated by dilution.<br />
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<a name="Equations_1_and_2"></a><h4> <span class="mw-headline"> <b>Equations (1) and (2)</b> </span></h4><br />
<div style='text-align:justify'><p>Equations (1) and (2) have identical structure, differing only in the parameters involved. They represent the synthesis, degradation and diluition of both the enzymes in the circuit, LuxI and AiiA, respectively in the first and second equation: in each of them both transcription and translation processes have been condensed. The mathematical formulation is analogous to the one used by <a href="#Pasotti"><i><b>Pasotti L</b> et al. 2011</i></a>, Suppl. Inf., even if we do not take LuxR-HSL complex formation into account, as explained below.</p><br />
<p>These equations are composed of 2 parts:</p><br />
<ol><br />
<li>The first term describes, through Hill's equation, the synthesis rate of the protein of interest (either LuxI or AiiA) depending on the concentration of the inducer (anhydrotetracicline -aTc- or HSL respectively), responsible for the activation of the regulatory element composed of promoter and RBS. In the parameter table (see below), &alpha; refers to the maximum activation of the promoter, while &delta; stands for its leakage activity (this means that the promoter is slightly active even if there is no induction). In particular, in equation (1), the almost entire inhibition of pTet promoter is given by the constitutive production of TetR by our MGZ1 strain. In equation (2), pLux is almost inactive in the absence of the complex LuxR-HSL. Furthermore, in both equations k stands for the dissociation constant of the promoter from the inducer (respectively aTc and HSL in eq. 1 and 2), while &eta; is the cooperativity constant.</p><br />
<p><li>The second term in equations (1) and (2) is in turn composed of 2 parts. The former one (<em>&gamma;</em>*LuxI or <em>&gamma;</em>*AiiA respectively) describes, with an exponential decay, the degradation rate per cell of the protein. The latter (&mu;*(Nmax-N)/Nmax)*LuxI or &mu;*(Nmax-N)/Nmax)*AiiA, respectively) takes into account the dilution factor against cell growth which is related to the cell replication process.</p><br />
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<a name="Equation_3"></a><h4> <span class="mw-headline"> <b>Equation (3)</b> </span></h4><br />
<div style='text-align:justify'><p>Here the kinetics of HSL is modeled, through enzymatic reactions either related to the production or the degradation of HSL. This equation is composed of 3 parts: </p><br />
<ol><br />
<p><li> The first term represents the production of HSL due to LuxI expression. We modeled this process with a saturation curve in which V<sub>max</sub> is the HSL maximum transcription rate, while k<sub>M,LuxI</sub> is LuxI dependent half-saturation constant.</p><br />
<p><li> The second term represents the degradation of HSL due to the AiiA expression. Similarly to LuxI, k<sub>cat</sub> represents the maximum degradation per unit of HSL concentration, while k<sub>M,AiiA</sub> is the concentration at which AiiA dependent HSL degradation rate is (k<sub>cat</sub>*HSL)/2. The formalism is similar to that found in the Supplementary Information of <a href="#Danino"><i><b>Danino T</b> et al 2010.</i></a></font></p><br />
<p><li> The third term (&gamma;<sub>HSL</sub>*HSL) is similar to the corresponding ones present in the first two equations and describes the intrinsic protein degradation.</div><br />
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<a name="Equation_4"></a><h4> <span class="mw-headline"> <b>Equation (4)</b> </span></h4><br />
<div style='text-align:justify'>This is the typical cells growth equation, depending on the rate &mu; and the maximum number N<sub>max</sub> of cells per well reachable <a href="#Pasotti">(<i><b>Pasotti L</b> et al. 2009</i>).</a></div><br />
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<a name="Table_of_parameters"></a><h2> <span class="mw-headline"> <b>Table of parameters and species</b> </span></h2><br />
<br><br />
<br />
<br />
<br />
<center><br />
<table class="data"><br />
<tr><br />
<td class="row" width='15%'><b>Parameter & Species</b></td><br />
<td class="row" width='50%'><b>Description</b></td><br />
<td class="row" width='15%'><b>Measurement Unit</b></td><br />
<td class="row" width='20%'><b>Value</b></td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row">&alpha;<sub>p<sub>Tet</sub></sub></td><br />
<td class="row">maximum transcription rate of pTet (dependent on <a href="#RBS">RBSx</a> efficiency)</td><br />
<td class="row">[(AUr/min)/cell]</td><br />
<td class="row">230.67 (RBS30)<br><br />
ND (RBS31)<br><br />
55.77 (RBS32)<br><br />
120 (RBS34)</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row">&delta;<sub>p<sub>Tet</sub></sub></td><br />
<td class="row">leakage factor of promoter pTet basic activity</td><br />
<td class="row">[-]</td><br />
<td class="row">0.028 (RBS30)<br><br />
ND (RBS31)<br><br />
1.53E-11 (RBS32)<br><br />
0.085 (RBS34)</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">&eta;<sub>p<sub>Tet</sub></sub></td><br />
<td class="row">Hill coefficient of pTet</td><br />
<td class="row">[-]</td><br />
<td class="row">4.61 (RBS30)<br><br />
ND (RBS31)<br><br />
4.98 (RBS32)<br><br />
24.85 (RBS34)</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">k<sub>p<sub>Tet</sub></sub></td><br />
<td class="row">dissociation constant of aTc from pTet</td><br />
<td class="row">[ng/ml]</td><br />
<td class="row">8.75 (RBS30)<br><br />
ND (RBS31)<br><br />
7.26 (RBS32)<br><br />
9 (RBS34)</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">&alpha;<sub>p<sub>Lux</sub></sub></td><br />
<td class="row">maximum transcription rate of pLux (dependent on <a href="#RBS">RBSx</a> efficiency)</td><br />
<td class="row">[(AUr/min)/cell]</td><br />
<td class="row">438 (RBS30)<br><br />
9.8 (RBS31)<br><br />
206 (RBS32)<br><br />
1105 (RBS34)</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row">&delta;<sub>p<sub>Lux</sub></sub></td><br />
<td class="row">leakage factor of promoter pLux basic activity</td><br />
<td class="row">[-]</td><br />
<td class="row">0.05 (RBS30)<br><br />
0.11 (RBS31)<br><br />
0 (RBS32)<br><br />
0.02 (RBS34)</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">&eta;<sub>p<sub>Lux</sub></sub></td><br />
<td class="row">Hill coefficient of pLux</td><br />
<td class="row">[-]</td><br />
<td class="row">2 (RBS30)<br><br />
1.2 (RBS31)<br><br />
1.36 (RBS32)<br><br />
1.33 (RBS34)</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">k<sub>p<sub>Lux</sub></sub></td><br />
<td class="row">dissociation constant of HSL from pLux</td><br />
<td class="row">[nM]</td><br />
<td class="row">1.88 (RBS30)<br><br />
1.5 (RBS31)<br><br />
1.87 (RBS32)<br><br />
2.34 (RBS34)</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">&gamma;<sub>LuxI</sub></td><br />
<td class="row">LuxI constant degradation</td><br />
<td class="row">[1/min]</td><br />
<td class="row">0.0173</td><br />
</tr><br />
<tr><br />
<td class="row">&gamma;<sub>AiiA</sub></td><br />
<td class="row">AiiA constant degradation</td><br />
<td class="row">[1/min]</td><br />
<td class="row">0.0173</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">&gamma;<sub>HSL</sub></td><br />
<td class="row">HSL constant degradation</td><br />
<td class="row">[1/min]</td><br />
<td class="row">0 (pH=6)</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">V<sub>max</sub></td><br />
<td class="row">maximum transcription rate of LuxI per cell</td><br />
<td class="row">[nM/(min*cell)]</td><br />
<td class="row">3.56*10-9</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">k<sub>M,LuxI</sub></td><br />
<td class="row">half-saturation constant of LuxI from HSL</td><br />
<td class="row">[AUr/cell]</td><br />
<td class="row">6.87*10<sup>3</sup></td><br />
</tr><br />
<tr><br />
<td class="row">k<sub>cat</sub></td><br />
<td class="row">maximum number of enzymatic reactions catalyzed per minute</td><br />
<td class="row">[1/(min*cell)]</td><br />
<td class="row">ND</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">k<sub>M,AiiA</sub></td><br />
<td class="row">half-saturation constant of AiiA from HSL</td><br />
<td class="row">[AUr/cell]</td><br />
<td class="row">ND</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">N<sub>max</sub></td><br />
<td class="row">maximum number of bacteria per well</td><br />
<td class="row">[cell]</td><br />
<td class="row">1*10<sup>9</sup></td><br />
</tr><br />
<br />
<tr><br />
<td class="row">&mu;</td><br />
<td class="row">rate of bacteria growth</td><br />
<td class="row">[1/min]</td><br />
<td class="row">0.004925</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row">LuxI</td><br />
<td class="row">kinetics of LuxI enzyme</td><br />
<td class="row">[<sup>AUr</sup>&frasl;<sub>cell</sub>]</td><br />
<td class="row">-</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">AiiA</td><br />
<td class="row">kinetics of AiiA enzyme</td><br />
<td class="row">[<sup>AUr</sup>&frasl;<sub>cell</sub>]</td><br />
<td class="row">-</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">HSL</td><br />
<td class="row">kinetics of HSL</b></td><br />
<td class="row">[<sup>nM</sup>&frasl;<sub>(min)</sub>]</td><br />
<td class="row">-</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">N</td><br />
<td class="row">number of cells</td><br />
<td class="row">cell</td><br />
<td class="row">-</td><br />
</tr><br />
</table><br />
</center><br />
<br><br />
<br><br />
<br />
<br />
<div align="justify"><b><a name="RBS">NOTE</a></b><p>In order to better investigate the range of dynamics of each subpart, every promoter has been studied with 4 different RBSs, so as to develop more knowledge about the state variables in several configurations of RBS' efficiency <a href="#Salis">(<i><b>Salis HM</b> et al. 2009</i>)</a>. Hereafter, referring to the notation "RBSx" we mean, respectively, <br />
<a href="http://partsregistry.org/Part:BBa_B0030">RBS30</a>, <br />
<a href="http://partsregistry.org/Part:BBa_B0031">RBS31</a>, <br />
<a href="http://partsregistry.org/Part:BBa_B0032">RBS32</a>, <br />
<a href="http://partsregistry.org/Part:BBa_B0034">RBS34</a>.<br />
</p></div><br />
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<a name="CV"></a><h4> <span class="mw-headline"> <b>Parameter CV</b> </span></h4><br />
<center><br />
<table class="data"><br />
<tr><br />
<td class="row"><b>Parameter & Species</b></td><br />
<td class="row"><b>BBa_B0030</b></td><br />
<td class="row"><b>BBa_B0031</b></td><br />
<td class="row"><b>BBa_B0032</b></td><br />
<td class="row"><b>BBa_B0034</b></td><br />
</tr><br />
<tr><br />
<td class="row">&alpha;<sub>p<sub>Tet</sub></sub></td><br />
<td class="row">3.7</td><br />
<td class="row">ND</td><br />
<td class="row">12</td><br />
<td class="row">5.94</td><br />
</tr><br />
<tr><br />
<td class="row">&delta;<sub>p<sub>Tet</sub></sub></td><br />
<td class="row">91.61</td><br />
<td class="row">>>100</td><br />
<td class="row">>100</td><br />
<td class="row">40.59</td><br />
</tr><br />
<tr><br />
<td class="row">&eta;<sub>p<sub>Tet</sub></sub></td><br />
<td class="row">23.72</td><br />
<td class="row">>>100</td><br />
<td class="row">57.62</td><br />
<td class="row">47.6</td><br />
</tr><br />
<tr><br />
<td class="row">k<sub>p<sub>Tet</sub></sub></td><br />
<td class="row">4.16</td><br />
<td class="row">>>100</td><br />
<td class="row">14.99</td><br />
<td class="row">5.43</td><br />
</tr><br />
<tr><br />
<td class="row">&alpha;<sub>p<sub>Lux</sub></sub></td><br />
<td class="row">10.14</td><br />
<td class="row">7.13</td><br />
<td class="row">2.78</td><br />
<td class="row">5.8</td><br />
</tr><br />
<tr><br />
<td class="row">&delta;<sub>p<sub>Lux</sub></sub></td><br />
<td class="row">179.7</td><br />
<td class="row">57.04</td><br />
<td class="row">1317.7</td><br />
<td class="row">187.2</td><br />
</tr><br />
<tr><br />
<td class="row">&eta;<sub>p<sub>Lux</sub></sub></td><br />
<td class="row">47.73</td><br />
<td class="row">29.13</td><br />
<td class="row">9.75</td><br />
<td class="row">19.3</td><br />
</tr><br />
<tr><br />
<td class="row">k<sub>p<sub>Lux</sub></sub></td><br />
<td class="row">27.5</td><br />
<td class="row">25.81</td><br />
<td class="row">8.46</td><br />
<td class="row">17.86</td><br />
</tr><br />
<br />
</table><br />
</center><br />
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<a name="Parameter_estimation"></a><h2> <span class="mw-headline"> <b>Parameter estimation</b></span></h2><br />
<div style='text-align:justify'>The aim of the model is to predict the behavior of the final closed loop circuit starting from the characterization of single BioBrick parts through a set of well-designed <em>ad hoc</em> experiments. This section presents the experiments performed.<br />
As explained before in <a href="#RBS"><span class="toctext"><b>NOTE</b></span></a>, considering a set of 4 RBSs for each subpart expands the range of dynamics and helps us to better understand the interactions between state variables.<br />
</div><br />
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<a name="Ptet_&_Plux"></a><h4> <span class="mw-headline"> <b>Promoter (PTet & pLux)</b> </span></h4><br />
<div style='text-align:justify'><br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><img alt="" src="https://static.igem.org/mediawiki/2011/9/91/Caratterizzazione_ptetN.jpg" class="thumbimage" width="33%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
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</div><br />
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<div style='text-align:justify'><br />
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<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><img alt="" src="https://static.igem.org/mediawiki/2011/7/79/Caratterizzazione_pluxN.jpg" class="thumbimage" width="70%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
</div><br />
<div style='text-align:justify'><p>These are the first parts tested, with the target of learning more about pTet and pLux promoters. In particular, as previously explained in <a href=#RBS>NOTE</a>, for each promoter, we tested four different combinations of promoter-RBS, providing us a set of fundamental building blocks for the subsequent assebly of the closed-loop circuit.</p><br />
<p>As shown in the figure below, we considered a range of inductions and we monitored, in time, absorbance (O.D. stands for "optical density") and fluorescence; the two vertical segments for each graph highlight the exponential phase of bacterial growth. S<sub>cell</sub> (namely, synthesis rate per cell) can be derived as a function of inducer concentration, thereby providing the desired input-output relation (inducer concentration versus promoter+RBS activity), which was modelled as a Hill curve:</p><br />
<br />
<div align="center"><div class="thumbinner" style="width: 600px;"><br />
<a href="https://static.igem.org/mediawiki/2011/5/58/Scell.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/5/58/Scell.jpg" class="thumbimage" width="45%"></a></div></div><br />
<br />
However, also Relative Promoter Unit (RPU, <a href="#Kelly"><i><b>Kelly JR</b> et al. 2009</i></a>) has been calculated as a ratio of S<sub>cell</sub> of promoter of interest and the S<sub>cell</sub> of <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a> (reference to <a href="#Hypothesis"><span class="toctext"><b><em>HP<sub>3</sub></em></b></span></a>).<br><br />
<br />
<div style='text-align:center'><div class="thumbinner" style="width: 600px;"><br />
<a href="https://static.igem.org/mediawiki/2011/2/26/Box2_new.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/2/26/Box2_new.jpg" class="thumbimage" height="48%" width="120%"></a></div></div><br><br />
<br />
<p>As shown in the figure, &alpha;, as already mentioned, represents the protein maximum synthesis rate, which is reached, in accordance with Hill equation, when the inducer concentration tends to infinite, and, more practically, when the inducer concentration is sufficiently higher than the dissociation constant. Meanwhile the product &alpha;*&delta; stands for the leakage activity (at no induction), liable for protein production (LuxI and AiiA respectively) even in the absence of inducer.</p><br />
<p>The paramenter &eta; is the Hill's cooperativity constant and it affects the ripidity of transition from the lower and upper boundary of the curve relating S<sub>cell</sub> to the inducer concentration.<br />
Lastly, k stands for the semi-saturation constant and, in case of &eta;=1, it indicates the concentration of substrate at which half the synthesis rate is achieved.<br />
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<a name="introduction_to_T9002"></a><h4> <span class="mw-headline"> <b>T9002 introduction</b> </span></h4><br />
<div style='text-align:justify'><br />
<em><br />
<p>LuxI and AiiA tests have been always performed exploiting the well-characterized BioBrick <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a>, by which it's possible to quantify exactly the concentration of HSL.</p><br />
<div align="center"><div class="thumbinner" style="width: 500px;"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c2/T9002.jpg" class="thumbimage" width="110%"></a></div></div><br />
<p>This is a biosensor which receives HSL concentration as input and returns GFP intensity (more precisely S<sub>cell</sub>) as output.<a href="#Canton"> (<i><b>Canton</b> et al. 2008</i>).</a><br />
According to this, it is necessary to understand the input-output relationship: so, a T9002 "calibration" curve is plotted for each test performed.</p><br><br><br />
</em><br />
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<a name="Enzymes"></a><h4> <span class="mw-headline"> <b>AiiA & LuxI</b> </span></h4><br />
<div style='text-align:justify'><p>This paragraph explains how parameters of equation (3) are estimated. The target is to learn the AiiA degradation and LuxI production mechanisms in addiction to HSL intrinsic degradation, in order to estimate V<sub>max</sub>, K<sub>M,LuxI</sub>, k<sub>cat</sub>, K<sub>M,AiiA</sub> and &gamma;<sub>HSL</sub> parameters. We adopt tests composed of two steps. In the first one, the following BioBrick parts are used:</p> <br />
</div><br />
<br />
<div style='text-align:justify'><br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><img alt="" src="https://static.igem.org/mediawiki/2011/8/88/Caratterizzazione_aiia.JPG" class="thumbimage" width="32%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div style='text-align:justify'><br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><img alt="" src="https://static.igem.org/mediawiki/2011/4/48/Caratterizzazione_luxIN.jpg" class="thumbimage" width="28%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div style='text-align:justify'><p>Based on our <a href="#Hypothesis"><span class="toctext"><b><em>HP<sub>3</sub></em></b></span></a> and <a href="#Hypothesis"><span class="toctext"><b><em>HP<sub>4</sub></em></b></span></a>, we are able to determine AiiA and LuxI concentrations, provided we have yet characterized pTet-RBSx contructs<a name='t9002'></a>. In particular, referring to <a href="#Hypothesis"><span class="toctext"><b><em>HP<sub>4</sub></em></b></span></a>, in exponential growth the equilibrium of the enzymes is conserved. Due to a known induction of aTc, the steady-state level per cell can be calculated:</p></div> <br />
<br />
<div style='text-align:justify'><div class="thumbinner" style="width: 500px;"><br />
<a href="https://static.igem.org/mediawiki/2011/7/74/Aiia_cost.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/7/74/Aiia_cost.jpg" class="thumbimage" width="120%"></a></div></div><br />
<br />
<p>Then, as a second step, we monitor in separate experiments HSL synthesis and degradation caused by the activities of the enzymes. In other words, our idea is to control the degradation of HSL versus time. ATc activates pTet and, later, a certain concentration of HSL is introduced. Then, at fixed times, O.D.<sub>600</sub> and HSL concentration are monitored using Tecan and T9002 biosensor.</p><p>For example for AiiA dependent HSL degradation, we have:</p><br />
<br />
<table align='center' width='100%'><br />
<div style='text-align:center'><div class="thumbinner" style="width: 70%;"><br />
<a href="https://static.igem.org/mediawiki/2011/9/99/Degradation.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/9/99/Degradation.jpg" class="thumbimage" width="140%"></a></div></div><br />
</table><br />
<br />
<br><p>Therefore, considering for a determined promoter-RBSx couple, several induction of aTc and, for each of them, several samples of HSL concentration during time, parameters V<sub>max</sub>, k<sub>M,LuxI</sub>, k<sub>cat</sub> and k<sub>M,AiiA</sub> can be estimated, through numerous iterations of an algorithm implemented in MATLAB.</p><br />
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<a name="N"></a><h4> <span class="mw-headline"> <b>N</b> </span></h4><br />
<div style='text-align:justify'>The parameters N<sub>max</sub> and μ can be calculated from the analysis of the OD<sub>600</sub> produced by our MGZ1 culture. In particular, μ is derived as the slope of the log(O.D.<sub>600</sub>) growth curve. Counting the number of cells of a saturated culture would be considerably complicated, so N<sub>max</sub> is determined with a proper procedure. The aim here is to derive the linear proportional coefficient &Theta; between O.D'.<sub>600</sub> and N: this constant can be estimated as the ratio between absorbance (read from TECAN) and the respective number of CFU on a petri plate. Finally, N<sub>max</sub> is calcultated as &Theta;*O.D'.<sub>600</sub><br />
<a href="#Pasotti">(<i><b>Pasotti L</b> et al. 2010</i>)</a>.<br />
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<a name="Degradation_rates"></a><h4> <span class="mw-headline"> <b>Degradation rates</b> </span></h4><br />
<div style='text-align:justify'>The parameters &gamma;<sub>LuxI</sub> and &gamma;<sub>AiiA</sub> are taken from literature since they contain LVA tag for rapid degradation. Instead, approximating HSL kinetics as a decaying exponential, &gamma;<sub>HSL</sub> can be derived as the slope of the log(concentration), which can be monitored through <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a>.<br />
</div><br />
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<a name="Simulations"></a><h1><span class="mw-headline"> <b>Simulations</b> </span></h1><br />
<div style='text-align:justify'><br />
<p>On a biological level, the ability to control the concentration of a given molecule reveals itself as fundamental in limiting the metabolic burden of the cell; moreover, in the particular case of HSL signalling molecules, this would give the possibility to regulate quorum sensing-based population behaviours. In this section we present some simulations of another circuit, which could validate the concept of the closed-loop model we have discussed so far.</p><br />
<p>In order to see that, we implemented and simulated in Matlab an open loop circuit, similar to <b>CTRL+<em>E</em></b>, except for the constitutive production of AiiA.</p><br />
<br />
<center><br />
<table><br />
<tr> <br />
<td><br />
<div style='text-align:justify'><div class="thumbinner" style="width: 500px;"><br />
<a href="https://static.igem.org/mediawiki/2011/b/b6/Sim_closed.jpg"><br />
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<a name="Sensitivity_Analysis"></a><h1> <span class="mw-headline"> <b>Sensitivity Analysis of the steady state of enzyme expression in exponential phase</b> </span></h1><br />
<br />
<p>In this paragraph we investigate the theoretical behaviour of our circuit in the cell culture exponential growth phase. According to this, we first derive, under feasible hypotheses, the steady state condition for the enzymes and HSL concentration in that phase. Then we perform a sensitivity analysis relating the output of our system (HSL) to input (aTc) and system parameters.</p><br />
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<a name="Steady state of enzyme expression"></a><h2> <span class="mw-headline"> <b>Steady state of enzyme expression</b> </span></h2><br />
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<br />
<p> Based on our <a href="#Hypothesis"><span class="toctext"><b><em>HP<sub>4</sub></em></b></span></a>, we can formulate the steady state expressions during the exponential growth phase. Adding other considerations about the involved processes, it is possible to add further simplifications to the steady state equations. In particular, one concern relates to the number of cells N (in the order of 3*10^3), which is far lower than N<sub>max</sub> (10^9). The other pertains to &gamma;*HSL parameter, whch can be neglected compared to the other two terms of the third equation. Based on this assumptions, equation (4) of the system becomes dN/dt=&mu;N. Moreover, from equation (3), after having removed the third term, we can simplify the N parameter, since it is common to the remaining two terms. On a biological point of view, this implies that AiiA, LuxI and HSL undergo only minor changes through time, thereby allowing to derive their steady state expressions:</p><br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width: 100%;"><br />
<a href="https://static.igem.org/mediawiki/2011/3/32/LuxI_SS.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/3/32/LuxI_SS.jpg" class="thumbimage" width="83%"></a></div></div><br />
</td><br />
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</table><br />
<br />
<p> The first equation is independent from the second and third ones, enabling us to directly determine LuxI steady state expression during the exponential growth phase. On the contrary, second and third equations depend each other in defining the value of AiiA and HSL respectively, because the former is a function of HSL, while the latter is a function of AiiA. So we could resolve a system of two equations, first by expliciting one of the two variables with respect to the other, and then substituting its expression in order to determine the other variable possible values. This would bring a complex mathematical formulation, which is not helpful in understanding the influence of the various model parameters on the output HSL.<br />
On the other hand, AiiA and HSL values can also be graphically determined from the intersection of the curves derived from these two equations, if we explicit HSL as a function of AiiA (or, alternatively, AiiA as a function of HSL). It is easy to discover that these two curves represent rectangular hyperbolae (the first one only under a simple approximation, explained below) whose tails intersect each other at a particular point, corresponding to the searched values for AiiA and HSL.</p> <br />
<div>For a rectangular hyperbola (RH), we have:</div><br />
<br />
<table align='center' width='100%'><br />
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<td><br />
<div style='text-align:center'><div class="thumbinner" style="width: 100%;"><a href="" class="image"><br />
<a href="https://static.igem.org/mediawiki/2011/7/75/UNIPV_Rectangular_hyperbola_general.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/7/75/UNIPV_Rectangular_hyperbola_general.jpg" class="thumbimage" width="14%"></a></div></div><br />
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</table><br />
<br />
<p>centered at O(-d/c;a/c), with the vertical asymptote x=-d/c and the horizontal asymptote y=a/c</p><br />
<p>From equation 2, we have:</p><br />
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<td><br />
<div style='text-align:center'><div class="thumbinner" style="width: 100%;"><a href="" class="image"><br />
<a href="https://static.igem.org/mediawiki/2011/3/3a/UNIPV_eta_root_HSL.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/3/3a/UNIPV_eta_root_HSL.jpg" class="thumbimage" width="62%"></a><br />
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</table><br />
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<p> We can introduce the simplification to remove the &eta;plux exponent to the entire expression in the right hand side of the equation, thereby obtaining a rectangular hyperbola; even if this leads to a slight change in the curve behaviour, it allows to more clearly understand the relation between HSL and AiiA. As pertains to equation 3, its steady state approximation during the exponential growth is more immediately identifiable as a rectangular hyperbola. Below the two RHs equations are provided, togheter with the table of parameters.</p><br />
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<td><br />
<div style='text-align:center'><div class="thumbinner" style="width: 100%;"><br />
<a href="https://static.igem.org/mediawiki/2011/1/11/RH1_UNIPV_HSL.jpg"><img src="https://static.igem.org/mediawiki/2011/1/11/RH1_UNIPV_HSL.jpg" class="thumbimage" width="50%"></a></div></div><br />
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<a href="https://static.igem.org/mediawiki/2011/4/4b/RH2_UNIPV_HSL.jpg" class="image"><br />
<img src="https://static.igem.org/mediawiki/2011/4/4b/RH2_UNIPV_HSL.jpg" class="thumbimage" width="70%"></a></div></div><br />
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<center><br />
<table class="data"><br />
<tr><br />
<td class="row"><b>Parameter</b></td><br />
<td class="row"><b>RH1</b></td><br />
<td class="row"><b>RH2</b></td><br />
</tr><br />
<br />
<tr><br />
<td class="row">a</sub></sub></td><br />
<td class="row">(&gamma;<sub>AiiA</sub>+&mu;)*(k<sub>pLux</sub>)<sup>&eta;pLux</td><br />
<td class="row"><span style="text-decoration:overline;" > V</span><sub>LuxI</sub></td><br />
</tr><br />
<br />
<tr><br />
<td class="row">b</td><br />
<td class="row">-&alpha;<sub>pLux</sub>*&delta;<sub>pLux</sub>*(k<sub>pLux</sub>)<sup>&eta;pLux</td><br />
<td class="row"><span style="text-decoration:overline;" > V</span><sub>LuxI</sub>*k<sub>M,LuxI</sub></td><br />
</tr><br />
<br />
<tr><br />
<td class="row">c</sub></sub></td><br />
<td class="row">-&gamma;<sub>AiiA</sub>+&mu;</td><br />
<td class="row">k<sub>cat</sub>*<span style="text-decoration:overline;" >AiiA</span></td><br />
</tr><br />
<br />
<tr><br />
<td class="row">d</td><br />
<td class="row">&alpha;<sub>pLux</sub></td><br />
<td class="row">0</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">Horizontal asymptote</td><br />
<td class="row">a/c=(k<sub>pLux</sub>)<sup>&eta;pLux</sup></td><br />
<td class="row"><span style="text-decoration:overline;" > V</span><sub>LuxI</sub>/k<sub>cat</sub></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td class="row">Vertical asymptote</td><br />
<td class="row">-d/c=&alpha;<sub>pLux</sub>/(&gamma;<sub>AiiA</sub>+&mu;)</td><br />
<td class="row">0</td><br />
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<a name="Sensitivity analysis"></a><h2><br><br />
<span class="mw-headline"> <b>Sensitivity analysis</b> </span></h2><br />
<br />
<p> Now, it is interesting to conduct some qualitative and quantitative considerations about our system sensitivity to its parameters and aTc input signal.</p><br />
<div> First of all, we analyze how the HSL output can be regulated by changing the characteristics of our RHs.</div><div>Referring to the first rectangular hyperbola, we recognize that its vertical asymptote could be varied by changing &alpha;<sub>p<sub>Lux</sub></sub></sub></sub> value (assuming fixed &gamma;<sub>AiiA</sub> and &mu;). In particular, thanks to the four Plux-RBSx constructs realized, we can vary &alpha;<sub>p<sub>Lux</sub></sub></sub></sub> more than a hundred factor. This can significantly shift the vertical asymptote, bringing this first RH farther or nearer the second one (whose vertical asymptote is the ordinate axis), thereby providing an intersection at higher AiiA and lower HSL values, or vice versa. The following two figures highlight this aspect.</div><br><br />
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<table align='center' width='100%'><br />
<div><div class="thumbinner"><a href="https://static.igem.org/mediawiki/2011/4/45/Iperbole_eq_3_as_ver2.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/4/45/Iperbole_eq_3_as_ver2.jpg" class="thumbimage" width="100%"></a></div></div><br />
</table><br />
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<table width="100%" align="center"><br />
<div style="WIDTH: 100%" class="thumbinner"><br />
<a class="image" href="https://static.igem.org/mediawiki/2011/9/97/UNIPV_inters_RH_ver_alfa_plux_acceptable_values2.jpg"><img class="thumbimage" src="https://static.igem.org/mediawiki/2011/9/97/UNIPV_inters_RH_ver_alfa_plux_acceptable_values2.jpg" width="100%"></a><br />
</div><br />
<tbody></tbody><br />
</table><br />
<br />
<br />
<p>From the above figures, it is also clear that HSL steady state value is not very sensitive to &alpha;<sub>p<sub>Lux</sub></sub></sub></sub>, at least when this parameter presents values greater than unity, because this brings the two curves to intersect in their low slope regions.</p><br />
<p>Referring to the second RH, the only adjustable asymptote is the horizontal one, that we can move upward or downward by altering VLuxI, which indirectly depends on aTc.</p><br />
<br />
<table align='center' width='100%'><br />
<div style='text-align:center'><div class="thumbinner" style="width: 100%;"><a href="https://static.igem.org/mediawiki/2011/0/04/UNIPV_RH2_eq2_as_or_corrected2.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/0/04/UNIPV_RH2_eq2_as_or_corrected2.jpg" class="thumbimage" width="95%"></a></div></div><br />
</table><br />
<br />
<p>A further deepening in the exponential phase analysis involves determining the characteristics of the input-output relation. Our closed loop system can be realized with two alternative purposes in mind:</p><br />
<ol><br />
<li> realizing a circuit able to adapt HSL output depending on aTc input concentration. This requires a good sensitivity between input and output.</li><br />
<li> designing a robust HSL concentration controller, which is immune to the input noise and offers a constant and defined amount of HSL. In this case HSL level should be appropriately tuned during the design stage, by choosing the correct strength of promoter-RBSx complexes.</li><br />
</ol><br />
<br />
<p>Now,again considering the system of equations, it is easy to observe that HSL dependence on aTc input passes through two Hill equations. The former describes aTc driven LuxI synthesis, while the latter models LuxI dependent HSL synthesis rate. Therefore, in order to achieve a high aTc sensitivity, it is advisable to tune aTc and LuxI levels so that they place outside the saturation regions of their Hill curves. In this regard, it is possible to determine a closed form expression relating HSL to aTc, if we hypothesize that both aTc and LuxI are far lower than their respective half-saturation constant (k_ptet and Km), thus simplifying the Hills with a first order relation:</p><br />
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<a name="References"></a><h1><span class="mw-headline"> <b>References</b> </span></h1><br />
<div style='text-align:justify'><br />
<br />
<ol type='1'><br />
<br />
<a name="Braun"></a><br />
<li>Braun D, Basu S, Weiss R (2005) <b>Parameter estimation for two synthetic gene networks: a case study </b> <br />
<i>ICASSP '05 </i> 5:v/769-v/772. </li> <br><br />
<br />
<a name="Canton"></a><br />
<li>Canton B, Labno A, Endy D. (2008) <b>Refinement and standardization of synthetic biological parts and devices. </b> <i> Nat Biotechnol. </i> 26(7):787-93. </li> <br><br />
<br />
<a name="Danino"></a><br />
<li>Danino T, Mondrag&oacute;n-Palomino O, Tsimring L et al. (2010) <b>A synchronized quorum of genetic clocks. </b> <i>Nature. </i> 463(7279):326-30. </li> <br><br />
<br />
<a name="Endler"></a><br />
<li>Endler L, Rodriguez N, Juty N et al. (2009) <b>Designing and encoding models for synthetic biology. </b> <i>J. R. Soc. Interface </i> 6:S405-S417. </li> <br><br />
<br />
<a name="Kelly"></a><br />
<li>Kelly JR, Rubin AJ, Davis JH et al. (2009) <b>Measuring the activity of BioBrick promoters using an in vivo reference standard.</b> <i> J. Biol. Eng.</i> 3:4.</li> <br> <br />
<br />
<a name="Pasotti"></a><br />
<li>Pasotti L, Quattrocelli M, Galli D et al. (2011) <b>Multiplexing and demultiplexing logic functions for computing signal processing tasks in synthetic biology. </b> <br />
<i>Biotechnol. J. </i>6(7):784-95. </li> <br><br />
<br />
<a name="Salis"></a><br />
<li>Salis H M, Mirsky E A, Voight C A (2009)<b> Automated design of synthetic ribosome binding sites to control protein expression. </b> <i>Nat. Biotechnol.</i>27:946-950. <br />
</li><br><br />
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</ol><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Project/ModellingTeam:UNIPV-Pavia/Project/Modelling2011-09-20T22:51:58Z<p>Nickpv: </p>
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<h2 class="art-postheader">Modelling</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<p><a name="indice"/> </p><br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#Mathematical_modelling_page"><span class="tocnumber"></span> <span class="toctext">Mathematical modelling: introduction</span></a> <br />
<ul> <br><br />
<li class="toclevel-2"><a href="#The importance of the mathematical model"><span class="tocnumber">1</span> <span class="toctext">The importance of mathematical modelling</span></a></li><br />
<li class="toclevel-2"><a href="#Equations_for_gene_networks"><span class="tocnumber">2</span> <span class="toctext">Equations for gene networks</span></a></li><br />
<ul><br />
<li class="toclevel-3"><a href="#Hypothesis"><span class="tocnumber">2.1</span> <span class="toctext">Hypotheses</span></a></li> <br />
<li class="toclevel-3"><a href="#Equations_1_and_2"><span class="tocnumber">2.2</span> <span class="toctext">Equations (1) and (2)</span></a></li><br />
<li class="toclevel-3"><a href="#Equation_3"><span class="tocnumber">2.3</span> <span class="toctext">Equation (3)</span></a></li><br />
<li class="toclevel-3"><a href="#Equation_4"><span class="tocnumber">2.4</span> <span class="toctext">Equation (4)</span></a></li><br />
</ul> <br />
<br />
<li class="toclevel-2"><a href="#Table_of_parameters"><span class="tocnumber">3</span> <span class="toctext">Table of parameters</span></a></li><br />
<ul><br />
<li class="toclevel-3"><a href="#CV"><span class="tocnumber">3</span> <span class="toctext">Table of parameter CV</span></a></li><br />
</ul><br />
<br />
<li class="toclevel-2"><a href="#Parameter_estimation"><span class="tocnumber">4</span> <span class="toctext">Parameter estimation</span></a></li> <br />
<ul> <br />
<li class="toclevel-3"><a href="#Ptet_&_Plux"><span class="tocnumber">4.1</span> <span class="toctext">pTet & pLux</span></a></li> <br />
<li class="toclevel-3"><a href="#introduction_to_T9002"><span class="tocnumber">4.2</span> <span class="toctext">T9002 introduction</span></a></li><br />
<li class="toclevel-3"><a href="#Enzymes"><span class="tocnumber">4.3</span> <span class="toctext"> AiiA & LuxI</span></a></li><br />
<li class="toclevel-3"><a href="#N"><span class="tocnumber">4.4</span> <span class="toctext">N</span></a></li><br />
<li class="toclevel-3"><a href="#Degradation_rates"><span class="tocnumber">4.5</span> <span class="toctext">Degradation rates</span></a></li></ul><br />
<br />
<br />
<li class="toclevel-2"><a href="#Simulations"><span class="tocnumber">5</span> <span class="toctext">Simulations</span></a></li> <li class="toclevel-1"><a href="#Sensitivity_Analysis"><span class="tocnumber">6</span> <span class="toctext">Sensitivity Analysis of the steady state of enzyme expression in exponential phase</span></a></li><br />
<ul><br />
<li class="toclevel-2"><a href="#Steady state of enzyme expression"><span class="tocnumber">6.1</span> <span class="toctext">Steady state of enzyme expression</span></a></li><br />
<li class="toclevel-2"><a href="#Sensitivity analysis"><span class="tocnumber">6.2</span> <span class="toctext">Sensitivity analysis</span></a></li><br />
</ul><br />
<li class="toclevel-1"><a href="#References"><span class="tocnumber">7</span> <span class="toctext">References</span></a></li> <br />
</ul> <br />
</li> <br />
</ul> <br />
</li> <br />
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<a name="Mathematical_modelling_page"></a><h1><span class="mw-headline"> <b>Mathematical modelling: introduction</b> </span></h1> <br />
<div style='text-align:justify'><p>Mathematical modelling plays a central role in Synthetic Biology, due to its ability to serve as a crucial link between the concept and realization of a biological circuit: what we propose in this page is a mathematical modelling approach to the entire project, which has proven extremely useful before and after the "wet lab" activities.</p><br />
<br />
<p>Thus, immediately at the beginning, when there was little knowledge, a mathematical model based on a system of differential equations was derived and implemented using a set of reasonable values of model parameters, to validate the feasibility of the project. Once this became clear, starting from the characterization of each simple subpart created in the wet lab, some of the parameters of the mathematical model were estimated thanks to several ad-hoc experiments we performed within the iGEM project (others were derived from literature) and they were used to predict the final behaviour of the whole engineered closed-loop circuit. This approach is consistent with the typical one adopted for the analysis and synthesis of a biological circuit, as exemplified by <a href="#Pasotti"><i><b>Pasotti L</b> et al. 2011.</i></a></p><br />
<br />
<p>After a brief overview on the importance of the mathematical modelling approach, we deeply analyze the system of equations, underlining the role and function of the parameters involved.</p><br />
<p>Experimental procedures for parameter estimation are discussed and, finally, a different type of circuit is presented. <font color="red">Simulations were performed, using <em>ODEs</em> with MATLAB and used to explain the difference between a closed-loop control system model and an open one.</font></div></p><br />
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<br />
<a name="The importance of the mathematical model"></a><h2> <span class="mw-headline"> <b>The importance of mathematical modelling</b> </span></h2> <br />
<div style='text-align:justify'><p>Mathematical modelling reveals fundamental in the challenge of understanding and engineering complex biological systems. Indeed, these are characterized by a high degree of interconnection among the single constituent parts, requiring a comprehensive analysis of their behavior through mathematical formalisms and computational tools.</p><br />
<div>Synthetically, we can identify two major roles concerning mathematical models:</div><br />
<br />
<ul><br />
<br />
<br />
<br />
<p><li><b>Simulation</b>: mathematical models allow to analyse complex system dynamics and to reveal the relationships between the involved variables, starting from the knowledge of the single subparts behavior and from simple hypotheses of their interconnection. <a href="#Endler">(<i><b>Endler L</b> et al. 2009</i>)</a></li></p><br />
<br />
<p><li><b>Knowledge elicitation</b>: mathematical models summarize into a small set of parameters the results of several experiments (parameter identification), allowing a robust comparison among different experimental conditions and providing an efficient way to synthesize knowledge about biological processes. Then, through the simulation process, they make possible the re-usability of the knowledge coming from different experiments, engineering complex systems from the composition of its constituent subparts under appropriate experimental/environmental conditions <a href="#Braun">(<i><b>Braun D</b> et al. 2005</a>;<a href="#Canton"> <b>Canton B</b> et al 2008</a></i>)</font>.</li><br />
</p><br />
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</ul><br />
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<a name="Equations_for_gene_networks"></a><h2> <span class="mw-headline"> <b>Equations for gene networks</b> </span></h2> <br />
<p>Below is provided the system of equations of our mathematical model. </p><br />
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<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/43/Model_new.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/4/43/Model_new.jpg" class="thumbimage" height="55%" width="80%"></a></div></div><br />
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<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><img alt="" src="https://static.igem.org/mediawiki/2011/5/5e/Circuito_finale.jpg" class="thumbimage" width="87%"></a></div></div><br />
</td><br />
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</table><br />
<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c7/QS_system_synthetic_circuit.png" class="thumbimage" width="85%"></a></div></div><br />
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<a name="Hypothesis"></a><h4> <span class="mw-headline"> <b>Hypotheses of the model</b> </span></h4><br />
<table class="data"><br />
<tr><br />
<td><br />
<div style='text-align:justify'><br />
<em><br />
<b>HP<sub>1</sub></b>: in equation (2) only HSL is considered as inducer, instead of the complex LuxR-HSL. <br />
This is motivated by the fact that our final device offers a constitutive LuxR production due to the upstream constitutive promoter P&lambda;. Assuming LuxR is abundant in the cytoplasm, we can understand this simplification of attributing pLux promoter induction only by HSL.<br />
<br><br />
<br><br />
<b>HP<sub>2</sub></b>: in system equation, LuxI and AiiA amounts are expressed per cell. For this reason, the whole equation (3), except for the <br />
term of intrinsic degradation of HSL, is multiplied by the number of cells N, due to the property of the lactone to diffuse freely inside/outside bacteria.<br />
<br><br />
<br><br />
<b>HP<sub>3</sub></b>: as regards promoters pTet and pLux, we assume their strengths (measured in PoPs), due to a given concentration of inducer (aTc and HSL for Ptet and Plux respectively), to be <br />
independent from the gene downstream.<br />
In other words, in our hypothesis, if the mRFP coding region is substituted with a region coding for another gene (in our case, AiiA or LuxI), we would obtain the same synthesis rate:<br />
this is the reason why the strength of the complex promoter-RBS is expressed in Arbitrary Units [AUr].<br />
<br><br />
<br><br />
<b>HP<sub>4</sub></b>: considering the exponential growth, the enzymes AiiA and LuxI concentration is supposed to be constant, because their production is equally compensated by dilution.<br />
</em><br />
</div><br />
</td><br />
</tr><br />
</table><br />
<br><br />
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<br><br />
<br><br />
<br />
<a name="Equations_1_and_2"></a><h4> <span class="mw-headline"> <b>Equations (1) and (2)</b> </span></h4><br />
<div style='text-align:justify'><p>Equations (1) and (2) have identical structure, differing only in the parameters involved. They represent the synthesis, degradation and diluition of both the enzymes in the circuit, LuxI and AiiA, respectively in the first and second equation: in each of them both transcription and translation processes have been condensed. The mathematical formulation is analogous to the one used by <a href="#Pasotti"><i><b>Pasotti L</b> et al. 2011</i></a>, Suppl. Inf., even if we do not take LuxR-HSL complex formation into account, as explained below.</p><br />
<p>These equations are composed of 2 parts:</p><br />
<ol><br />
<li>The first term describes, through Hill's equation, the synthesis rate of the protein of interest (either LuxI or AiiA) depending on the concentration of the inducer (anhydrotetracicline -aTc- or HSL respectively), responsible for the activation of the regulatory element composed of promoter and RBS. In the parameter table (see below), &alpha; refers to the maximum activation of the promoter, while &delta; stands for its leakage activity (this means that the promoter is slightly active even if there is no induction). In particular, in equation (1), the almost entire inhibition of pTet promoter is given by the constitutive production of TetR by our MGZ1 strain. In equation (2), pLux is almost inactive in the absence of the complex LuxR-HSL. Furthermore, in both equations k stands for the dissociation constant of the promoter from the inducer (respectively aTc and HSL in eq. 1 and 2), while &eta; is the cooperativity constant.</p><br />
<p><li>The second term in equations (1) and (2) is in turn composed of 2 parts. The former one (<em>&gamma;</em>*LuxI or <em>&gamma;</em>*AiiA respectively) describes, with an exponential decay, the degradation rate per cell of the protein. The latter (&mu;*(Nmax-N)/Nmax)*LuxI or &mu;*(Nmax-N)/Nmax)*AiiA, respectively) takes into account the dilution factor against cell growth which is related to the cell replication process.</p><br />
</ol><br />
</div><br />
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<br />
<br />
<a name="Equation_3"></a><h4> <span class="mw-headline"> <b>Equation (3)</b> </span></h4><br />
<div style='text-align:justify'><p>Here the kinetics of HSL is modeled, through enzymatic reactions either related to the production or the degradation of HSL. This equation is composed of 3 parts: </p><br />
<ol><br />
<p><li> The first term represents the production of HSL due to LuxI expression. We modeled this process with a saturation curve in which V<sub>max</sub> is the HSL maximum transcription rate, while k<sub>M,LuxI</sub> is LuxI dependent half-saturation constant.</p><br />
<p><li> The second term represents the degradation of HSL due to the AiiA expression. Similarly to LuxI, k<sub>cat</sub> represents the maximum degradation per unit of HSL concentration, while k<sub>M,AiiA</sub> is the concentration at which AiiA dependent HSL degradation rate is (k<sub>cat</sub>*HSL)/2. The formalism is similar to that found in the Supplementary Information of <a href="#Danino"><i><b>Danino T</b> et al 2010.</i></a></font></p><br />
<p><li> The third term (&gamma;<sub>HSL</sub>*HSL) is similar to the corresponding ones present in the first two equations and describes the intrinsic protein degradation.</div><br />
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<br><br />
<br />
<br />
<a name="Equation_4"></a><h4> <span class="mw-headline"> <b>Equation (4)</b> </span></h4><br />
<div style='text-align:justify'>This is the typical cells growth equation, depending on the rate &mu; and the maximum number N<sub>max</sub> of cells per well reachable <a href="#Pasotti">(<i><b>Pasotti L</b> et al. 2009</i>).</a></div><br />
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<br><br><br />
<br />
<br />
<a name="Table_of_parameters"></a><h2> <span class="mw-headline"> <b>Table of parameters and species</b> </span></h2><br />
<br><br />
<br />
<br />
<br />
<center><br />
<table class="data"><br />
<tr><br />
<td class="row"><b>Parameter & Species</b></td><br />
<td class="row"><b>Description</b></td><br />
<td class="row"><b>Measurement Unit</b></td><br />
<td class="row"><b>Value</b></td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row">&alpha;<sub>p<sub>Tet</sub></sub></td><br />
<td class="row">maximum transcription rate of pTet (dependent on <a href="#RBS">RBSx</a> efficiency)</td><br />
<td class="row">[(AUr/min)/cell]</td><br />
<td class="row">230.67 (RBS30)<br><br />
ND (RBS31)<br><br />
55.77 (RBS32)<br><br />
120 (RBS34)</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row">&delta;<sub>p<sub>Tet</sub></sub></td><br />
<td class="row">leakage factor of promoter pTet basic activity</td><br />
<td class="row">[-]</td><br />
<td class="row">0.028 (RBS30)<br><br />
ND (RBS31)<br><br />
1.53E-11 (RBS32)<br><br />
0.085 (RBS34)</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">&eta;<sub>p<sub>Tet</sub></sub></td><br />
<td class="row">Hill coefficient of pTet</td><br />
<td class="row">[-]</td><br />
<td class="row">4.61 (RBS30)<br><br />
ND (RBS31)<br><br />
4.98 (RBS32)<br><br />
24.85 (RBS34)</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">k<sub>p<sub>Tet</sub></sub></td><br />
<td class="row">dissociation constant of aTc from pTet</td><br />
<td class="row">[ng/ml]</td><br />
<td class="row">8.75 (RBS30)<br><br />
ND (RBS31)<br><br />
7.26 (RBS32)<br><br />
9 (RBS34)</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">&alpha;<sub>p<sub>Lux</sub></sub></td><br />
<td class="row">maximum transcription rate of pLux (dependent on <a href="#RBS">RBSx</a> efficiency)</td><br />
<td class="row">[(AUr/min)/cell]</td><br />
<td class="row">438 (RBS30)<br><br />
9.8 (RBS31)<br><br />
206 (RBS32)<br><br />
1105 (RBS34)</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row">&delta;<sub>p<sub>Lux</sub></sub></td><br />
<td class="row">leakage factor of promoter pLux basic activity</td><br />
<td class="row">[-]</td><br />
<td class="row">0.05 (RBS30)<br><br />
0.11 (RBS31)<br><br />
0 (RBS32)<br><br />
0.02 (RBS34)</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">&eta;<sub>p<sub>Lux</sub></sub></td><br />
<td class="row">Hill coefficient of pLux</td><br />
<td class="row">[-]</td><br />
<td class="row">2 (RBS30)<br><br />
1.2 (RBS31)<br><br />
1.36 (RBS32)<br><br />
1.33 (RBS34)</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">k<sub>p<sub>Lux</sub></sub></td><br />
<td class="row">dissociation constant of HSL from pLux</td><br />
<td class="row">[nM]</td><br />
<td class="row">1.88 (RBS30)<br><br />
1.5 (RBS31)<br><br />
1.87 (RBS32)<br><br />
2.34 (RBS34)</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">&gamma;<sub>LuxI</sub></td><br />
<td class="row">LuxI constant degradation</td><br />
<td class="row">[1/min]</td><br />
<td class="row">0.0173</td><br />
</tr><br />
<tr><br />
<td class="row">&gamma;<sub>AiiA</sub></td><br />
<td class="row">AiiA constant degradation</td><br />
<td class="row">[1/min]</td><br />
<td class="row">0.0173</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">&gamma;<sub>HSL</sub></td><br />
<td class="row">HSL constant degradation</td><br />
<td class="row">[1/min]</td><br />
<td class="row">0 (pH=6)</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">V<sub>max</sub></td><br />
<td class="row">maximum transcription rate of LuxI per cell</td><br />
<td class="row">[nM/(min*cell)]</td><br />
<td class="row">3.56*10-9</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">k<sub>M,LuxI</sub></td><br />
<td class="row">half-saturation constant of LuxI from HSL</td><br />
<td class="row">[AUr/cell]</td><br />
<td class="row">6.87*10<sup>3</sup></td><br />
</tr><br />
<tr><br />
<td class="row">k<sub>cat</sub></td><br />
<td class="row">maximum number of enzymatic reactions catalyzed per minute</td><br />
<td class="row">[1/(min*cell)]</td><br />
<td class="row">ND</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">k<sub>M,AiiA</sub></td><br />
<td class="row">half-saturation constant of AiiA from HSL</td><br />
<td class="row">[AUr/cell]</td><br />
<td class="row">ND</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">N<sub>max</sub></td><br />
<td class="row">maximum number of bacteria per well</td><br />
<td class="row">[cell]</td><br />
<td class="row">1*10<sup>9</sup></td><br />
</tr><br />
<br />
<tr><br />
<td class="row">&mu;</td><br />
<td class="row">rate of bacteria growth</td><br />
<td class="row">[1/min]</td><br />
<td class="row">0.004925</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td class="row">LuxI</td><br />
<td class="row">kinetics of LuxI enzyme</td><br />
<td class="row">[<sup>AUr</sup>&frasl;<sub>cell</sub>]</td><br />
<td class="row">-</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">AiiA</td><br />
<td class="row">kinetics of AiiA enzyme</td><br />
<td class="row">[<sup>AUr</sup>&frasl;<sub>cell</sub>]</td><br />
<td class="row">-</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">HSL</td><br />
<td class="row">kinetics of HSL</b></td><br />
<td class="row">[<sup>nM</sup>&frasl;<sub>(min)</sub>]</td><br />
<td class="row">-</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">N</td><br />
<td class="row">number of cells</td><br />
<td class="row">cell</td><br />
<td class="row">-</td><br />
</tr><br />
</table><br />
</center><br />
<br><br />
<br><br />
<br />
<br />
<div align="justify"><b><a name="RBS">NOTE</a></b><p>In order to better investigate the range of dynamics of each subpart, every promoter has been studied with 4 different RBSs, so as to develop more knowledge about the state variables in several configurations of RBS' efficiency <a href="#Salis">(<i><b>Salis HM</b> et al. 2009</i>)</a>. Hereafter, referring to the notation "RBSx" we mean, respectively, <br />
<a href="http://partsregistry.org/Part:BBa_B0030">RBS30</a>, <br />
<a href="http://partsregistry.org/Part:BBa_B0031">RBS31</a>, <br />
<a href="http://partsregistry.org/Part:BBa_B0032">RBS32</a>, <br />
<a href="http://partsregistry.org/Part:BBa_B0034">RBS34</a>.<br />
</p></div><br />
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<br />
<a name="CV"></a><h4> <span class="mw-headline"> <b>Parameter CV</b> </span></h4><br />
<center><br />
<table class="data"><br />
<tr><br />
<td class="row"><b>Parameter & Species</b></td><br />
<td class="row"><b>BBa_B0030</b></td><br />
<td class="row"><b>BBa_B0031</b></td><br />
<td class="row"><b>BBa_B0032</b></td><br />
<td class="row"><b>BBa_B0034</b></td><br />
</tr><br />
<tr><br />
<td class="row">&alpha;<sub>p<sub>Tet</sub></sub></td><br />
<td class="row">3.7</td><br />
<td class="row">ND</td><br />
<td class="row">12</td><br />
<td class="row">5.94</td><br />
</tr><br />
<tr><br />
<td class="row">&delta;<sub>p<sub>Tet</sub></sub></td><br />
<td class="row">91.61</td><br />
<td class="row">>>100</td><br />
<td class="row">>100</td><br />
<td class="row">40.59</td><br />
</tr><br />
<tr><br />
<td class="row">&eta;<sub>p<sub>Tet</sub></sub></td><br />
<td class="row">23.72</td><br />
<td class="row">>>100</td><br />
<td class="row">57.62</td><br />
<td class="row">47.6</td><br />
</tr><br />
<tr><br />
<td class="row">k<sub>p<sub>Tet</sub></sub></td><br />
<td class="row">4.16</td><br />
<td class="row">>>100</td><br />
<td class="row">14.99</td><br />
<td class="row">5.43</td><br />
</tr><br />
<tr><br />
<td class="row">&alpha;<sub>p<sub>Lux</sub></sub></td><br />
<td class="row">10.14</td><br />
<td class="row">7.13</td><br />
<td class="row">2.78</td><br />
<td class="row">5.8</td><br />
</tr><br />
<tr><br />
<td class="row">&delta;<sub>p<sub>Lux</sub></sub></td><br />
<td class="row">179.7</td><br />
<td class="row">57.04</td><br />
<td class="row">1317.7</td><br />
<td class="row">187.2</td><br />
</tr><br />
<tr><br />
<td class="row">&eta;<sub>p<sub>Lux</sub></sub></td><br />
<td class="row">47.73</td><br />
<td class="row">29.13</td><br />
<td class="row">9.75</td><br />
<td class="row">19.3</td><br />
</tr><br />
<tr><br />
<td class="row">k<sub>p<sub>Lux</sub></sub></td><br />
<td class="row">27.5</td><br />
<td class="row">25.81</td><br />
<td class="row">8.46</td><br />
<td class="row">17.86</td><br />
</tr><br />
<br />
</table><br />
</center><br />
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<a name="Parameter_estimation"></a><h2> <span class="mw-headline"> <b>Parameter estimation</b></span></h2><br />
<div style='text-align:justify'>The aim of the model is to predict the behavior of the final closed loop circuit starting from the characterization of single BioBrick parts through a set of well-designed <em>ad hoc</em> experiments. This section presents the experiments performed.<br />
As explained before in <a href="#RBS"><span class="toctext"><b>NOTE</b></span></a>, considering a set of 4 RBSs for each subpart expands the range of dynamics and helps us to better understand the interactions between state variables.<br />
</div><br />
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<br><br />
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<br />
<br />
<a name="Ptet_&_Plux"></a><h4> <span class="mw-headline"> <b>Promoter (PTet & pLux)</b> </span></h4><br />
<div style='text-align:justify'><br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><img alt="" src="https://static.igem.org/mediawiki/2011/9/91/Caratterizzazione_ptetN.jpg" class="thumbimage" width="33%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
</div><br />
<br />
<div style='text-align:justify'><br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><img alt="" src="https://static.igem.org/mediawiki/2011/7/79/Caratterizzazione_pluxN.jpg" class="thumbimage" width="70%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
</div><br />
<div style='text-align:justify'><p>These are the first parts tested, with the target of learning more about pTet and pLux promoters. In particular, as previously explained in <a href=#RBS>NOTE</a>, for each promoter, we tested four different combinations of promoter-RBS, providing us a set of fundamental building blocks for the subsequent assebly of the closed-loop circuit.</p><br />
<p>As shown in the figure below, we considered a range of inductions and we monitored, in time, absorbance (O.D. stands for "optical density") and fluorescence; the two vertical segments for each graph highlight the exponential phase of bacterial growth. S<sub>cell</sub> (namely, synthesis rate per cell) can be derived as a function of inducer concentration, thereby providing the desired input-output relation (inducer concentration versus promoter+RBS activity), which was modelled as a Hill curve:</p><br />
<br />
<div align="center"><div class="thumbinner" style="width: 600px;"><br />
<a href="https://static.igem.org/mediawiki/2011/5/58/Scell.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/5/58/Scell.jpg" class="thumbimage" width="45%"></a></div></div><br />
<br />
However, also Relative Promoter Unit (RPU, <a href="#Kelly"><i><b>Kelly JR</b> et al. 2009</i></a>) has been calculated as a ratio of S<sub>cell</sub> of promoter of interest and the S<sub>cell</sub> of <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a> (reference to <a href="#Hypothesis"><span class="toctext"><b><em>HP<sub>3</sub></em></b></span></a>).<br><br />
<br />
<div style='text-align:center'><div class="thumbinner" style="width: 600px;"><br />
<a href="https://static.igem.org/mediawiki/2011/2/26/Box2_new.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/2/26/Box2_new.jpg" class="thumbimage" height="48%" width="120%"></a></div></div><br><br />
<br />
<p>As shown in the figure, &alpha;, as already mentioned, represents the protein maximum synthesis rate, which is reached, in accordance with Hill equation, when the inducer concentration tends to infinite, and, more practically, when the inducer concentration is sufficiently higher than the dissociation constant. Meanwhile the product &alpha;*&delta; stands for the leakage activity (at no induction), liable for protein production (LuxI and AiiA respectively) even in the absence of inducer.</p><br />
<p>The paramenter &eta; is the Hill's cooperativity constant and it affects the ripidity of transition from the lower and upper boundary of the curve relating S<sub>cell</sub> to the inducer concentration.<br />
Lastly, k stands for the semi-saturation constant and, in case of &eta;=1, it indicates the concentration of substrate at which half the synthesis rate is achieved.<br />
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<br />
<a name="introduction_to_T9002"></a><h4> <span class="mw-headline"> <b>T9002 introduction</b> </span></h4><br />
<div style='text-align:justify'><br />
<em><br />
<p>LuxI and AiiA tests have been always performed exploiting the well-characterized BioBrick <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a>, by which it's possible to quantify exactly the concentration of HSL.</p><br />
<div align="center"><div class="thumbinner" style="width: 500px;"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c2/T9002.jpg" class="thumbimage" width="110%"></a></div></div><br />
<p>This is a biosensor which receives HSL concentration as input and returns GFP intensity (more precisely S<sub>cell</sub>) as output.<a href="#Canton"> (<i><b>Canton</b> et al. 2008</i>).</a><br />
According to this, it is necessary to understand the input-output relationship: so, a T9002 "calibration" curve is plotted for each test performed.</p><br><br><br />
</em><br />
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<br />
<a name="Enzymes"></a><h4> <span class="mw-headline"> <b>AiiA & LuxI</b> </span></h4><br />
<div style='text-align:justify'><p>This paragraph explains how parameters of equation (3) are estimated. The target is to learn the AiiA degradation and LuxI production mechanisms in addiction to HSL intrinsic degradation, in order to estimate V<sub>max</sub>, K<sub>M,LuxI</sub>, k<sub>cat</sub>, K<sub>M,AiiA</sub> and &gamma;<sub>HSL</sub> parameters. We adopt tests composed of two steps. In the first one, the following BioBrick parts are used:</p> <br />
</div><br />
<br />
<div style='text-align:justify'><br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><img alt="" src="https://static.igem.org/mediawiki/2011/8/88/Caratterizzazione_aiia.JPG" class="thumbimage" width="32%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div style='text-align:justify'><br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width:100%;"><img alt="" src="https://static.igem.org/mediawiki/2011/4/48/Caratterizzazione_luxIN.jpg" class="thumbimage" width="28%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div style='text-align:justify'><p>Based on our <a href="#Hypothesis"><span class="toctext"><b><em>HP<sub>3</sub></em></b></span></a> and <a href="#Hypothesis"><span class="toctext"><b><em>HP<sub>4</sub></em></b></span></a>, we are able to determine AiiA and LuxI concentrations, provided we have yet characterized pTet-RBSx contructs<a name='t9002'></a>. In particular, referring to <a href="#Hypothesis"><span class="toctext"><b><em>HP<sub>4</sub></em></b></span></a>, in exponential growth the equilibrium of the enzymes is conserved. Due to a known induction of aTc, the steady-state level per cell can be calculated:</p></div> <br />
<br />
<div style='text-align:justify'><div class="thumbinner" style="width: 500px;"><br />
<a href="https://static.igem.org/mediawiki/2011/7/74/Aiia_cost.jpg"><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/7/74/Aiia_cost.jpg" class="thumbimage" width="120%"></a></div></div><br />
<br />
<p>Then, as a second step, we monitor in separate experiments HSL synthesis and degradation caused by the activities of the enzymes. In other words, our idea is to control the degradation of HSL versus time. ATc activates pTet and, later, a certain concentration of HSL is introduced. Then, at fixed times, O.D.<sub>600</sub> and HSL concentration are monitored using Tecan and T9002 biosensor.</p><p>For example for AiiA dependent HSL degradation, we have:</p><br />
<br />
<table align='center' width='100%'><br />
<div style='text-align:center'><div class="thumbinner" style="width: 70%;"><br />
<a href="https://static.igem.org/mediawiki/2011/9/99/Degradation.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/9/99/Degradation.jpg" class="thumbimage" width="140%"></a></div></div><br />
</table><br />
<br />
<br><p>Therefore, considering for a determined promoter-RBSx couple, several induction of aTc and, for each of them, several samples of HSL concentration during time, parameters V<sub>max</sub>, k<sub>M,LuxI</sub>, k<sub>cat</sub> and k<sub>M,AiiA</sub> can be estimated, through numerous iterations of an algorithm implemented in MATLAB.</p><br />
<div align="right"><small><a href="#indice">^top</a></small></div><br />
<br><br />
<br><br />
<br />
<a name="N"></a><h4> <span class="mw-headline"> <b>N</b> </span></h4><br />
<div style='text-align:justify'>The parameters N<sub>max</sub> and μ can be calculated from the analysis of the OD<sub>600</sub> produced by our MGZ1 culture. In particular, μ is derived as the slope of the log(O.D.<sub>600</sub>) growth curve. Counting the number of cells of a saturated culture would be considerably complicated, so N<sub>max</sub> is determined with a proper procedure. The aim here is to derive the linear proportional coefficient &Theta; between O.D'.<sub>600</sub> and N: this constant can be estimated as the ratio between absorbance (read from TECAN) and the respective number of CFU on a petri plate. Finally, N<sub>max</sub> is calcultated as &Theta;*O.D'.<sub>600</sub><br />
<a href="#Pasotti">(<i><b>Pasotti L</b> et al. 2010</i>)</a>.<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br />
</div><br />
<br />
<br><br />
<br><br />
<br />
<br />
<a name="Degradation_rates"></a><h4> <span class="mw-headline"> <b>Degradation rates</b> </span></h4><br />
<div style='text-align:justify'>The parameters &gamma;<sub>LuxI</sub> and &gamma;<sub>AiiA</sub> are taken from literature since they contain LVA tag for rapid degradation. Instead, approximating HSL kinetics as a decaying exponential, &gamma;<sub>HSL</sub> can be derived as the slope of the log(concentration), which can be monitored through <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a>.<br />
</div><br />
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<br />
<br />
<a name="Simulations"></a><h1><span class="mw-headline"> <b>Simulations</b> </span></h1><br />
<div style='text-align:justify'><br />
<p>On a biological level, the ability to control the concentration of a given molecule reveals itself as fundamental in limiting the metabolic burden of the cell; moreover, in the particular case of HSL signalling molecules, this would give the possibility to regulate quorum sensing-based population behaviours. In this section we present some simulations of another circuit, which could validate the concept of the closed-loop model we have discussed so far.</p><br />
<p>In order to see that, we implemented and simulated in Matlab an open loop circuit, similar to <b>CTRL+<em>E</em></b>, except for the constitutive production of AiiA.</p><br />
<br />
<center><br />
<table><br />
<tr> <br />
<td><br />
<div style='text-align:justify'><div class="thumbinner" style="width: 500px;"><br />
<a href="https://static.igem.org/mediawiki/2011/b/b6/Sim_closed.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/b/b6/Sim_closed.jpg" class="thumbimage" width="100%"></a></div></div><br />
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<tr><br />
<td><br />
<div style='text-align:justify'><div class="thumbinner" style="width: 500px;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/4b/Sim_open.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/4/4b/Sim_open.jpg" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
</center><br />
</div><br />
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<br><br />
<br><br />
<br />
<a name="Sensitivity_Analysis"></a><h1> <span class="mw-headline"> <b>Sensitivity Analysis of the steady state of enzyme expression in exponential phase</b> </span></h1><br />
<br />
<p>In this paragraph we investigate the theoretical behaviour of our circuit in the cell culture exponential growth phase. According to this, we first derive, under feasible hypotheses, the steady state condition for the enzymes and HSL concentration in that phase. Then we perform a sensitivity analysis relating the output of our system (HSL) to input (aTc) and system parameters.</p><br />
<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br />
<br />
<a name="Steady state of enzyme expression"></a><h2> <span class="mw-headline"> <b>Steady state of enzyme expression</b> </span></h2><br />
<br />
<br />
<p> Based on our <a href="#Hypothesis"><span class="toctext"><b><em>HP<sub>4</sub></em></b></span></a>, we can formulate the steady state expressions during the exponential growth phase. Adding other considerations about the involved processes, it is possible to add further simplifications to the steady state equations. In particular, one concern relates to the number of cells N (in the order of 3*10^3), which is far lower than N<sub>max</sub> (10^9). The other pertains to &gamma;*HSL parameter, whch can be neglected compared to the other two terms of the third equation. Based on this assumptions, equation (4) of the system becomes dN/dt=&mu;N. Moreover, from equation (3), after having removed the third term, we can simplify the N parameter, since it is common to the remaining two terms. On a biological point of view, this implies that AiiA, LuxI and HSL undergo only minor changes through time, thereby allowing to derive their steady state expressions:</p><br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width: 100%;"><br />
<a href="https://static.igem.org/mediawiki/2011/3/32/LuxI_SS.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/3/32/LuxI_SS.jpg" class="thumbimage" width="83%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<p> The first equation is independent from the second and third ones, enabling us to directly determine LuxI steady state expression during the exponential growth phase. On the contrary, second and third equations depend each other in defining the value of AiiA and HSL respectively, because the former is a function of HSL, while the latter is a function of AiiA. So we could resolve a system of two equations, first by expliciting one of the two variables with respect to the other, and then substituting its expression in order to determine the other variable possible values. This would bring a complex mathematical formulation, which is not helpful in understanding the influence of the various model parameters on the output HSL.<br />
On the other hand, AiiA and HSL values can also be graphically determined from the intersection of the curves derived from these two equations, if we explicit HSL as a function of AiiA (or, alternatively, AiiA as a function of HSL). It is easy to discover that these two curves represent rectangular hyperbolae (the first one only under a simple approximation, explained below) whose tails intersect each other at a particular point, corresponding to the searched values for AiiA and HSL.</p> <br />
<div>For a rectangular hyperbola (RH), we have:</div><br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width: 100%;"><a href="" class="image"><br />
<a href="https://static.igem.org/mediawiki/2011/7/75/UNIPV_Rectangular_hyperbola_general.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/7/75/UNIPV_Rectangular_hyperbola_general.jpg" class="thumbimage" width="14%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<p>centered at O(-d/c;a/c), with the vertical asymptote x=-d/c and the horizontal asymptote y=a/c</p><br />
<p>From equation 2, we have:</p><br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width: 100%;"><a href="" class="image"><br />
<a href="https://static.igem.org/mediawiki/2011/3/3a/UNIPV_eta_root_HSL.jpg"><br />
<img src="https://static.igem.org/mediawiki/2011/3/3a/UNIPV_eta_root_HSL.jpg" class="thumbimage" width="62%"></a><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<p> We can introduce the simplification to remove the &eta;plux exponent to the entire expression in the right hand side of the equation, thereby obtaining a rectangular hyperbola; even if this leads to a slight change in the curve behaviour, it allows to more clearly understand the relation between HSL and AiiA. As pertains to equation 3, its steady state approximation during the exponential growth is more immediately identifiable as a rectangular hyperbola. Below the two RHs equations are provided, togheter with the table of parameters.</p><br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width: 100%;"><br />
<a href="https://static.igem.org/mediawiki/2011/1/11/RH1_UNIPV_HSL.jpg"><img src="https://static.igem.org/mediawiki/2011/1/11/RH1_UNIPV_HSL.jpg" class="thumbimage" width="50%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<table align='center' width='100%'><br />
<tr><br />
<td><br />
<div style='text-align:center'><div class="thumbinner" style="width: 100%;"><br />
<a href="https://static.igem.org/mediawiki/2011/4/4b/RH2_UNIPV_HSL.jpg" class="image"><br />
<img src="https://static.igem.org/mediawiki/2011/4/4b/RH2_UNIPV_HSL.jpg" class="thumbimage" width="70%"></a></div></div><br />
</td><br />
</tr><br />
</table><br />
<br><br />
<br />
<br />
<center><br />
<table class="data"><br />
<tr><br />
<td class="row"><b>Parameter</b></td><br />
<td class="row"><b>RH1</b></td><br />
<td class="row"><b>RH2</b></td><br />
</tr><br />
<br />
<tr><br />
<td class="row">a</sub></sub></td><br />
<td class="row">(&gamma;<sub>AiiA</sub>+&mu;)*(k<sub>pLux</sub>)<sup>&eta;pLux</td><br />
<td class="row"><span style="text-decoration:overline;" > V</span><sub>LuxI</sub></td><br />
</tr><br />
<br />
<tr><br />
<td class="row">b</td><br />
<td class="row">-&alpha;<sub>pLux</sub>*&delta;<sub>pLux</sub>*(k<sub>pLux</sub>)<sup>&eta;pLux</td><br />
<td class="row"><span style="text-decoration:overline;" > V</span><sub>LuxI</sub>*k<sub>M,LuxI</sub></td><br />
</tr><br />
<br />
<tr><br />
<td class="row">c</sub></sub></td><br />
<td class="row">-&gamma;<sub>AiiA</sub>+&mu;</td><br />
<td class="row">k<sub>cat</sub>*<span style="text-decoration:overline;" >AiiA</span></td><br />
</tr><br />
<br />
<tr><br />
<td class="row">d</td><br />
<td class="row">&alpha;<sub>pLux</sub></td><br />
<td class="row">0</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">Horizontal asymptote</td><br />
<td class="row">a/c=(k<sub>pLux</sub>)<sup>&eta;pLux</sup></td><br />
<td class="row"><span style="text-decoration:overline;" > V</span><sub>LuxI</sub>/k<sub>cat</sub></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td class="row">Vertical asymptote</td><br />
<td class="row">-d/c=&alpha;<sub>pLux</sub>/(&gamma;<sub>AiiA</sub>+&mu;)</td><br />
<td class="row">0</td><br />
</tr><br />
</table><br />
<br />
</center><br />
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<br />
<a name="Sensitivity analysis"></a><h2><br><br />
<span class="mw-headline"> <b>Sensitivity analysis</b> </span></h2><br />
<br />
<p> Now, it is interesting to conduct some qualitative and quantitative considerations about our system sensitivity to its parameters and aTc input signal.</p><br />
<div> First of all, we analyze how the HSL output can be regulated by changing the characteristics of our RHs.</div><div>Referring to the first rectangular hyperbola, we recognize that its vertical asymptote could be varied by changing &alpha;<sub>p<sub>Lux</sub></sub></sub></sub> value (assuming fixed &gamma;<sub>AiiA</sub> and &mu;). In particular, thanks to the four Plux-RBSx constructs realized, we can vary &alpha;<sub>p<sub>Lux</sub></sub></sub></sub> more than a hundred factor. This can significantly shift the vertical asymptote, bringing this first RH farther or nearer the second one (whose vertical asymptote is the ordinate axis), thereby providing an intersection at higher AiiA and lower HSL values, or vice versa. The following two figures highlight this aspect.</div><br><br />
<br />
<table align='center' width='100%'><br />
<div><div class="thumbinner"><a href="https://static.igem.org/mediawiki/2011/4/45/Iperbole_eq_3_as_ver2.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/4/45/Iperbole_eq_3_as_ver2.jpg" class="thumbimage" width="100%"></a></div></div><br />
</table><br />
<br />
<table width="100%" align="center"><br />
<div style="WIDTH: 100%" class="thumbinner"><br />
<a class="image" href="https://static.igem.org/mediawiki/2011/9/97/UNIPV_inters_RH_ver_alfa_plux_acceptable_values2.jpg"><img class="thumbimage" src="https://static.igem.org/mediawiki/2011/9/97/UNIPV_inters_RH_ver_alfa_plux_acceptable_values2.jpg" width="100%"></a><br />
</div><br />
<tbody></tbody><br />
</table><br />
<br />
<br />
<p>From the above figures, it is also clear that HSL steady state value is not very sensitive to &alpha;<sub>p<sub>Lux</sub></sub></sub></sub>, at least when this parameter presents values greater than unity, because this brings the two curves to intersect in their low slope regions.</p><br />
<p>Referring to the second RH, the only adjustable asymptote is the horizontal one, that we can move upward or downward by altering VLuxI, which indirectly depends on aTc.</p><br />
<br />
<table align='center' width='100%'><br />
<div style='text-align:center'><div class="thumbinner" style="width: 100%;"><a href="https://static.igem.org/mediawiki/2011/0/04/UNIPV_RH2_eq2_as_or_corrected2.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/0/04/UNIPV_RH2_eq2_as_or_corrected2.jpg" class="thumbimage" width="95%"></a></div></div><br />
</table><br />
<br />
<p>A further deepening in the exponential phase analysis involves determining the characteristics of the input-output relation. Our closed loop system can be realized with two alternative purposes in mind:</p><br />
<ol><br />
<li> realizing a circuit able to adapt HSL output depending on aTc input concentration. This requires a good sensitivity between input and output.</li><br />
<li> designing a robust HSL concentration controller, which is immune to the input noise and offers a constant and defined amount of HSL. In this case HSL level should be appropriately tuned during the design stage, by choosing the correct strength of promoter-RBSx complexes.</li><br />
</ol><br />
<br />
<p>Now,again considering the system of equations, it is easy to observe that HSL dependence on aTc input passes through two Hill equations. The former describes aTc driven LuxI synthesis, while the latter models LuxI dependent HSL synthesis rate. Therefore, in order to achieve a high aTc sensitivity, it is advisable to tune aTc and LuxI levels so that they place outside the saturation regions of their Hill curves. In this regard, it is possible to determine a closed form expression relating HSL to aTc, if we hypothesize that both aTc and LuxI are far lower than their respective half-saturation constant (k_ptet and Km), thus simplifying the Hills with a first order relation:</p><br />
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<br><br />
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<table align='center' width='100%'><br />
<div style='text-align:center'><div class="thumbinner" style="width: 100%;"><a href="https://static.igem.org/mediawiki/2011/d/dd/UNIPV_Imply_sensitivity.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/dd/UNIPV_Imply_sensitivity.jpg" class="thumbimage" width="80%"></a></div></div><br />
</table><br />
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<br />
<a name="References"></a><h1><span class="mw-headline"> <b>References</b> </span></h1><br />
<div style='text-align:justify'><br />
<br />
<ol type='1'><br />
<br />
<a name="Braun"></a><br />
<li>Braun D, Basu S, Weiss R (2005) <b>Parameter estimation for two synthetic gene networks: a case study </b> <br />
<i>ICASSP '05 </i> 5:v/769-v/772. </li> <br><br />
<br />
<a name="Canton"></a><br />
<li>Canton B, Labno A, Endy D. (2008) <b>Refinement and standardization of synthetic biological parts and devices. </b> <i> Nat Biotechnol. </i> 26(7):787-93. </li> <br><br />
<br />
<a name="Danino"></a><br />
<li>Danino T, Mondrag&oacute;n-Palomino O, Tsimring L et al. (2010) <b>A synchronized quorum of genetic clocks. </b> <i>Nature. </i> 463(7279):326-30. </li> <br><br />
<br />
<a name="Endler"></a><br />
<li>Endler L, Rodriguez N, Juty N et al. (2009) <b>Designing and encoding models for synthetic biology. </b> <i>J. R. Soc. Interface </i> 6:S405-S417. </li> <br><br />
<br />
<a name="Kelly"></a><br />
<li>Kelly JR, Rubin AJ, Davis JH et al. (2009) <b>Measuring the activity of BioBrick promoters using an in vivo reference standard.</b> <i> J. Biol. Eng.</i> 3:4.</li> <br> <br />
<br />
<a name="Pasotti"></a><br />
<li>Pasotti L, Quattrocelli M, Galli D et al. (2011) <b>Multiplexing and demultiplexing logic functions for computing signal processing tasks in synthetic biology. </b> <br />
<i>Biotechnol. J. </i>6(7):784-95. </li> <br><br />
<br />
<a name="Salis"></a><br />
<li>Salis H M, Mirsky E A, Voight C A (2009)<b> Automated design of synthetic ribosome binding sites to control protein expression. </b> <i>Nat. Biotechnol.</i>27:946-950. <br />
</li><br><br />
<br />
<br />
</ol><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Project/ResultsTeam:UNIPV-Pavia/Project/Results2011-09-17T23:29:27Z<p>Nickpv: </p>
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<div>{{main}}<br />
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<h2 class="art-postheader"><br />
Results<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
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<a name='indice'></a><br />
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#assembly"><span class="tocnumber">1</span> <span class="toctext">Part assembly</span></a> <br />
<li class="toclevel-1"><a href="#characterization"><span class="tocnumber">1</span> <span class="toctext">Characterization of basic modules</span></a> <br />
<ul><br />
<li class="toclevel-2"><a href="#promoters"><span class="tocnumber">2.1</span> <span class="toctext">Promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#enzymes"><span class="tocnumber">2.2</span> <span class="toctext">Characterization of the activity of the enzymes AiiA and LuxI</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.3</span> <span class="toctext">Characterization of RBS efficiency</span></a></li><br />
</ul><br />
<li class="toclevel-1"><a href="#growth"><span class="tocnumber">3</span> <span class="toctext">Identification of bacterial growth parameters</span></a></li><br />
<li class="toclevel-1"><a href="#HSL"><span class="tocnumber">4</span> <span class="toctext">Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</span></a></li><br />
<li class="toclevel-1"><a href="#t9002"><span class="tocnumber">5</span> <span class="toctext">Characterization of BBa_T9002 biosensor</span></a></li><br />
</ul></td></tr></table><br />
</div><br />
<br />
<br />
<br><br />
<br />
<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C. For the cloning of the parts, <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#TOP10'><em>E. coli</em> TOP10</a> was used. <br />
</em><br />
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<a name='assembly'></a><h1>Parts assembly</h1><br />
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<a name='characterization'></a><h1>Characterization of basic modules</h1><br />
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<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------pTet and pLux-----------><br />
<!--------------------------------><br />
<!--------------------------------><br />
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<br />
<a name='promoters'></a><h2>Characterization of promoters pTet and pLux</h2><br />
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<!--------------------------------><br />
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<!--------AiiA and LuxI-----------><br />
<!--------------------------------><br />
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<a name='enzymes'></a><h2>Characterization of enzymes AiiA and LuxI</h2><br />
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<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!--------------------------------><br />
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<!--------------RBS---------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='rbs'></a><h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-----------growth---------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<a name='growth'></a><h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
The bacterial growth curve has been modelled as a logistic curve and is represented by the following equation:<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br><br />
N(0)=n<sub>0</sub><br />
<br><br><br />
<br />
where &mu; represents the growth rate of the cells (<em>E. coli</em> MGZ1 in M9 supplemented medium) and N<sub>max</sub> represents the maximum number of cells in the well. For a detailed description of the parameters, see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>modelling section</a>. For details on parameters identification, see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#N'>identification section.</a> <br />
The growth curves in all the performed experiments are measured in O.D.<sub>600</sub>. Since the <em>N</em> species in the model is expressed in <em>cell number</em>, a conversion factor has been estimated. The conversion factor <b>K<sub>O.D.toC.F.U.</sub></b> has been estimated as follows.<br />
<ol><ul><br />
<li>Two cultures C1 and C2 (MGZ1 cells) were grown in 1ml M9 medium till saturation (ON liquid culture, 37°C, 220 rpm).</li><br />
<li>Next morning, both C1 and C2 were diluted in M9 medium with a final volume of 1ml with the following dilution factors:<br />
<ul><li>1:1</li><li>1:10</li><li>1:100</li><li>1:1000</li><br />
</ul> in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm.<br />
</li><br />
<li>After 1 hour, O.D.<sub>600</sub> was measured using TECAN microplate reader (don't forget to measure a M9 sample for blanking!)<br><br />
<em>NB: from now on, cultures must be placed in ice to stop cell growth.</em></li><br />
<li>At the same time, proper dilution of the cultures were plated on LB agar plates.<br><br />
<em>NB: All the dilutions are performed moving 100 &mu;l of culture in previously ice-chilled 900 &mu;l fresch M9. 100 &mu;l of the final dilution are plated (It still represents a 1:10 dilution!)</em></li><br />
<li>Plates were grown overnight and next morning C.F.U. were counted.</li><br />
<li>C.F.U. values were corrected by the dilution factor and a linear regression (N vs O.D.<sub>600</sub>) was performed in order to evaluate the conversion factor <b>K<sub>O.D.toC.F.U.</sub></b>. </li><br />
<li><b>K<sub>O.D.toC.F.U.</sub></b> was used as conversion factor to multiply the O.D.<sub>600</sub> value of saturation in the growth curves (~0,5). </li><br />
<br />
</ul></ol><br />
<br />
<p align='center'><br />
The results are summarized in the table and in the figure below.<br />
</p><br />
<table class='data'><tr><td class='row'><b> Culture </b></td><td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><td class='row'><b> C.F.U. </b></td><td class='row'><b> Dilution Factor (10^) </b></td><td class='row'><b> N </b></td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,367950002 </td><td class='row'> 0,758004416 </td><td class='row'> 990 </td><td class='row'> 5 </td><td class='row'> 990000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,044649998 </td><td class='row'> 0,091982322 </td><td class='row'> 141 </td><td class='row'> 6 </td><td class='row'> 1410000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,004799999 </td><td class='row'> 0,009888356 </td><td class='row'> 136 </td><td class='row'> 5 </td><td class='row'> 136000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,000549998 </td><td class='row'> 0,001133037 </td><td class='row'> 20 </td><td class='row'> 6 </td><td class='row'> 200000000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,54840003 </td><td class='row'> 1,129744917 </td><td class='row'> 165 </td><td class='row'> 4 </td><td class='row'> 16500000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,058200002 </td><td class='row'> 0,119896339 </td><td class='row'> 23 </td><td class='row'> 5 </td><td class='row'> 23000000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,008700002 </td><td class='row'> 0,017922652 </td><td class='row'> 251 </td><td class='row'> 3 </td><td class='row'> 2510000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,000100002 </td><td class='row'> 0,000206011 </td><td class='row'> 24 </td><td class='row'> 4 </td><td class='row'> 2400000 </td> </tr><br />
</table><br><br />
</p><br />
<br />
<table align='center><tr><td align='center'><br />
<div style='text-align:justify'><div class="thumbinner" style="width: 90%;"><img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"></a></div></div><br />
</td></tr></table><br />
<br><br />
The estimation of &mu; parameter was performed by determining the slope of the logarithmic curve of O.D.<sub>600</sub> in exponential phase. Exponential phase was determined by visual inspection as the linear phase of the logarithmic curve of O.D.<sub>600</sub>. <br />
<br><br><br />
The estimated parameters are summarized in the table below:<br />
<br><br><br />
<br />
<table width='50%' class='data'><tr><br />
<td class='row'><b>N<sub>max</sub> [cell number]</b></td><br />
<td class='row'><b>&mu; [min<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><td class='row'>1*10<sup>9</sup></td><br />
<td class='row'>0.004925</td><br />
</tr><br />
</table><br />
<br />
The reported value of &mu; corresponds to a doubling time of 142 minutes.<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------HSL---------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<a name='HSL'></a><h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'><br />
In order to estimate the spontaneous degradation rate of HSL in M9 medium and in a culture of MGZ1 cells as a function of pH, two simple tests have been performed.<br><br />
Two different M9 media were prepared, one with the nominal pH (7.0) and one with pH=6.0.<br><br />
These media, now named respectively M9<sub>pH 7</sub> and M9<sub>pH 6</sub>, were added with a known concentration of HSL (100 nM) and then incubated at 37°C, 220 rpm (NB: the media were not infected with any culture but the standard growth conditions were reproduced). The amount of HSL present in the medium was assayed through the BBa_T9002 biosensor at 4 time points: <br><br />
<ul><br />
<li>t=0 h;</li><br />
<li>t=1 h;</li><br />
<li>t=2 h;</li><br />
<li>t=4 h;</li><br />
<li>t=8 h;</li><br />
</ul><br />
The obtained time series of HSL amounts were processed to evaluate the time constant governing the dynamic of HSL degradation, supposing an exponential decay. The results are reported in the table below:<br />
<br />
<table class='data'><br />
<tr><br />
<td class='row'><br />
</td><br />
<td class='row'><br />
<b>t<sub>1/2</sub><sup>*</sup> [h]</b><br />
</td><td class='row'><br />
<b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b><br />
</td><br />
</tr><br />
<tr><td class='row'><b>M9<sub>pH 6</sub></b><br />
</td><br />
<td class='row'>32<br />
</td><br />
<td class='row'>0.022<br />
</td><br />
</tr><br />
<tr><td class='row'><b>M9<sub>pH 7</sub></b><br />
</td><br />
<td class='row'>8<br />
</td><br />
<td class='row'>0.087<br />
</td><br />
</tr><br />
</table><br />
<br><br />
The described experiment was repeated with a MGZ1 culture in order to evaluate the effect of culture on HSL stability. The estimated values are reported in the table below:<br><br />
<br />
<table class='data'><br />
<tr><br />
<td class='row'><br />
</td><br />
<td class='row'><br />
<b>t<sub>1/2</sub><sup>*</sup> [h]</b><br />
</td><td class='row'><br />
<b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b><br />
</td><br />
</tr><br />
<tr><td class='row'><b>Culture<sub>pH 6</sub></b><br />
</td><br />
<td class='row'>&inf;<br />
</td><br />
<td class='row'>0<br />
</td><br />
</tr><br />
<tr><td class='row'><b>Culture<sub>pH 7</sub></b><br />
</td><br />
<td class='row'>19<br />
</td><br />
<td class='row'>0.037<br />
</td><br />
</tr><br />
</table><br />
<br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-------------t9002--------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='t9002'></a><h2>Characterization of BBa_T9002 biosensor</h2><br />
As described in the <a href="https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002">modelling section</a>, BioBrick <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> is an HSL biosensor, which provides a non lineaer relationship between HSL input and S<sub>cell</sub> output. More precisely, the characteristic sigmoidal curve requires synthetic parameters for its accurate identification. These are the minimum and maximum values, the swtich point (i.e., the curve inflection point), and the upper and lower boundaries of linearity. This biosensor revealed greatly reliable, providing measurement repeatability and minimal experimental noise.<br><br><br />
<br />
<table width='50%' class='data'><tr><br />
<td class='row'><b>Minimum</b></td><br />
<td class='row'><b>Maximum</b></td><br />
<td class='row'><b>Switch point</b></td><br />
<td class='row'><b>Lower boundary of linearity</b></td><br />
<td class='row'><b>Upper boundary of linearity</b></td><br />
</tr><br />
<br />
<br />
<tr><td class='row'><b>Minimum</b></td><br />
<td class='row'><b>Maximum</b></td><br />
<td class='row'><b>Switch point</b></td><br />
<td class='row'><b>Lower boundary of linearity</b></td><br />
<td class='row'><b>Upper boundary of linearity</b></td><br />
</tr><br />
</table><br />
<br />
<br><br><br />
In order to determine the threshold sensitivity of T9002 biosensor, experiments were performed with several HSL inductions minimally interspaced in the region of low detectability. These allowed to reveal that the minimum detectable HSL concentration is ? nM. <br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
</html><br />
<br />
{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Project/ResultsTeam:UNIPV-Pavia/Project/Results2011-09-17T23:26:17Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<html><br />
<h2 class="art-postheader"><br />
Results<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-----------MENU-----------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='indice'></a><br />
<br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#assembly"><span class="tocnumber">1</span> <span class="toctext">Part assembly</span></a> <br />
<li class="toclevel-1"><a href="#characterization"><span class="tocnumber">1</span> <span class="toctext">Characterization of basic modules</span></a> <br />
<ul><br />
<li class="toclevel-2"><a href="#promoters"><span class="tocnumber">2.1</span> <span class="toctext">Promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#enzymes"><span class="tocnumber">2.2</span> <span class="toctext">Characterization of the activity of the enzymes AiiA and LuxI</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.3</span> <span class="toctext">Characterization of RBS efficiency</span></a></li><br />
</ul><br />
<li class="toclevel-1"><a href="#growth"><span class="tocnumber">3</span> <span class="toctext">Identification of bacterial growth parameters</span></a></li><br />
<li class="toclevel-1"><a href="#HSL"><span class="tocnumber">4</span> <span class="toctext">Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</span></a></li><br />
<li class="toclevel-1"><a href="#t9002"><span class="tocnumber">5</span> <span class="toctext">Characterization of BBa_T9002 biosensor</span></a></li><br />
</ul></td></tr></table><br />
</div><br />
<br />
<br />
<br><br />
<br />
<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C. For the cloning of the parts, <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#TOP10'><em>E. coli</em> TOP10</a> was used. <br />
</em><br />
<br><br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-----------ASSEMBLY-------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<br />
<a name='assembly'></a><h1>Parts assembly</h1><br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-------CHARACTERIZATION---------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='characterization'></a><h1>Characterization of basic modules</h1><br />
<br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------pTet and pLux-----------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<a name='promoters'></a><h2>Characterization of promoters pTet and pLux</h2><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------AiiA and LuxI-----------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='enzymes'></a><h2>Characterization of enzymes AiiA and LuxI</h2><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------RBS---------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='rbs'></a><h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-----------growth---------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<a name='growth'></a><h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
The bacterial growth curve has been modelled as a logistic curve and is represented by the following equation:<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br><br />
N(0)=n<sub>0</sub><br />
<br><br><br />
<br />
where &mu; represents the growth rate of the cells (<em>E. coli</em> MGZ1 in M9 supplemented medium) and N<sub>max</sub> represents the maximum number of cells in the well. For a detailed description of the parameters, see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>modelling section</a>. For details on parameters identification, see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#N'>identification section.</a> <br />
The growth curves in all the performed experiments are measured in O.D.<sub>600</sub>. Since the <em>N</em> species in the model is expressed in <em>cell number</em>, a conversion factor has been estimated. The conversion factor <b>K<sub>O.D.toC.F.U.</sub></b> has been estimated as follows.<br />
<ol><ul><br />
<li>Two cultures C1 and C2 (MGZ1 cells) were grown in 1ml M9 medium till saturation (ON liquid culture, 37°C, 220 rpm).</li><br />
<li>Next morning, both C1 and C2 were diluted in M9 medium with a final volume of 1ml with the following dilution factors:<br />
<ul><li>1:1</li><li>1:10</li><li>1:100</li><li>1:1000</li><br />
</ul> in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm.<br />
</li><br />
<li>After 1 hour, O.D.<sub>600</sub> was measured using TECAN microplate reader (don't forget to measure a M9 sample for blanking!)<br><br />
<em>NB: from now on, cultures must be placed in ice to stop cell growth.</em></li><br />
<li>At the same time, proper dilution of the cultures were plated on LB agar plates.<br><br />
<em>NB: All the dilutions are performed moving 100 &mu;l of culture in previously ice-chilled 900 &mu;l fresch M9. 100 &mu;l of the final dilution are plated (It still represents a 1:10 dilution!)</em></li><br />
<li>Plates were grown overnight and next morning C.F.U. were counted.</li><br />
<li>C.F.U. values were corrected by the dilution factor and a linear regression (N vs O.D.<sub>600</sub>) was performed in order to evaluate the conversion factor <b>K<sub>O.D.toC.F.U.</sub></b>. </li><br />
<li><b>K<sub>O.D.toC.F.U.</sub></b> was used as conversion factor to multiply the O.D.<sub>600</sub> value of saturation in the growth curves (~0,5). </li><br />
<br />
</ul></ol><br />
<br />
<p align='center'><br />
The results are summarized in the table and in the figure below.<br />
</p><br />
<table class='data'><tr><td class='row'><b> Culture </b></td><td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><td class='row'><b> C.F.U. </b></td><td class='row'><b> Dilution Factor (10^) </b></td><td class='row'><b> N </b></td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,367950002 </td><td class='row'> 0,758004416 </td><td class='row'> 990 </td><td class='row'> 5 </td><td class='row'> 990000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,044649998 </td><td class='row'> 0,091982322 </td><td class='row'> 141 </td><td class='row'> 6 </td><td class='row'> 1410000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,004799999 </td><td class='row'> 0,009888356 </td><td class='row'> 136 </td><td class='row'> 5 </td><td class='row'> 136000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,000549998 </td><td class='row'> 0,001133037 </td><td class='row'> 20 </td><td class='row'> 6 </td><td class='row'> 200000000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,54840003 </td><td class='row'> 1,129744917 </td><td class='row'> 165 </td><td class='row'> 4 </td><td class='row'> 16500000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,058200002 </td><td class='row'> 0,119896339 </td><td class='row'> 23 </td><td class='row'> 5 </td><td class='row'> 23000000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,008700002 </td><td class='row'> 0,017922652 </td><td class='row'> 251 </td><td class='row'> 3 </td><td class='row'> 2510000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,000100002 </td><td class='row'> 0,000206011 </td><td class='row'> 24 </td><td class='row'> 4 </td><td class='row'> 2400000 </td> </tr><br />
</table><br><br />
</p><br />
<br />
<table align='center><tr><td align='center'><br />
<div style='text-align:justify'><div class="thumbinner" style="width: 90%;"><img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"></a></div></div><br />
</td></tr></table><br />
<br><br />
The estimation of &mu; parameter was performed by determining the slope of the logarithmic curve of O.D.<sub>600</sub> in exponential phase. Exponential phase was determined by visual inspection as the linear phase of the logarithmic curve of O.D.<sub>600</sub>. <br />
<br><br><br />
The estimated parameters are summarized in the table below:<br />
<br><br><br />
<br />
<table width='50%' class='data'><tr><br />
<td class='row'><b>N<sub>max</sub> [cell number]</b></td><br />
<td class='row'><b>&mu; [min<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><td class='row'>1*10<sup>9</sup></td><br />
<td class='row'>0.004925</td><br />
</tr><br />
</table><br />
<br />
The reported value of &mu; corresponds to a doubling time of 142 minutes.<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------HSL---------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<a name='HSL'></a><h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'><br />
In order to estimate the spontaneous degradation rate of HSL in M9 medium and in a culture of MGZ1 cells as a function of pH, two simple tests have been performed.<br><br />
Two different M9 media were prepared, one with the nominal pH (7.0) and one with pH=6.0.<br><br />
These media, now named respectively M9<sub>pH 7</sub> and M9<sub>pH 6</sub>, were added with a known concentration of HSL (100 nM) and then incubated at 37°C, 220 rpm (NB: the media were not infected with any culture but the standard growth conditions were reproduced). The amount of HSL present in the medium was assayed through the BBa_T9002 biosensor at 4 time points: <br><br />
<ul><br />
<li>t=0 h;</li><br />
<li>t=1 h;</li><br />
<li>t=2 h;</li><br />
<li>t=4 h;</li><br />
<li>t=8 h;</li><br />
</ul><br />
The obtained time series of HSL amounts were processed to evaluate the time constant governing the dynamic of HSL degradation, supposing an exponential decay. The results are reported in the table below:<br />
<br />
<table class='data'><br />
<tr><br />
<td class='row'><br />
</td><br />
<td class='row'><br />
<b>t<sub>1/2</sub><sup>*</sup> [h]</b><br />
</td><td class='row'><br />
<b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b><br />
</td><br />
</tr><br />
<tr><td class='row'><b>M9<sub>pH 6</sub></b><br />
</td><br />
<td class='row'>32<br />
</td><br />
<td class='row'>0.022<br />
</td><br />
</tr><br />
<tr><td class='row'><b>M9<sub>pH 7</sub></b><br />
</td><br />
<td class='row'>8<br />
</td><br />
<td class='row'>0.087<br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
</p><br />
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<!--------------------------------><br />
<!-------------t9002--------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='t9002'></a><h2>Characterization of BBa_T9002 biosensor</h2><br />
As described in the <a href="https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002">modelling section</a>, BioBrick <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> is an HSL biosensor, which provides a non lineaer relationship between HSL input and S<sub>cell</sub> output. More precisely, the characteristic sigmoidal curve requires synthetic parameters for its accurate identification. These are the minimum and maximum values, the swtich point (i.e., the curve inflection point), and the upper and lower boundaries of linearity. This biosensor revealed greatly reliable, providing measurement repeatability and minimal experimental noise.<br><br><br />
<br />
<table width='50%' class='data'><tr><br />
<td class='row'><b>Minimum</b></td><br />
<td class='row'><b>Maximum</b></td><br />
<td class='row'><b>Switch point</b></td><br />
<td class='row'><b>Lower boundary of linearity</b></td><br />
<td class='row'><b>Upper boundary of linearity</b></td><br />
</tr><br />
<br />
<br />
<tr><td class='row'><b>Minimum</b></td><br />
<td class='row'><b>Maximum</b></td><br />
<td class='row'><b>Switch point</b></td><br />
<td class='row'><b>Lower boundary of linearity</b></td><br />
<td class='row'><b>Upper boundary of linearity</b></td><br />
</tr><br />
</table><br />
<br />
<br><br><br />
In order to determine the threshold sensitivity of T9002 biosensor, experiments were performed with several HSL inductions minimally interspaced in the region of low detectability. These allowed to reveal that the minimum detectable HSL concentration is ? nM. <br />
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</html><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Parts/CharacterizedTeam:UNIPV-Pavia/Parts/Characterized2011-09-17T23:11:53Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<html><br />
<a name='indice'></a><br />
<h2 class="art-postheader"><br />
Parts<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#new"><span class="tocnumber">1</span> <span class="toctext">New Parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#J101"><span class="tocnumber">1.1</span> <span class="toctext">J23101x series with different RBSs</span></a></li><br />
<li class="toclevel-2"><a href="#pTetAiiA"><span class="tocnumber">1.2</span> <span class="toctext">AiiA Expression cassette driven by aTc</span></a></li><br />
<li class="toclevel-2"><a href="#pTetLuxI"><span class="tocnumber">1.3</span> <span class="toctext">LuxI Expression cassette driven by aTc</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Existing"><span class="tocnumber">2</span> <span class="toctext">Characterization of existing parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#note"><span class="tocnumber">2.1</span> <span class="toctext">Notes on promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.2</span> <span class="toctext">RBSs from the community collection</span></a></li><br />
<li class="toclevel-2"><a href="#pLux"><span class="tocnumber">2.3</span> <span class="toctext">pLux - a 3OC6-HSL-in PoPs-out device</span></a></li><br />
<li class="toclevel-2"><a href="#pTet"><span class="tocnumber">2.4</span> <span class="toctext">pTet - BBa_R0040</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.5</span> <span class="toctext">AiiA - BBa_C0060</span></a></li><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.6</span> <span class="toctext">LuxI - BBa_C0061</span></a></li><br />
<li class="toclevel-2"><a href="#K999"><span class="tocnumber">2.7</span> <span class="toctext">pSB1C3 vector with mRFP between S and P for easy cloning</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Improvement"><span class="tocnumber">3</span> <span class="toctext">Sequence debugging</span></a></li><br />
</ul></td></tr></table><br />
</div><br />
<br />
<br />
<a name='new'></a><h1>New parts</h1><br />
<a name='J101'></a><h2>J23101x series</h2><br />
<br />
<ol type='1'><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> (wiki name: J101-E5 ) J101-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> (wiki name: J101-31 ) J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> (wiki name: J101-E7 ) J101-RBS32-mRFP-TT </li><br />
</ol><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- J23101x description --><br />
<!-- ----------------- --><br />
<p align='justify'><br />
BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in '<a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>data analysis</a>' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene. <br><br />
The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. <br>It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P.<br> <br />
This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts. <br><br />
Salis et al. [Nat Biotec, 2009] stated that<br />
<em> 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels'</em><br><br><br />
and again<br />
<em>'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.'</em> <br><br><br />
<br />
<br />
For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS.<br><br />
<br />
In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.<br />
<br />
<br><br><em><br />
<b>NB</b>: in the RPU computation, the J23101-RBS34-mRFP-TT construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency. </em><br />
<br />
<table align='center'><tr><td width='50%' ><br />
<table class='data'><br />
<tr><br />
<td class='row'><b>J23101 promoter with RBS</b><br />
</td><br />
<td class='row'><b>S<sub>cell</sub></b><br />
</td><br />
<td class='row'><b>R.P.U.s</b><br />
</td><br />
</tr><br />
<tr><td class='row'>RBS30<br />
</td><br />
<td class='row'>122 [13.23]</td><br />
<td class='row'>3.17</td><br />
</tr><br />
<tr><td class='row'>RBS31<br />
</td><br />
<td class='row'>2 [0.25]</td><br />
<td class='row'>0.05</td><br />
</tr><br />
<tr><td class='row'>RBS32<br />
</td><br />
<td class='row'>20 [1.7]</td><br />
<td class='row'>0.37</td><br />
</tr><br />
<tr><td class='row'>RBS34<br />
</td><br />
<td class='row'>50 [1.19]</td><br />
<td class='row'>1</td><br />
</tr><br />
</table></td><br />
<td width='50%' ><br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:UNIPV_RPU_J101.png" class="image"><img alt="RPUs of J23101 promoter with different RBSs" src="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="thumbimage" width="80%"></a></div></div><br />
</td><br />
</tr></table><br />
<br><br><br />
On the other hand, the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.<br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- pLuxAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetAiiA'></a><h2>AiiA expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> (wiki name: E24 ) pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> (wiki name: E25 ) pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- pTetAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetLuxI'></a><h2>LuxI expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> (wiki name: E13 ) pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> (wiki name: E14 ) pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> (wiki name: E15 ) pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<a name='Existing'></a><h1>Existing parts</h1><br />
<br />
<a name='note'></a><h2>Notes for promoter characterization</h2><br />
<p align='justify'><br />
Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection. <br />
<br><br><br />
The assembled RBSs are:<br />
<br><br><br />
<div align='center'><br />
<table class='data'><br />
<tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr><br />
<tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr><br />
<tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr><br />
<tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr><br />
<tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr><br />
</table></div><br />
<br><br />
For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.<br />
<br><br><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. <br />
RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. <br><br />
It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP). <br><br><br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element.<br />
Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.<br />
<br />
</p><br />
<br />
<p><br />
Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least sqares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs :<br />
<br><br />
<ol><ul><li><b><br />
RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity,<br />
</li><li><b><br />
RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity,<br />
</li><li><br />
<b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and it is representative of the position of linear region,<br />
</li><br />
<li><br />
<b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</li><br />
</ul></ol><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --RBS description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='rbs'></a><h2>RBSs</h2><br />
<p align='justify'><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of mRFP produced. <br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. <br />
The evaluation of RBS efficiency can be performed in a very intuitive fashion:<br><br />
<ul><br />
<li>1. select the RBSs you want to study, </li><br />
<li>2. assemble them in a Promoter - XX - Coding sequence circuit, </li></ul></ol><br />
<br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:Vettore_base.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d1/Vettore_base.jpg" class="thumbimage" width="50%"></a></div></div><br />
<br />
<ul><br />
<li>3. measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS. </li><br />
</ul></ol><br><br />
This simple measurement system allows the quantification of RBS efficiency depending on the whole measurement system (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in a complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments. <br><br />
To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.<br><br />
In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, pTet, pLux). Measuring the system output and evaluating the RBS efficiency. The results are summarized in the table below:<br><br><br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>pLux</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>pTet</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>J23101</sub></b><sup>**</sup></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>0.40</td><td class='row'>1.6814</td><td class='row'>3.17</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.01</td><td class='row'>ND</td><td class='row'>0.05</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.19</td><td class='row'>0.4193</td><td class='row'>0.37</td><td class='row'>0,3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<br />
<br><br />
On the other end, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (pTet-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below:<br><br><br />
<br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>mRFP</sub></b><sup>**</sup></td><br />
<td class='row'><b>eff<sub>AiiA</sub></b><sup>***</sup></td><br />
<td class='row'><b>eff<sub>LuxI</sub><sup>****</sup></b></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>1.72</td><td class='row'>0.53</td><td class='row'>0.45</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.03</td><td class='row'>0.83</td><td class='row'>0.028</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.37</td><td class='row'>0.50</td><td class='row'>N.D.</td><td class='row'>0.3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<p align='justify'><br />
<sup>*</sup> The RBS efficiency for inducible devices expressing mRFP was estimated as the ratio of the AUCs (Area under the curve) of the induction curve of the system with the studied RBS and the B0034 reference: AUC<sub>P, RBSx</sub>/AUC<sub>P, B0034</sub><br><br />
<sup>**</sup> The RBS efficiency for constitutive promoters expressing mRFP was computed as the ratio between S<sub>cell<sub>P, RBSx</sub></sub>/S<sub>cell<sub>P, B0034</sub></sub><br><br />
<sup>***</sup> The RBS efficiency for pTet promoter driving the expression of AiiA enzyme was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated for the measurement system pTet-RBSx-AiiA-TT. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#AiiA'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<sup>****</sup>The RBS efficiency for promoters driving the expression of LuxI was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated from the measurement systems pTet-RBSx-LuxI. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#LuxI'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<br />
<br><br />
</p><br />
<br><br><br />
The parts we used to characterize the RBSs are listed here:<br />
<ol><br />
<ul><li>mRFP expression with different promoters</li><br />
<ul><br />
<li>J23101</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> J101-RBS30-RFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> J101-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_J23101 "> BBa_J23101 </a> J101-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pTet</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pLux</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li><br />
</ul><br />
</ul><br />
<li>pTet driving the expression of different genes</li><br />
<ul><li>mRFP</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>AiiA</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> pTet-RBS34-AiiA-TT </li><br />
</ul><br />
<li>LuxI</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> pTet-RBS34-LuxI </li><br />
</ul></ul><br />
</ul></ol><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --pLux description- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<a name='pLux'></a><h2>pLux promoter</h2><br />
<p align='justify'><br />
<br />
<br />
</p><br />
<ol><ul><br />
<br />
<br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> (wiki name: E17 ) pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> (wiki name: E18 ) pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> (wiki name: E19 ) pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> (wiki name: E20 ) pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li></ul><br />
</ol><br />
<br />
<p align='justify'><br />
The estimated parameters for the Hill functions are summarized in the table below. For more details on parameter estimation, see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>model section</a>. <br />
</p><br />
<br />
<table class='data' width='100%'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Lux</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">438 [10]</td><br />
<td class="row">0.05 [>100]</td><br />
<td class="row">2 [47]</td><br />
<td class="row">1.88 [27]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">9.8 [7]</td><br />
<td class="row">0.11 [57]</td><br />
<td class="row">1.2 [29]</td><br />
<td class="row">1.5 [26]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">206 [3]</td><br />
<td class="row">0 [>>100]</td><br />
<td class="row">1.36 [10]</td><br />
<td class="row">1.87 [9]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">1105 [6]</td><br />
<td class="row">0.02 [>100]</td><br />
<td class="row">1.33 [19]</td><br />
<td class="row">2.34 [18]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br />
<br />
<p align='justify'><br />
From this table, it is evident that, whilst &alpha;<sub>pLux</sub> assumes significantly different values for different RBSs, &eta;<sub>pLux</sub> and k<sub>pLux</sub> assume very similar values. This result shows that RBS variation modulates the amplitude of Hill function, not affecting the shape of the curve. The four induction curves result to be the same Hill function modulated in amplitude by a parameter, that is the estimated RBS efficiency for this measurement system.<br><br />
These results are quite encouraging, because suggest that, given the non-modular behavior of RBS dpending on the encoded gene, the RBS has a modular behaviour respect to the promoter. <br />
<br />
</p><br />
<br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>4.28</td><td class='row'>0.20</td><td class='row'>1.08</td><td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>4.93</td><td class='row'>0.55</td><td class='row'>0.25</td><td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>9.49</td><td class='row'>0.02</td><td class='row'>0.47</td><td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>21.53</td><td class='row'>0.51</td><td class='row'>0.53</td><td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<p align='justify'><br />
<br />
These operative parameters provide useful information on the behavior of this 3OC6-HSL inducible device. RPU<sub>max</sub> assumes very different values, showing that the modulation in amplitude of the Hill can't be explained by a linear dependance on the RBS efficiency (in this case, in fact, the same RPUs should be observed for every RBS, since the standard reference used for RPUs computation)<br />
<br />
</p><br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- -pTet description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='pTet'></a><h2>pTet promoter</h2><br />
<br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> (wiki name: E21 ) pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> (wiki name: E22 ) pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT<br />
</li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT <br />
</li></ul><br />
</ul><br />
</ol><br />
<p align='justify'><br />
<br />
The protocols for the characterization of pTet promoter are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#pTet_protocol'>pTet measurement section</a>. <br><br />
This promoter is widely studied and characterized usually using the strong RBS BBa_B0034. Here we have characterized its transcriptional strength as a function of aTc induction (ng/ul) for different RBSs. Four different induction curves were obtained and are reported in figure:<br><br><br />
<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
The data collected from the mRFP measurement systems were processed as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'> data analysis section</a>. The induction curves were obtained by fitting a Hill function as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>modelling section</a> and the estimated <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>parameters</a> for pTet are reported in the pictures and in table below. </p><br />
<p align='justify'><br />
The estimated parameters of the Hill curves described in the figures are summarized in the table below:<br />
</p><br />
<br><br><br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">230.67 [3.7]</td><br />
<td class="row">0.028 [91.61]</td><br />
<td class="row">4.61 [23.73]</td><br />
<td class="row">8.75 [4.16]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">55.77 [12]</td><br />
<td class="row">1.53E-11 [>>100]</td><br />
<td class="row">4.98 [57.62]</td><br />
<td class="row">7.26 [14.98]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">120 [5.95]</td><br />
<td class="row">0.085 [40.6]</td><br />
<td class="row">24.85 [47.6]</td><br />
<td class="row">9 [5.43]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br><br><br />
<br />
<p align='justify'><br />
The measurement system pTet-B0031-mRFP-TT couldn't be assayed because its fluorescence output is under the detectability threshold of our measurement instrument. For this reason, the parameters of the corresponding Hill curve couldn't be estimated and are reported as 'Not Determined' ND. <br />
</p><br />
<br />
<p align='justify'><br />
&alpha; parameter (representing the maximum trascriptional rate in the studied range of induction) varies as expected with the RBS variation and also the &delta; and &eta; parameters are quite different among the RBS variations.<br><br />
The K<sub>pTet</sub> parameter is quite constant among the RBS variations, thus suggesting that in this case the RBS variation doesn't substantially affect the switch point of the Hill curve, even if the amplitude and the maximum slope are not quite maintained (for the &eta; parameter, maybe fitting problems). <br />
<br />
</p><br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [ng/&mu;]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [ng/&mu;]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>1.53</td><td class='row'>~0</td><td class='row'>7.95</td><td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>3.16</td><td class='row'>~0</td><td class='row'>6.7</td><td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>2.73</td><td class='row'>0.23</td><td class='row'>8.96</td><td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
From these parameters, it is evident that whilst the switch-point is almost maintained for all the RBSs, the linear boundaries are similar for RBS30 and RBS32 but for RBS34 are moved on the right of one order of magnitude. <br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --AiiA description-- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<br />
<a name='AiiA'></a><h2>AiiA gene - BBa_C0060</h2><br />
<br />
<p align='justify'><br />
The activity of AiiA enzyme has been evaluated by testing the measurement systems <a href='<a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a>. <br />
The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
</p><br />
<br />
<br />
<br><br><br />
dAiiA/dt=-&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>AiiA</sub>*AiiA<br><br><br />
d[HSL]/dt=N*K<sub>cat</sub>*AiiA*HSL/(K<sub>M, AiiA</sub>+AiiA))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
<p align='justify'><br />
<br />
Since the measurement systems are only assayed in the exponential growth phase, the third equation can be modified as follows:<br />
</p><br />
dN/dt=N*&mu;<br />
<br />
<p align='justify'><br />
because N&lt;N<sub>max</sub>. <br><br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.003152</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
<br />
<br />
<br />
<p align='justify'><br />
<br />
The parameters K<sub>cat</sub>, K<sub>M,AiiA</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of AiiA are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>cat</sub></b></td><br />
<td class='row'><b>K<sub>M, AiiA</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'></td><br />
<td class='row'></td><br />
<td class='row'></td><br />
<td class='row'></td><br />
<td class='row'></td><br />
<td class='row'></td><br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
<br />
<br />
</p><br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --LuxI description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='LuxI'></a><h2>LuxI gene - BBa_C0061</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#t9002'>modeling section</a>). <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
<br />
<br><br><br />
dLuxI/dt=&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>LuxI</sub>*LuxI<br><br><br />
d[HSL]/dt=N*V<sub>max</sub>*1/(1+(K<sub>M, LuxI</sub>/LuxI))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
<br />
<p align='justify'><br />
<br />
Since the measurement systems are only assayed in the exponential growth phase, the third equation can be modified as follows:<br />
</p><br />
dN/dt=N*&mu;<br />
<br />
<p align='justify'><br />
because N&lt;N<sub>max</sub>. <br><br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:</p><br />
<br />
<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.003152</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
<br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, K<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>pTet-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of LuxI are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>M, LuxI</sub></b></td><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>1.5*10<sup>4</sup></td><br />
<td class='row'>3.63*10<sup>-9</sup></td><br />
<td class='row'>247</td><br />
<td class='row'>15</td><br />
<td class='row'>ND</td><br />
<td class='row'>553</td><br />
<br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
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<!-- ----------------- --><br />
<!-- ---pSB1C3-mRFP-- --><br />
<!-- ----------------- --><br />
<br />
<a name='K999'></a><h2>pSB1C3 plasmid with mRFP between S and P bearing pTet (easy-to-clone)</h2><br />
This vector was designed and realized in order to facilitate the cloning of coding sequences downstream of the strong promoter pTet. This vector was assembled by ligating S-P excided mRFP coding sequence from BBa_J61002 and ligating it in BBa_R0040 cut with S and P. Thus, the resulting vector contains mRFP between S and P. pTet cn be easily excided (E-P) and moved in the desired vector (E-P) and then the desired coding sequence can be easily assembled by digesting S-P the vector and X-P the coding sequence, thus obtaining a final part that is standard10-compatible. <br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<a name='Improvement'></a><h1>Existing parts: sequence debugging</h1><br />
<ol><ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516021 "> BBa_K516021 </a> (wiki name: E10 ) RBS31-AiiA-TT Rebuilt existing part from BBa_I13914 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516022 "> BBa_K516022 </a> (wiki name: E11 ) RBS32-AiiA-TT Rebuilt existing part from BBa_I13912 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516030 "> BBa_K516030 </a> (wiki name: E5 ) RBS30-mRFP-TT Rebuilt existing part from BBa_S04180 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516032 "> BBa_K516032 </a> (wiki name: E7 ) RBS32-mRFP-TT Rebuilt existing part from BBa_ J133000 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI Rebuilt existing part from BBa_S03623 (DNA available, only 2008 kit, inconsistent) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT Rebuilt existing part from BBa_ J22071 (2008 only, Bad sequencing) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT Rebuilt existing part from BBa_K077047 (Part deleted) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT Rebuilt existing part from BBa_I20252 (DNA planning) </li></ul></ol><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Project/ResultsTeam:UNIPV-Pavia/Project/Results2011-09-17T23:08:03Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<html><br />
<h2 class="art-postheader"><br />
Results<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
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<!-----------MENU-----------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='indice'></a><br />
<br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#assembly"><span class="tocnumber">1</span> <span class="toctext">Part assembly</span></a> <br />
<li class="toclevel-1"><a href="#characterization"><span class="tocnumber">1</span> <span class="toctext">Characterization of basic modules</span></a> <br />
<ul><br />
<li class="toclevel-2"><a href="#promoters"><span class="tocnumber">2.1</span> <span class="toctext">Promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#enzymes"><span class="tocnumber">2.2</span> <span class="toctext">Characterization of the activity of the enzymes AiiA and LuxI</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.3</span> <span class="toctext">Characterization of RBS efficiency</span></a></li><br />
</ul><br />
<li class="toclevel-1"><a href="#growth"><span class="tocnumber">3</span> <span class="toctext">Identification of bacterial growth parameters</span></a></li><br />
<li class="toclevel-1"><a href="#HSL"><span class="tocnumber">4</span> <span class="toctext">Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</span></a></li><br />
<li class="toclevel-1"><a href="#t9002"><span class="tocnumber">5</span> <span class="toctext">Characterization of BBa_T9002 biosensor</span></a></li><br />
</ul></td></tr></table><br />
</div><br />
<br />
<br />
<br><br />
<br />
<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C. For the cloning of the parts, <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#TOP10'><em>E. coli</em> TOP10</a> was used. <br />
</em><br />
<br><br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-----------ASSEMBLY-------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<br />
<a name='assembly'></a><h1>Parts assembly</h1><br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-------CHARACTERIZATION---------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='characterization'></a><h1>Characterization of basic modules</h1><br />
<br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------pTet and pLux-----------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<a name='promoters'></a><h2>Characterization of promoters pTet and pLux</h2><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------AiiA and LuxI-----------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='enzymes'></a><h2>Characterization of enzymes AiiA and LuxI</h2><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------RBS---------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='rbs'></a><h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-----------growth---------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<a name='growth'></a><h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
The bacterial growth curve has been modelled as a logistic curve and is represented by the following equation:<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br><br />
N(0)=n<sub>0</sub><br />
<br><br><br />
<br />
where &mu; represents the growth rate of the cells (<em>E. coli</em> MGZ1 in M9 supplemented medium) and N<sub>max</sub> represents the maximum number of cells in the well. For a detailed description of the parameters, see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>modelling section</a>. For details on parameters identification, see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#N'>identification section.</a> <br />
The growth curves in all the performed experiments are measured in O.D.<sub>600</sub>. Since the <em>N</em> species in the model is expressed in <em>cell number</em>, a conversion factor has been estimated. The conversion factor <b>K<sub>O.D.toC.F.U.</sub></b> has been estimated as follows.<br />
<ol><ul><br />
<li>Two cultures C1 and C2 (MGZ1 cells) were grown in 1ml M9 medium till saturation (ON liquid culture, 37°C, 220 rpm).</li><br />
<li>Next morning, both C1 and C2 were diluted in M9 medium with a final volume of 1ml with the following dilution factors:<br />
<ul><li>1:1</li><li>1:10</li><li>1:100</li><li>1:1000</li><br />
</ul> in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm.<br />
</li><br />
<li>After 1 hour, O.D.<sub>600</sub> was measured using TECAN microplate reader (don't forget to measure a M9 sample for blanking!)<br><br />
<em>NB: from now on, cultures must be placed in ice to stop cell growth.</em></li><br />
<li>At the same time, proper dilution of the cultures were plated on LB agar plates.<br><br />
<em>NB: All the dilutions are performed moving 100 &mu;l of culture in previously ice-chilled 900 &mu;l fresch M9. 100 &mu;l of the final dilution are plated (It still represents a 1:10 dilution!)</em></li><br />
<li>Plates were grown overnight and next morning C.F.U. were counted.</li><br />
<li>C.F.U. values were corrected by the dilution factor and a linear regression (N vs O.D.<sub>600</sub>) was performed in order to evaluate the conversion factor <b>K<sub>O.D.toC.F.U.</sub></b>. </li><br />
<li><b>K<sub>O.D.toC.F.U.</sub></b> was used as conversion factor to multiply the O.D.<sub>600</sub> value of saturation in the growth curves (~0,5). </li><br />
<br />
</ul></ol><br />
<br />
<p align='center'><br />
The results are summarized in the table and in the figure below.<br />
</p><br />
<table class='data'><tr><td class='row'><b> Culture </b></td><td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><td class='row'><b> C.F.U. </b></td><td class='row'><b> Dilution Factor (10^) </b></td><td class='row'><b> N </b></td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,367950002 </td><td class='row'> 0,758004416 </td><td class='row'> 990 </td><td class='row'> 5 </td><td class='row'> 990000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,044649998 </td><td class='row'> 0,091982322 </td><td class='row'> 141 </td><td class='row'> 6 </td><td class='row'> 1410000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,004799999 </td><td class='row'> 0,009888356 </td><td class='row'> 136 </td><td class='row'> 5 </td><td class='row'> 136000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,000549998 </td><td class='row'> 0,001133037 </td><td class='row'> 20 </td><td class='row'> 6 </td><td class='row'> 200000000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,54840003 </td><td class='row'> 1,129744917 </td><td class='row'> 165 </td><td class='row'> 4 </td><td class='row'> 16500000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,058200002 </td><td class='row'> 0,119896339 </td><td class='row'> 23 </td><td class='row'> 5 </td><td class='row'> 23000000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,008700002 </td><td class='row'> 0,017922652 </td><td class='row'> 251 </td><td class='row'> 3 </td><td class='row'> 2510000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,000100002 </td><td class='row'> 0,000206011 </td><td class='row'> 24 </td><td class='row'> 4 </td><td class='row'> 2400000 </td> </tr><br />
</table><br><br />
</p><br />
<br />
<table align='center><tr><td align='center'><br />
<div style='text-align:justify'><div class="thumbinner" style="width: 90%;"><img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"></a></div></div><br />
</td></tr></table><br />
<br><br />
The estimation of &mu; parameter was performed by determining the slope of the logarithmic curve of O.D.<sub>600</sub> in exponential phase. Exponential phase was determined by visual inspection as the linear phase of the logarithmic curve of O.D.<sub>600</sub>. <br />
<br><br><br />
The estimated parameters are summarized in the table below:<br />
<br><br><br />
<br />
<table width='50%' class='data'><tr><br />
<td class='row'><b>N<sub>max</sub> [cell number]</b></td><br />
<td class='row'><b>&mu; [min<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><td class='row'>1*10<sup>9</sup></td><br />
<td class='row'>0.004925</td><br />
</tr><br />
</table><br />
<br />
The reported value of &mu; corresponds to a doubling time of 142 minutes.<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------HSL---------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<a name='HSL'></a><h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'><br />
In order to estimate the spontaneous degradation rate of HSL in M9 medium and in a culture of MGZ1 cells as a function of pH, two simple tests have been performed.<br><br />
Two different M9 media were prepared, one with the nominal pH (7.0) and one with pH=6.0.<br><br />
These media, now named respectively M9<sub>pH 7</sub> and M9<sub>pH 6</sub>, were added with a known concentration of HSL (100 nM) and then incubated at 37°C, 220 rpm (NB: the media were not infected with any culture but the standard growth conditions were reproduced). The amount of HSL present in the medium was assayed through the BBa_T9002 biosensor at 4 time points: <br><br />
<ul><br />
<li>t=0 h;</li><br />
<li>t=1 h;</li><br />
<li>t=2 h;</li><br />
<li>t=4 h;</li><br />
</ul><br />
The obtained time series of HSL amounts were processed to evaluate the time constant governing the dynamic of HSL degradation, supposing an exponential decay. The results are reported in the table below:<br />
<br />
<table class='data'><br />
<tr><br />
<td class='row'><br />
<b>t<sub>1/2</sub><sup>*</sup> [h]</b><br />
<b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
</p><br />
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<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-------------t9002--------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='t9002'></a><h2>Characterization of BBa_T9002 biosensor</h2><br />
As described in the <a href="https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002">modelling section</a>, BioBrick <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> is an HSL biosensor, which provides a non lineaer relationship between HSL input and S<sub>cell</sub> output. More precisely, the characteristic sigmoidal curve requires synthetic parameters for its accurate identification. These are the minimum and maximum values, the swtich point (i.e., the curve inflection point), and the upper and lower boundaries of linearity. This biosensor revealed greatly reliable, providing measurement repeatability and minimal experimental noise.<br><br><br />
<br />
<table width='50%' class='data'><tr><br />
<td class='row'><b>Minimum</b></td><br />
<td class='row'><b>Maximum</b></td><br />
<td class='row'><b>Switch point</b></td><br />
<td class='row'><b>Lower boundary of linearity</b></td><br />
<td class='row'><b>Upper boundary of linearity</b></td><br />
</tr><br />
<br />
<br />
<tr><td class='row'><b>Minimum</b></td><br />
<td class='row'><b>Maximum</b></td><br />
<td class='row'><b>Switch point</b></td><br />
<td class='row'><b>Lower boundary of linearity</b></td><br />
<td class='row'><b>Upper boundary of linearity</b></td><br />
</tr><br />
</table><br />
<br />
<br><br><br />
In order to determine the threshold sensitivity of T9002 biosensor, experiments were performed with several HSL inductions minimally interspaced in the region of low detectability. These allowed to reveal that the minimum detectable HSL concentration is ? nM. <br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Project/ResultsTeam:UNIPV-Pavia/Project/Results2011-09-17T23:06:30Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<html><br />
<h2 class="art-postheader"><br />
Results<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
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<a name='indice'></a><br />
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#assembly"><span class="tocnumber">1</span> <span class="toctext">Part assembly</span></a> <br />
<li class="toclevel-1"><a href="#characterization"><span class="tocnumber">1</span> <span class="toctext">Characterization of basic modules</span></a> <br />
<ul><br />
<li class="toclevel-2"><a href="#promoters"><span class="tocnumber">2.1</span> <span class="toctext">Promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#enzymes"><span class="tocnumber">2.2</span> <span class="toctext">Characterization of the activity of the enzymes AiiA and LuxI</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.3</span> <span class="toctext">Characterization of RBS efficiency</span></a></li><br />
</ul><br />
<li class="toclevel-1"><a href="#growth"><span class="tocnumber">3</span> <span class="toctext">Identification of bacterial growth parameters</span></a></li><br />
<li class="toclevel-1"><a href="#HSL"><span class="tocnumber">4</span> <span class="toctext">Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</span></a></li><br />
<li class="toclevel-1"><a href="#t9002"><span class="tocnumber">5</span> <span class="toctext">Characterization of BBa_T9002 biosensor</span></a></li><br />
</ul></td></tr></table><br />
</div><br />
<br />
<br />
<br><br />
<br />
<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C. For the cloning of the parts, <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#TOP10'><em>E. coli</em> TOP10</a> was used. <br />
</em><br />
<br><br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-----------ASSEMBLY-------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<br />
<a name='assembly'></a><h1>Parts assembly</h1><br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-------CHARACTERIZATION---------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='characterization'></a><h1>Characterization of basic modules</h1><br />
<br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------pTet and pLux-----------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<a name='promoters'></a><h2>Characterization of promoters pTet and pLux</h2><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------AiiA and LuxI-----------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='enzymes'></a><h2>Characterization of enzymes AiiA and LuxI</h2><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------RBS---------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='rbs'></a><h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-----------growth---------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<a name='growth'></a><h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
The bacterial growth curve has been modelled as a logistic curve and is represented by the following equation:<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br><br />
N(0)=n<sub>0</sub><br />
<br><br><br />
<br />
where &mu; represents the growth rate of the cells (<em>E. coli</em> MGZ1 in M9 supplemented medium) and N<sub>max</sub> represents the maximum number of cells in the well. For a detailed description of the parameters, see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>modelling section</a>. For details on parameters identification, see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#N'>identification section.</a> <br />
The growth curves in all the performed experiments are measured in O.D.<sub>600</sub>. Since the <em>N</em> species in the model is expressed in <em>cell number</em>, a conversion factor has been estimated. The conversion factor <b>K<sub>O.D.toC.F.U.</sub></b> has been estimated as follows.<br />
<ol><ul><br />
<li>Two cultures C1 and C2 (MGZ1 cells) were grown in 1ml M9 medium till saturation (ON liquid culture, 37°C, 220 rpm).</li><br />
<li>Next morning, both C1 and C2 were diluted in M9 medium with a final volume of 1ml with the following dilution factors:<br />
<ul><li>1:1</li><li>1:10</li><li>1:100</li><li>1:1000</li><br />
</ul> in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm.<br />
</li><br />
<li>After 1 hour, O.D.<sub>600</sub> was measured using TECAN microplate reader (don't forget to measure a M9 sample for blanking!)<br><br />
<em>NB: from now on, cultures must be placed in ice to stop cell growth.</em></li><br />
<li>At the same time, proper dilution of the cultures were plated on LB agar plates.<br><br />
<em>NB: All the dilutions are performed moving 100 &mu;l of culture in previously ice-chilled 900 &mu;l fresch M9. 100 &mu;l of the final dilution are plated (It still represents a 1:10 dilution!)</em></li><br />
<li>Plates were grown overnight and next morning C.F.U. were counted.</li><br />
<li>C.F.U. values were corrected by the dilution factor and a linear regression (N vs O.D.<sub>600</sub>) was performed in order to evaluate the conversion factor <b>K<sub>O.D.toC.F.U.</sub></b>. </li><br />
<li><b>K<sub>O.D.toC.F.U.</sub></b> was used as conversion factor to multiply the O.D.<sub>600</sub> value of saturation in the growth curves (~0,5). </li><br />
<br />
</ul></ol><br />
<br />
<p align='center'><br />
The results are summarized in the table and in the figure below.<br />
</p><br />
<table class='data'><tr><td class='row'><b> Culture </b></td><td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><td class='row'><b> C.F.U. </b></td><td class='row'><b> Dilution Factor (10^) </b></td><td class='row'><b> N </b></td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,367950002 </td><td class='row'> 0,758004416 </td><td class='row'> 990 </td><td class='row'> 5 </td><td class='row'> 990000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,044649998 </td><td class='row'> 0,091982322 </td><td class='row'> 141 </td><td class='row'> 6 </td><td class='row'> 1410000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,004799999 </td><td class='row'> 0,009888356 </td><td class='row'> 136 </td><td class='row'> 5 </td><td class='row'> 136000000 </td> </tr><br />
<tr><td class='row'> C1 </td><td class='row'> 0,000549998 </td><td class='row'> 0,001133037 </td><td class='row'> 20 </td><td class='row'> 6 </td><td class='row'> 200000000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,54840003 </td><td class='row'> 1,129744917 </td><td class='row'> 165 </td><td class='row'> 4 </td><td class='row'> 16500000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,058200002 </td><td class='row'> 0,119896339 </td><td class='row'> 23 </td><td class='row'> 5 </td><td class='row'> 23000000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,008700002 </td><td class='row'> 0,017922652 </td><td class='row'> 251 </td><td class='row'> 3 </td><td class='row'> 2510000 </td> </tr><br />
<tr><td class='row'> C2 </td><td class='row'> 0,000100002 </td><td class='row'> 0,000206011 </td><td class='row'> 24 </td><td class='row'> 4 </td><td class='row'> 2400000 </td> </tr><br />
</table><br><br />
</p><br />
<br />
<table align='center><tr><td align='center'><br />
<div style='text-align:justify'><div class="thumbinner" style="width: 90%;"><img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"></a></div></div><br />
</td></tr></table><br />
<br><br />
The estimation of &mu; parameter was performed by determining the slope of the logarithmic curve of O.D.<sub>600</sub> in exponential phase. Exponential phase was determined by visual inspection as the linear phase of the logarithmic curve of O.D.<sub>600</sub>. <br />
<br><br><br />
The estimated parameters are summarized in the table below:<br />
<br><br><br />
<br />
<table width='50%' class='data'><tr><br />
<td class='row'><b>N<sub>max</sub> [cell number]</b></td><br />
<td class='row'><b>&mu; [min<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><td class='row'>1*10<sup>9</sup></td><br />
<td class='row'>0.004925</td><br />
</tr><br />
</table><br />
<br />
The reported value of &mu; corresponds to a doubling time of 142 minutes.<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------HSL---------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<br />
<a name='HSL'></a><h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'><br />
In order to estimate the spontaneous degradation rate of HSL in M9 medium and in a culture of MGZ1 cells as a function of pH, two simple tests have been performed.<br><br />
Two different M9 media were prepared, one with the nominal pH (7.0) and one with pH=6.0.<br><br />
These media, now named respectively M9<sub>pH 7</sub> and M9<sub>pH 6</sub>, were added with a known concentration of HSL (100 nM) and then incubated at 37°C, 220 rpm (NB: the media were not infected with any culture but the standard growth conditions were reproduced). The amount of HSL present in the medium was assayed through the BBa_T9002 biosensor at 4 time points: <br><br />
<ul><br />
<li>t=0 h;</li><br />
<li>t=1 h;</li><br />
<li>t=2 h;</li><br />
<li>t=4 h;</li><br />
</ul><br />
The obtained time series of HSL amounts were processed to evaluate the time constant governing the dynamic of HSL degradation, supposing an exponential decay. The results are reported in the table below:<br />
<br />
<table class='data'><br />
<tr><br />
<td class='row'><br />
<b>t<sub>1/2</sub><sup>*</sup> [h]</b><br />
<b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b><br />
</td><br />
</tr><br />
<br />
<br />
<br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!-------------t9002--------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
<br />
<a name='t9002'></a><h2>Characterization of BBa_T9002 biosensor</h2><br />
As described in the <a href="https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#introduction_to_T9002">modelling section</a>, BioBrick <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> is an HSL biosensor, which provides a non lineaer relationship between HSL input and S<sub>cell</sub> output. More precisely, the characteristic sigmoidal curve requires synthetic parameters for its accurate identification. These are the minimum and maximum values, the swtich point (i.e., the curve inflection point), and the upper and lower boundaries of linearity. This biosensor revealed greatly reliable, providing measurement repeatability and minimal experimental noise.<br><br><br />
<br />
<table width='50%' class='data'><tr><br />
<td class='row'><b>Minimum</b></td><br />
<td class='row'><b>Maximum</b></td><br />
<td class='row'><b>Switch point</b></td><br />
<td class='row'><b>Lower boundary of linearity</b></td><br />
<td class='row'><b>Upper boundary of linearity</b></td><br />
</tr><br />
<br />
<br />
<tr><td class='row'><b>Minimum</b></td><br />
<td class='row'><b>Maximum</b></td><br />
<td class='row'><b>Switch point</b></td><br />
<td class='row'><b>Lower boundary of linearity</b></td><br />
<td class='row'><b>Upper boundary of linearity</b></td><br />
</tr><br />
</table><br />
<br />
<br><br><br />
In order to determine the threshold sensitivity of T9002 biosensor, experiments were performed with several HSL inductions minimally interspaced in the region of low detectability. These allowed to reveal that the minimum detectable HSL concentration is ? nM. <br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
</html><br />
<br />
{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Parts/CharacterizedTeam:UNIPV-Pavia/Parts/Characterized2011-09-17T22:41:45Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<html><br />
<a name='indice'></a><br />
<h2 class="art-postheader"><br />
Parts<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#new"><span class="tocnumber">1</span> <span class="toctext">New Parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#J101"><span class="tocnumber">1.1</span> <span class="toctext">J23101x series with different RBSs</span></a></li><br />
<li class="toclevel-2"><a href="#pTetAiiA"><span class="tocnumber">1.2</span> <span class="toctext">AiiA Expression cassette driven by aTc</span></a></li><br />
<li class="toclevel-2"><a href="#pTetLuxI"><span class="tocnumber">1.3</span> <span class="toctext">LuxI Expression cassette driven by aTc</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Existing"><span class="tocnumber">2</span> <span class="toctext">Characterization of existing parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#note"><span class="tocnumber">2.1</span> <span class="toctext">Notes on promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.2</span> <span class="toctext">RBSs from the community collection</span></a></li><br />
<li class="toclevel-2"><a href="#pLux"><span class="tocnumber">2.3</span> <span class="toctext">pLux - a 3OC6-HSL-in PoPs-out device</span></a></li><br />
<li class="toclevel-2"><a href="#pTet"><span class="tocnumber">2.4</span> <span class="toctext">pTet - BBa_R0040</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.5</span> <span class="toctext">AiiA - BBa_C0060</span></a></li><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.6</span> <span class="toctext">LuxI - BBa_C0061</span></a></li><br />
<li class="toclevel-2"><a href="#K999"><span class="tocnumber">2.7</span> <span class="toctext">pSB1C3 vector with mRFP between S and P for easy cloning</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Improvement"><span class="tocnumber">3</span> <span class="toctext">Sequence debugging</span></a></li><br />
</ul></td></tr></table><br />
</div><br />
<br />
<br />
<a name='new'></a><h1>New parts</h1><br />
<a name='J101'></a><h2>J23101x series</h2><br />
<br />
<ol type='1'><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> (wiki name: J101-E5 ) J101-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> (wiki name: J101-31 ) J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> (wiki name: J101-E7 ) J101-RBS32-mRFP-TT </li><br />
</ol><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- J23101x description --><br />
<!-- ----------------- --><br />
<p align='justify'><br />
BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in '<a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>data analysis</a>' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene. <br><br />
The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. <br>It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P.<br> <br />
This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts. <br><br />
Salis et al. [Nat Biotec, 2009] stated that<br />
<em> 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels'</em><br><br><br />
and again<br />
<em>'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.'</em> <br><br><br />
<br />
<br />
For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS.<br><br />
<br />
In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.<br />
<br />
<br><br><em><br />
<b>NB</b>: in the RPU computation, the J23101-RBS34-mRFP-TT construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency. </em><br />
<br />
<table align='center'><tr><td width='50%' ><br />
<table class='data'><br />
<tr><br />
<td class='row'><b>J23101 promoter with RBS</b><br />
</td><br />
<td class='row'><b>S<sub>cell</sub></b><br />
</td><br />
<td class='row'><b>R.P.U.s</b><br />
</td><br />
</tr><br />
<tr><td class='row'>RBS30<br />
</td><br />
<td class='row'>122 [13.23]</td><br />
<td class='row'>3.17</td><br />
</tr><br />
<tr><td class='row'>RBS31<br />
</td><br />
<td class='row'>2 [0.25]</td><br />
<td class='row'>0.05</td><br />
</tr><br />
<tr><td class='row'>RBS32<br />
</td><br />
<td class='row'>20 [1.7]</td><br />
<td class='row'>0.37</td><br />
</tr><br />
<tr><td class='row'>RBS34<br />
</td><br />
<td class='row'>50 [1.19]</td><br />
<td class='row'>1</td><br />
</tr><br />
</table></td><br />
<td width='50%' ><br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:UNIPV_RPU_J101.png" class="image"><img alt="RPUs of J23101 promoter with different RBSs" src="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="thumbimage" width="80%"></a></div></div><br />
</td><br />
</tr></table><br />
<br><br><br />
On the other hand, the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.<br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- pLuxAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetAiiA'></a><h2>AiiA expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> (wiki name: E24 ) pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> (wiki name: E25 ) pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- pTetAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetLuxI'></a><h2>LuxI expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> (wiki name: E13 ) pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> (wiki name: E14 ) pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> (wiki name: E15 ) pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<a name='Existing'></a><h1>Existing parts</h1><br />
<br />
<a name='note'></a><h2>Notes for promoter characterization</h2><br />
<p align='justify'><br />
Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection. <br />
<br><br><br />
The assembled RBSs are:<br />
<br><br><br />
<div align='center'><br />
<table class='data'><br />
<tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr><br />
<tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr><br />
<tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr><br />
<tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr><br />
<tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr><br />
</table></div><br />
<br><br />
For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.<br />
<br><br><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. <br />
RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. <br><br />
It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP). <br><br><br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element.<br />
Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.<br />
<br />
</p><br />
<br />
<p><br />
Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least sqares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs :<br />
<br><br />
<ol><ul><li><b><br />
RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity,<br />
</li><li><b><br />
RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity,<br />
</li><li><br />
<b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and it is representative of the position of linear region,<br />
</li><br />
<li><br />
<b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</li><br />
</ul></ol><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --RBS description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='rbs'></a><h2>RBSs</h2><br />
<p align='justify'><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of mRFP produced. <br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. <br />
The evaluation of RBS efficiency can be performed in a very intuitive fashion:<br><br />
<ul><br />
<li>1. select the RBSs you want to study, </li><br />
<li>2. assemble them in a Promoter - XX - Coding sequence circuit, </li></ul></ol><br />
<br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:Vettore_base.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d1/Vettore_base.jpg" class="thumbimage" width="50%"></a></div></div><br />
<br />
<ul><br />
<li>3. measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS. </li><br />
</ul></ol><br><br />
This simple measurement system allows the quantification of RBS efficiency depending on the whole measurement system (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in a complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments. <br><br />
To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.<br><br />
In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, pTet, pLux). Measuring the system output and evaluating the RBS efficiency. The results are summarized in the table below:<br><br><br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>pLux</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>pTet</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>J23101</sub></b><sup>**</sup></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>0.40</td><td class='row'>1.6814</td><td class='row'>3.17</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.01</td><td class='row'>ND</td><td class='row'>0.05</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.19</td><td class='row'>0.4193</td><td class='row'>0.37</td><td class='row'>0,3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<br />
<br><br />
On the other end, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (pTet-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below:<br><br><br />
<br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>mRFP</sub></b><sup>**</sup></td><br />
<td class='row'><b>eff<sub>AiiA</sub></b><sup>***</sup></td><br />
<td class='row'><b>eff<sub>LuxI</sub><sup>****</sup></b></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>1.72</td><td class='row'>0.53</td><td class='row'>0.18</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.03</td><td class='row'>0.83</td><td class='row'>0.001</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.37</td><td class='row'>0.50</td><td class='row'>N.D.</td><td class='row'>0.3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<p align='justify'><br />
<sup>*</sup> The RBS efficiency for inducible devices expressing mRFP was estimated as the ratio of the AUCs (Area under the curve) of the induction curve of the system with the studied RBS and the B0034 reference: AUC<sub>P, RBSx</sub>/AUC<sub>P, B0034</sub><br><br />
<sup>**</sup> The RBS efficiency for constitutive promoters expressing mRFP was computed as the ratio between S<sub>cell<sub>P, RBSx</sub></sub>/S<sub>cell<sub>P, B0034</sub></sub><br><br />
<sup>***</sup> The RBS efficiency for pTet promoter driving the expression of AiiA enzyme was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated for the measurement system pTet-RBSx-AiiA-TT. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#AiiA'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<sup>****</sup>The RBS efficiency for promoters driving the expression of LuxI was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated from the measurement systems pTet-RBSx-LuxI. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#LuxI'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<br />
<br><br />
</p><br />
<br><br><br />
The parts we used to characterize the RBSs are listed here:<br />
<ol><br />
<ul><li>mRFP expression with different promoters</li><br />
<ul><br />
<li>J23101</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> J101-RBS30-RFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> J101-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_J23101 "> BBa_J23101 </a> J101-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pTet</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pLux</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li><br />
</ul><br />
</ul><br />
<li>pTet driving the expression of different genes</li><br />
<ul><li>mRFP</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>AiiA</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> pTet-RBS34-AiiA-TT </li><br />
</ul><br />
<li>LuxI</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> pTet-RBS34-LuxI </li><br />
</ul></ul><br />
</ul></ol><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --pLux description- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<a name='pLux'></a><h2>pLux promoter</h2><br />
<p align='justify'><br />
<br />
<br />
</p><br />
<ol><ul><br />
<br />
<br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> (wiki name: E17 ) pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> (wiki name: E18 ) pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> (wiki name: E19 ) pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> (wiki name: E20 ) pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li></ul><br />
</ol><br />
<br />
<p align='justify'><br />
The estimated parameters for the Hill functions are summarized in the table below. For more details on parameter estimation, see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>model section</a>. <br />
</p><br />
<br />
<table class='data' width='100%'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Lux</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">438 [10]</td><br />
<td class="row">0.05 [>100]</td><br />
<td class="row">2 [47]</td><br />
<td class="row">1.88 [27]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">9.8 [7]</td><br />
<td class="row">0.11 [57]</td><br />
<td class="row">1.2 [29]</td><br />
<td class="row">1.5 [26]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">206 [3]</td><br />
<td class="row">0 [>>100]</td><br />
<td class="row">1.36 [10]</td><br />
<td class="row">1.87 [9]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">1105 [6]</td><br />
<td class="row">0.02 [>100]</td><br />
<td class="row">1.33 [19]</td><br />
<td class="row">2.34 [18]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br />
<br />
<p align='justify'><br />
From this table, it is evident that, whilst &alpha;<sub>pLux</sub> assumes significantly different values for different RBSs, &eta;<sub>pLux</sub> and k<sub>pLux</sub> assume very similar values. This result shows that RBS variation modulates the amplitude of Hill function, not affecting the shape of the curve. The four induction curves result to be the same Hill function modulated in amplitude by a parameter, that is the estimated RBS efficiency for this measurement system.<br><br />
These results are quite encouraging, because suggest that, given the non-modular behavior of RBS dpending on the encoded gene, the RBS has a modular behaviour respect to the promoter. <br />
<br />
</p><br />
<br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>4.28</td><td class='row'>0.20</td><td class='row'>1.08</td><td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>4.93</td><td class='row'>0.55</td><td class='row'>0.25</td><td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>9.49</td><td class='row'>0.02</td><td class='row'>0.47</td><td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>21.53</td><td class='row'>0.51</td><td class='row'>0.53</td><td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<p align='justify'><br />
<br />
These operative parameters provide useful information on the behavior of this 3OC6-HSL inducible device. RPU<sub>max</sub> assumes very different values, showing that the modulation in amplitude of the Hill can't be explained by a linear dependance on the RBS efficiency (in this case, in fact, the same RPUs should be observed for every RBS, since the standard reference used for RPUs computation)<br />
<br />
</p><br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- -pTet description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='pTet'></a><h2>pTet promoter</h2><br />
<br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> (wiki name: E21 ) pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> (wiki name: E22 ) pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT<br />
</li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT <br />
</li></ul><br />
</ul><br />
</ol><br />
<p align='justify'><br />
<br />
The protocols for the characterization of pTet promoter are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#pTet_protocol'>pTet measurement section</a>. <br><br />
This promoter is widely studied and characterized usually using the strong RBS BBa_B0034. Here we have characterized its transcriptional strength as a function of aTc induction (ng/ul) for different RBSs. Four different induction curves were obtained and are reported in figure:<br><br><br />
<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
The data collected from the mRFP measurement systems were processed as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'> data analysis section</a>. The induction curves were obtained by fitting a Hill function as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>modelling section</a> and the estimated <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>parameters</a> for pTet are reported in the pictures and in table below. </p><br />
<p align='justify'><br />
The estimated parameters of the Hill curves described in the figures are summarized in the table below:<br />
</p><br />
<br><br><br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">230.67 [3.7]</td><br />
<td class="row">0.028 [91.61]</td><br />
<td class="row">4.61 [23.73]</td><br />
<td class="row">8.75 [4.16]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">55.77 [12]</td><br />
<td class="row">1.53E-11 [>>100]</td><br />
<td class="row">4.98 [57.62]</td><br />
<td class="row">7.26 [14.98]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">120 [5.95]</td><br />
<td class="row">0.085 [40.6]</td><br />
<td class="row">24.85 [47.6]</td><br />
<td class="row">9 [5.43]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br><br><br />
<br />
<p align='justify'><br />
The measurement system pTet-B0031-mRFP-TT couldn't be assayed because its fluorescence output is under the detectability threshold of our measurement instrument. For this reason, the parameters of the corresponding Hill curve couldn't be estimated and are reported as 'Not Determined' ND. <br />
</p><br />
<br />
<p align='justify'><br />
&alpha; parameter (representing the maximum trascriptional rate in the studied range of induction) varies as expected with the RBS variation and also the &delta; and &eta; parameters are quite different among the RBS variations.<br><br />
The K<sub>pTet</sub> parameter is quite constant among the RBS variations, thus suggesting that in this case the RBS variation doesn't substantially affect the switch point of the Hill curve, even if the amplitude and the maximum slope are not quite maintained (for the &eta; parameter, maybe fitting problems). <br />
<br />
</p><br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [ng/&mu;]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [ng/&mu;]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>1.53</td><td class='row'>~0</td><td class='row'>7.95</td><td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>3.16</td><td class='row'>~0</td><td class='row'>6.7</td><td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>2.73</td><td class='row'>0.23</td><td class='row'>8.96</td><td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
From these parameters, it is evident that whilst the switch-point is almost maintained for all the RBSs, the linear boundaries are similar for RBS30 and RBS32 but for RBS34 are moved on the right of one order of magnitude. <br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --AiiA description-- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<br />
<a name='AiiA'></a><h2>AiiA gene - BBa_C0060</h2><br />
<br />
<p align='justify'><br />
The activity of AiiA enzyme has been evaluated by testing the measurement systems <a href='<a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a>. <br />
The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
</p><br />
<br />
<br />
<br><br><br />
dAiiA/dt=-&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>AiiA</sub>*AiiA<br><br><br />
d[HSL]/dt=N*K<sub>cat</sub>*AiiA*HSL/(K<sub>M, AiiA</sub>+AiiA))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
<p align='justify'><br />
<br />
Since the measurement systems are only assayed in the exponential growth phase, the third equation can be modified as follows:<br />
</p><br />
dN/dt=N*&mu;<br />
<br />
<p align='justify'><br />
because N&lt;N<sub>max</sub>. <br><br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.003152</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
<br />
<br />
<br />
<p align='justify'><br />
<br />
The parameters K<sub>cat</sub>, K<sub>M,AiiA</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of AiiA are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>cat</sub></b></td><br />
<td class='row'><b>K<sub>M, AiiA</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'></td><br />
<td class='row'></td><br />
<td class='row'></td><br />
<td class='row'></td><br />
<td class='row'></td><br />
<td class='row'></td><br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
<br />
<br />
</p><br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --LuxI description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='LuxI'></a><h2>LuxI gene - BBa_C0061</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#t9002'>modeling section</a>). <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
<br />
<br><br><br />
dLuxI/dt=&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>LuxI</sub>*LuxI<br><br><br />
d[HSL]/dt=N*V<sub>max</sub>*1/(1+(K<sub>M, LuxI</sub>/LuxI))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
<br />
<p align='justify'><br />
<br />
Since the measurement systems are only assayed in the exponential growth phase, the third equation can be modified as follows:<br />
</p><br />
dN/dt=N*&mu;<br />
<br />
<p align='justify'><br />
because N&lt;N<sub>max</sub>. <br><br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:</p><br />
<br />
<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.003152</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
<br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, K<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>pTet-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of LuxI are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>M, LuxI</sub></b></td><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>9.575*10<sup>2</sup></td><br />
<td class='row'>2.975*10<sup>-9</sup></td><br />
<td class='row'>14.08</td><br />
<td class='row'>1.67</td><br />
<td class='row'>ND</td><br />
<td class='row'>76.73</td><br />
<br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
<br />
</p><br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- ---pSB1C3-mRFP-- --><br />
<!-- ----------------- --><br />
<br />
<a name='K999'></a><h2>pSB1C3 plasmid with mRFP between S and P bearing pTet (easy-to-clone)</h2><br />
This vector was designed and realized in order to facilitate the cloning of coding sequences downstream of the strong promoter pTet. This vector was assembled by ligating S-P excided mRFP coding sequence from BBa_J61002 and ligating it in BBa_R0040 cut with S and P. Thus, the resulting vector contains mRFP between S and P. pTet cn be easily excided (E-P) and moved in the desired vector (E-P) and then the desired coding sequence can be easily assembled by digesting S-P the vector and X-P the coding sequence, thus obtaining a final part that is standard10-compatible. <br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<a name='Improvement'></a><h1>Existing parts: sequence debugging</h1><br />
<ol><ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516021 "> BBa_K516021 </a> (wiki name: E10 ) RBS31-AiiA-TT Rebuilt existing part from BBa_I13914 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516022 "> BBa_K516022 </a> (wiki name: E11 ) RBS32-AiiA-TT Rebuilt existing part from BBa_I13912 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516030 "> BBa_K516030 </a> (wiki name: E5 ) RBS30-mRFP-TT Rebuilt existing part from BBa_S04180 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516032 "> BBa_K516032 </a> (wiki name: E7 ) RBS32-mRFP-TT Rebuilt existing part from BBa_ J133000 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI Rebuilt existing part from BBa_S03623 (DNA available, only 2008 kit, inconsistent) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT Rebuilt existing part from BBa_ J22071 (2008 only, Bad sequencing) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT Rebuilt existing part from BBa_K077047 (Part deleted) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT Rebuilt existing part from BBa_I20252 (DNA planning) </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
</html><br />
<br />
{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Parts/CharacterizedTeam:UNIPV-Pavia/Parts/Characterized2011-09-17T22:39:54Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<html><br />
<a name='indice'></a><br />
<h2 class="art-postheader"><br />
Parts<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#new"><span class="tocnumber">1</span> <span class="toctext">New Parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#J101"><span class="tocnumber">1.1</span> <span class="toctext">J23101x series with different RBSs</span></a></li><br />
<li class="toclevel-2"><a href="#pTetAiiA"><span class="tocnumber">1.2</span> <span class="toctext">AiiA Expression cassette driven by aTc</span></a></li><br />
<li class="toclevel-2"><a href="#pTetLuxI"><span class="tocnumber">1.3</span> <span class="toctext">LuxI Expression cassette driven by aTc</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Existing"><span class="tocnumber">2</span> <span class="toctext">Characterization of existing parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#note"><span class="tocnumber">2.1</span> <span class="toctext">Notes on promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.2</span> <span class="toctext">RBSs from the community collection</span></a></li><br />
<li class="toclevel-2"><a href="#pLux"><span class="tocnumber">2.3</span> <span class="toctext">pLux - a 3OC6-HSL-in PoPs-out device</span></a></li><br />
<li class="toclevel-2"><a href="#pTet"><span class="tocnumber">2.4</span> <span class="toctext">pTet - BBa_R0040</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.5</span> <span class="toctext">AiiA - BBa_C0060</span></a></li><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.6</span> <span class="toctext">LuxI - BBa_C0061</span></a></li><br />
<li class="toclevel-2"><a href="#K999"><span class="tocnumber">2.7</span> <span class="toctext">pSB1C3 vector with mRFP between S and P for easy cloning</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Improvement"><span class="tocnumber">3</span> <span class="toctext">Sequence debugging</span></a></li><br />
</ul></td></tr></table><br />
</div><br />
<br />
<br />
<a name='new'></a><h1>New parts</h1><br />
<a name='J101'></a><h2>J23101x series</h2><br />
<br />
<ol type='1'><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> (wiki name: J101-E5 ) J101-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> (wiki name: J101-31 ) J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> (wiki name: J101-E7 ) J101-RBS32-mRFP-TT </li><br />
</ol><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- J23101x description --><br />
<!-- ----------------- --><br />
<p align='justify'><br />
BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in '<a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>data analysis</a>' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene. <br><br />
The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. <br>It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P.<br> <br />
This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts. <br><br />
Salis et al. [Nat Biotec, 2009] stated that<br />
<em> 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels'</em><br><br><br />
and again<br />
<em>'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.'</em> <br><br><br />
<br />
<br />
For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS.<br><br />
<br />
In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.<br />
<br />
<br><br><em><br />
<b>NB</b>: in the RPU computation, the J23101-RBS34-mRFP-TT construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency. </em><br />
<br />
<table align='center'><tr><td width='50%' ><br />
<table class='data'><br />
<tr><br />
<td class='row'><b>J23101 promoter with RBS</b><br />
</td><br />
<td class='row'><b>S<sub>cell</sub></b><br />
</td><br />
<td class='row'><b>R.P.U.s</b><br />
</td><br />
</tr><br />
<tr><td class='row'>RBS30<br />
</td><br />
<td class='row'>122 [13.23]</td><br />
<td class='row'>3.17</td><br />
</tr><br />
<tr><td class='row'>RBS31<br />
</td><br />
<td class='row'>2 [0.25]</td><br />
<td class='row'>0.05</td><br />
</tr><br />
<tr><td class='row'>RBS32<br />
</td><br />
<td class='row'>20 [1.7]</td><br />
<td class='row'>0.37</td><br />
</tr><br />
<tr><td class='row'>RBS34<br />
</td><br />
<td class='row'>50 [1.19]</td><br />
<td class='row'>1</td><br />
</tr><br />
</table></td><br />
<td width='50%' ><br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:UNIPV_RPU_J101.png" class="image"><img alt="RPUs of J23101 promoter with different RBSs" src="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="thumbimage" width="80%"></a></div></div><br />
</td><br />
</tr></table><br />
<br><br><br />
On the other hand, the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.<br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- pLuxAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetAiiA'></a><h2>AiiA expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> (wiki name: E24 ) pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> (wiki name: E25 ) pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- pTetAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetLuxI'></a><h2>LuxI expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> (wiki name: E13 ) pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> (wiki name: E14 ) pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> (wiki name: E15 ) pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<a name='Existing'></a><h1>Existing parts</h1><br />
<br />
<a name='note'></a><h2>Notes for promoter characterization</h2><br />
<p align='justify'><br />
Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection. <br />
<br><br><br />
The assembled RBSs are:<br />
<br><br><br />
<div align='center'><br />
<table class='data'><br />
<tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr><br />
<tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr><br />
<tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr><br />
<tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr><br />
<tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr><br />
</table></div><br />
<br><br />
For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.<br />
<br><br><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. <br />
RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. <br><br />
It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP). <br><br><br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element.<br />
Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.<br />
<br />
</p><br />
<br />
<p><br />
Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least sqares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs :<br />
<br><br />
<ol><ul><li><b><br />
RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity,<br />
</li><li><b><br />
RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity,<br />
</li><li><br />
<b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and it is representative of the position of linear region,<br />
</li><br />
<li><br />
<b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</li><br />
</ul></ol><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --RBS description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='rbs'></a><h2>RBSs</h2><br />
<p align='justify'><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of mRFP produced. <br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. <br />
The evaluation of RBS efficiency can be performed in a very intuitive fashion:<br><br />
<ul><br />
<li>1. select the RBSs you want to study, </li><br />
<li>2. assemble them in a Promoter - XX - Coding sequence circuit, </li></ul></ol><br />
<br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:Vettore_base.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d1/Vettore_base.jpg" class="thumbimage" width="50%"></a></div></div><br />
<br />
<ul><br />
<li>3. measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS. </li><br />
</ul></ol><br><br />
This simple measurement system allows the quantification of RBS efficiency depending on the whole measurement system (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in a complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments. <br><br />
To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.<br><br />
In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, pTet, pLux). Measuring the system output and evaluating the RBS efficiency. The results are summarized in the table below:<br><br><br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>pLux</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>pTet</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>J23101</sub></b><sup>**</sup></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>0.40</td><td class='row'>1.6814</td><td class='row'>3.17</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.01</td><td class='row'>ND</td><td class='row'>0.05</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.19</td><td class='row'>0.4193</td><td class='row'>0.37</td><td class='row'>0,3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<br />
<br><br />
On the other end, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (pTet-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below:<br><br><br />
<br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>mRFP</sub></b><sup>**</sup></td><br />
<td class='row'><b>eff<sub>AiiA</sub></b><sup>***</sup></td><br />
<td class='row'><b>eff<sub>LuxI</sub><sup>****</sup></b></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>1.72</td><td class='row'>0.53</td><td class='row'>0.18</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.03</td><td class='row'>0.83</td><td class='row'>0.001</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.37</td><td class='row'>0.50</td><td class='row'>N.D.</td><td class='row'>0.3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<p align='justify'><br />
<sup>*</sup> The RBS efficiency for inducible devices expressing mRFP was estimated as the ratio of the AUCs (Area under the curve) of the induction curve of the system with the studied RBS and the B0034 reference: AUC<sub>P, RBSx</sub>/AUC<sub>P, B0034</sub><br><br />
<sup>**</sup> The RBS efficiency for constitutive promoters expressing mRFP was computed as the ratio between S<sub>cell<sub>P, RBSx</sub></sub>/S<sub>cell<sub>P, B0034</sub></sub><br><br />
<sup>***</sup> The RBS efficiency for pTet promoter driving the expression of AiiA enzyme was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated for the measurement system pTet-RBSx-AiiA-TT. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#AiiA'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<sup>****</sup>The RBS efficiency for promoters driving the expression of LuxI was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated from the measurement systems pTet-RBSx-LuxI. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#LuxI'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<br />
<br><br />
</p><br />
<br><br><br />
The parts we used to characterize the RBSs are listed here:<br />
<ol><br />
<ul><li>mRFP expression with different promoters</li><br />
<ul><br />
<li>J23101</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> J101-RBS30-RFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> J101-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_J23101 "> BBa_J23101 </a> J101-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pTet</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pLux</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li><br />
</ul><br />
</ul><br />
<li>pTet driving the expression of different genes</li><br />
<ul><li>mRFP</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>AiiA</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> pTet-RBS34-AiiA-TT </li><br />
</ul><br />
<li>LuxI</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> pTet-RBS34-LuxI </li><br />
</ul></ul><br />
</ul></ol><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --pLux description- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<a name='pLux'></a><h2>pLux promoter</h2><br />
<p align='justify'><br />
<br />
<br />
</p><br />
<ol><ul><br />
<br />
<br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> (wiki name: E17 ) pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> (wiki name: E18 ) pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> (wiki name: E19 ) pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> (wiki name: E20 ) pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li></ul><br />
</ol><br />
<br />
<p align='justify'><br />
The estimated parameters for the Hill functions are summarized in the table below. For more details on parameter estimation, see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>model section</a>. <br />
</p><br />
<br />
<table class='data' width='100%'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Lux</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">438 [10]</td><br />
<td class="row">0.05 [>100]</td><br />
<td class="row">2 [47]</td><br />
<td class="row">1.88 [27]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">9.8 [7]</td><br />
<td class="row">0.11 [57]</td><br />
<td class="row">1.2 [29]</td><br />
<td class="row">1.5 [26]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">206 [3]</td><br />
<td class="row">0 [>>100]</td><br />
<td class="row">1.36 [10]</td><br />
<td class="row">1.87 [9]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">1105 [6]</td><br />
<td class="row">0.02 [>100]</td><br />
<td class="row">1.33 [19]</td><br />
<td class="row">2.34 [18]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br />
<br />
<p align='justify'><br />
From this table, it is evident that, whilst &alpha;<sub>pLux</sub> assumes significantly different values for different RBSs, &eta;<sub>pLux</sub> and k<sub>pLux</sub> assume very similar values. This result shows that RBS variation modulates the amplitude of Hill function, not affecting the shape of the curve. The four induction curves result to be the same Hill function modulated in amplitude by a parameter, that is the estimated RBS efficiency for this measurement system.<br><br />
These results are quite encouraging, because suggest that, given the non-modular behavior of RBS dpending on the encoded gene, the RBS has a modular behaviour respect to the promoter. <br />
<br />
</p><br />
<br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>4.28</td><td class='row'>0.20</td><td class='row'>1.08</td><td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>4.93</td><td class='row'>0.55</td><td class='row'>0.25</td><td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>9.49</td><td class='row'>0.02</td><td class='row'>0.47</td><td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>21.53</td><td class='row'>0.51</td><td class='row'>0.53</td><td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<p align='justify'><br />
<br />
These operative parameters provide useful information on the behavior of this 3OC6-HSL inducible device. RPU<sub>max</sub> assumes very different values, showing that the modulation in amplitude of the Hill can't be explained by a linear dependance on the RBS efficiency (in this case, in fact, the same RPUs should be observed for every RBS, since the standard reference used for RPUs computation)<br />
<br />
</p><br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- -pTet description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='pTet'></a><h2>pTet promoter</h2><br />
<br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> (wiki name: E21 ) pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> (wiki name: E22 ) pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT<br />
</li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT <br />
</li></ul><br />
</ul><br />
</ol><br />
<p align='justify'><br />
<br />
The protocols for the characterization of pTet promoter are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#pTet_protocol'>pTet measurement section</a>. <br><br />
This promoter is widely studied and characterized usually using the strong RBS BBa_B0034. Here we have characterized its transcriptional strength as a function of aTc induction (ng/ul) for different RBSs. Four different induction curves were obtained and are reported in figure:<br><br><br />
<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
The data collected from the mRFP measurement systems were processed as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'> data analysis section</a>. The induction curves were obtained by fitting a Hill function as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>modelling section</a> and the estimated <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>parameters</a> for pTet are reported in the pictures and in table below. </p><br />
<p align='justify'><br />
The estimated parameters of the Hill curves described in the figures are summarized in the table below:<br />
</p><br />
<br><br><br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">230.67 [3.7]</td><br />
<td class="row">0.028 [91.61]</td><br />
<td class="row">4.61 [23.73]</td><br />
<td class="row">8.75 [4.16]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">55.77 [12]</td><br />
<td class="row">1.53E-11 [>>100]</td><br />
<td class="row">4.98 [57.62]</td><br />
<td class="row">7.26 [14.98]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">120 [5.95]</td><br />
<td class="row">0.085 [40.6]</td><br />
<td class="row">24.85 [47.6]</td><br />
<td class="row">9 [5.43]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br><br><br />
<br />
<p align='justify'><br />
The measurement system pTet-B0031-mRFP-TT couldn't be assayed because its fluorescence output is under the detectability threshold of our measurement instrument. For this reason, the parameters of the corresponding Hill curve couldn't be estimated and are reported as 'Not Determined' ND. <br />
</p><br />
<br />
<p align='justify'><br />
&alpha; parameter (representing the maximum trascriptional rate in the studied range of induction) varies as expected with the RBS variation and also the &delta; and &eta; parameters are quite different among the RBS variations.<br><br />
The K<sub>pTet</sub> parameter is quite constant among the RBS variations, thus suggesting that in this case the RBS variation doesn't substantially affect the switch point of the Hill curve, even if the amplitude and the maximum slope are not quite maintained (for the &eta; parameter, maybe fitting problems). <br />
<br />
</p><br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [ng/&mu;]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [ng/&mu;]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>1.53</td><td class='row'>~0</td><td class='row'>7.95</td><td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>3.16</td><td class='row'>~0</td><td class='row'>6.7</td><td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>2.73</td><td class='row'>0.23</td><td class='row'>8.96</td><td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
From these parameters, it is evident that whilst the switch-point is almost maintained for all the RBSs, the linear boundaries are similar for RBS30 and RBS32 but for RBS34 are moved on the right of one order of magnitude. <br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --AiiA description-- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<br />
<a name='AiiA'></a><h2>AiiA gene - BBa_C0060</h2><br />
<br />
<p align='justify'><br />
The activity of AiiA enzyme has been evaluated by testing the measurement systems <a href='<a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a>. <br />
The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
</p><br />
<br />
<br />
<br><br><br />
dAiiA/dt=-&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>AiiA</sub>*AiiA<br><br><br />
d[HSL]/dt=N*K<sub>cat</sub>*AiiA*HSL/(K<sub>M, AiiA</sub>+AiiA))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
<p align='justify'><br />
<br />
Since the measurement systems are only assayed in the exponential growth phase, the third equation can be modified as follows:<br />
</p><br />
dN/dt=N*&mu;<br />
<br />
<p align='justify'><br />
because N&lt;N<sub>max</sub>. <br><br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.003152</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
<br />
<br />
<br />
<p align='justify'><br />
<br />
The parameters K<sub>cat</sub>, K<sub>M,AiiA</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of AiiA are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>cat</sub></b></td><br />
<td class='row'><b>K<sub>M, AiiA</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'></td><br />
<td class='row'></td><br />
<td class='row'></td><br />
<td class='row'></td><br />
<td class='row'></td><br />
<td class='row'></td><br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
<br />
<br />
</p><br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --LuxI description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='LuxI'></a><h2>LuxI gene - BBa_C0061</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#t9002'>modeling section</a>). <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
<br />
<br><br><br />
dLuxI/dt=&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>LuxI</sub>*LuxI<br><br><br />
d[HSL]/dt=N*V<sub>max</sub>*1/(1+(K<sub>M, LuxI</sub>/LuxI))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
<br />
<p align='justify'><br />
<br />
Since the measurement systems are only assayed in the exponential growth phase, the third equation can be modified as follows:<br />
</p><br />
dN/dt=N*&mu;<br />
<br />
<p align='justify'><br />
because N&lt;N<sub>max</sub>. <br><br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:</p><br />
<br />
<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.003152</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
<br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, K<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>pTet-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of LuxI are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>M, LuxI</sub></b></td><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>9.575*10<sup>2</sup></td><br />
<td class='row'>2.975*10<sup>-9</sup></td><br />
<td class='row'>14.08</td><br />
<td class='row'>1.67</td><br />
<td class='row'>ND</td><br />
<td class='row'>76.73</td><br />
<br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
<br />
Explain that in all these evaluations we have estimated Nmax, mu, gamma_HSL from previous experiments - see measurement and modelling sections. When do we talk about HSL stability as a function of pH?<br />
Provide parameters of HSL. How do we present these data? It would be nice also to say what's the amount of HSL produced by a liquid 5 ml culture at a given OD600 in M9 medium after tot hours starting from 1:1000 dilution of a saturated ON culture..<br />
We nee a synthetic parameter to express LuxI activity as a function of PoPS in. I suggest to report the HSL vs pH analysis here AND in the AiiA section of registry and, for what concerns our wiki, to add an appendix to the 'measurement section' to wich link when explaining.. <br />
Decide figures! <br />
</p><br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- ---pSB1C3-mRFP-- --><br />
<!-- ----------------- --><br />
<br />
<a name='K999'></a><h2>pSB1C3 plasmid with mRFP between S and P bearing pTet (easy-to-clone)</h2><br />
This vector was designed and realized in order to facilitate the cloning of coding sequences downstream of the strong promoter pTet. This vector was assembled by ligating S-P excided mRFP coding sequence from BBa_J61002 and ligating it in BBa_R0040 cut with S and P. Thus, the resulting vector contains mRFP between S and P. pTet cn be easily excided (E-P) and moved in the desired vector (E-P) and then the desired coding sequence can be easily assembled by digesting S-P the vector and X-P the coding sequence, thus obtaining a final part that is standard10-compatible. <br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<a name='Improvement'></a><h1>Existing parts: sequence debugging</h1><br />
<ol><ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516021 "> BBa_K516021 </a> (wiki name: E10 ) RBS31-AiiA-TT Rebuilt existing part from BBa_I13914 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516022 "> BBa_K516022 </a> (wiki name: E11 ) RBS32-AiiA-TT Rebuilt existing part from BBa_I13912 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516030 "> BBa_K516030 </a> (wiki name: E5 ) RBS30-mRFP-TT Rebuilt existing part from BBa_S04180 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516032 "> BBa_K516032 </a> (wiki name: E7 ) RBS32-mRFP-TT Rebuilt existing part from BBa_ J133000 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI Rebuilt existing part from BBa_S03623 (DNA available, only 2008 kit, inconsistent) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT Rebuilt existing part from BBa_ J22071 (2008 only, Bad sequencing) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT Rebuilt existing part from BBa_K077047 (Part deleted) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT Rebuilt existing part from BBa_I20252 (DNA planning) </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
</html><br />
<br />
{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Parts/CharacterizedTeam:UNIPV-Pavia/Parts/Characterized2011-09-17T22:33:19Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<html><br />
<a name='indice'></a><br />
<h2 class="art-postheader"><br />
Parts<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#new"><span class="tocnumber">1</span> <span class="toctext">New Parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#J101"><span class="tocnumber">1.1</span> <span class="toctext">J23101x series with different RBSs</span></a></li><br />
<li class="toclevel-2"><a href="#pTetAiiA"><span class="tocnumber">1.2</span> <span class="toctext">AiiA Expression cassette driven by aTc</span></a></li><br />
<li class="toclevel-2"><a href="#pTetLuxI"><span class="tocnumber">1.3</span> <span class="toctext">LuxI Expression cassette driven by aTc</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Existing"><span class="tocnumber">2</span> <span class="toctext">Characterization of existing parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#note"><span class="tocnumber">2.1</span> <span class="toctext">Notes on promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.2</span> <span class="toctext">RBSs from the community collection</span></a></li><br />
<li class="toclevel-2"><a href="#pLux"><span class="tocnumber">2.3</span> <span class="toctext">pLux - a 3OC6-HSL-in PoPs-out device</span></a></li><br />
<li class="toclevel-2"><a href="#pTet"><span class="tocnumber">2.4</span> <span class="toctext">pTet - BBa_R0040</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.5</span> <span class="toctext">AiiA - BBa_C0060</span></a></li><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.6</span> <span class="toctext">LuxI - BBa_C0061</span></a></li><br />
<li class="toclevel-2"><a href="#K999"><span class="tocnumber">2.7</span> <span class="toctext">pSB1C3 vector with mRFP between S and P for easy cloning</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Improvement"><span class="tocnumber">3</span> <span class="toctext">Sequence debugging</span></a></li><br />
</ul></td></tr></table><br />
</div><br />
<br />
<br />
<a name='new'></a><h1>New parts</h1><br />
<a name='J101'></a><h2>J23101x series</h2><br />
<br />
<ol type='1'><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> (wiki name: J101-E5 ) J101-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> (wiki name: J101-31 ) J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> (wiki name: J101-E7 ) J101-RBS32-mRFP-TT </li><br />
</ol><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- J23101x description --><br />
<!-- ----------------- --><br />
<p align='justify'><br />
BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in '<a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>data analysis</a>' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene. <br><br />
The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. <br>It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P.<br> <br />
This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts. <br><br />
Salis et al. [Nat Biotec, 2009] stated that<br />
<em> 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels'</em><br><br><br />
and again<br />
<em>'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.'</em> <br><br><br />
<br />
<br />
For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS.<br><br />
<br />
In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.<br />
<br />
<br><br><em><br />
<b>NB</b>: in the RPU computation, the J23101-RBS34-mRFP-TT construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency. </em><br />
<br />
<table align='center'><tr><td width='50%' ><br />
<table class='data'><br />
<tr><br />
<td class='row'><b>J23101 promoter with RBS</b><br />
</td><br />
<td class='row'><b>S<sub>cell</sub></b><br />
</td><br />
<td class='row'><b>R.P.U.s</b><br />
</td><br />
</tr><br />
<tr><td class='row'>RBS30<br />
</td><br />
<td class='row'>122 [13.23]</td><br />
<td class='row'>3.17</td><br />
</tr><br />
<tr><td class='row'>RBS31<br />
</td><br />
<td class='row'>2 [0.25]</td><br />
<td class='row'>0.05</td><br />
</tr><br />
<tr><td class='row'>RBS32<br />
</td><br />
<td class='row'>20 [1.7]</td><br />
<td class='row'>0.37</td><br />
</tr><br />
<tr><td class='row'>RBS34<br />
</td><br />
<td class='row'>50 [1.19]</td><br />
<td class='row'>1</td><br />
</tr><br />
</table></td><br />
<td width='50%' ><br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:UNIPV_RPU_J101.png" class="image"><img alt="RPUs of J23101 promoter with different RBSs" src="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="thumbimage" width="80%"></a></div></div><br />
</td><br />
</tr></table><br />
<br><br><br />
On the other hand, the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.<br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- pLuxAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetAiiA'></a><h2>AiiA expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> (wiki name: E24 ) pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> (wiki name: E25 ) pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- pTetAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetLuxI'></a><h2>LuxI expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> (wiki name: E13 ) pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> (wiki name: E14 ) pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> (wiki name: E15 ) pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<a name='Existing'></a><h1>Existing parts</h1><br />
<br />
<a name='note'></a><h2>Notes for promoter characterization</h2><br />
<p align='justify'><br />
Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection. <br />
<br><br><br />
The assembled RBSs are:<br />
<br><br><br />
<div align='center'><br />
<table class='data'><br />
<tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr><br />
<tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr><br />
<tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr><br />
<tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr><br />
<tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr><br />
</table></div><br />
<br><br />
For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.<br />
<br><br><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. <br />
RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. <br><br />
It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP). <br><br><br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element.<br />
Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.<br />
<br />
</p><br />
<br />
<p><br />
Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least sqares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs :<br />
<br><br />
<ol><ul><li><b><br />
RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity,<br />
</li><li><b><br />
RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity,<br />
</li><li><br />
<b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and it is representative of the position of linear region,<br />
</li><br />
<li><br />
<b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</li><br />
</ul></ol><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --RBS description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='rbs'></a><h2>RBSs</h2><br />
<p align='justify'><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of mRFP produced. <br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. <br />
The evaluation of RBS efficiency can be performed in a very intuitive fashion:<br><br />
<ul><br />
<li>1. select the RBSs you want to study, </li><br />
<li>2. assemble them in a Promoter - XX - Coding sequence circuit, </li></ul></ol><br />
<br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:Vettore_base.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d1/Vettore_base.jpg" class="thumbimage" width="50%"></a></div></div><br />
<br />
<ul><br />
<li>3. measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS. </li><br />
</ul></ol><br><br />
This simple measurement system allows the quantification of RBS efficiency depending on the whole measurement system (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in a complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments. <br><br />
To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.<br><br />
In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, pTet, pLux). Measuring the system output and evaluating the RBS efficiency. The results are summarized in the table below:<br><br><br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>pLux</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>pTet</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>J23101</sub></b><sup>**</sup></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>0.40</td><td class='row'>1.6814</td><td class='row'>3.17</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.01</td><td class='row'>ND</td><td class='row'>0.05</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.19</td><td class='row'>0.4193</td><td class='row'>0.37</td><td class='row'>0,3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<br />
<br><br />
On the other end, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (pTet-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below:<br><br><br />
<br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>mRFP</sub></b><sup>**</sup></td><br />
<td class='row'><b>eff<sub>AiiA</sub></b><sup>***</sup></td><br />
<td class='row'><b>eff<sub>LuxI</sub><sup>****</sup></b></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>1.72</td><td class='row'>0.53</td><td class='row'>0.18</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.03</td><td class='row'>0.83</td><td class='row'>0.001</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.37</td><td class='row'>0.50</td><td class='row'>N.D.</td><td class='row'>0.3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<p align='justify'><br />
<sup>*</sup> The RBS efficiency for inducible devices expressing mRFP was estimated as the ratio of the AUCs (Area under the curve) of the induction curve of the system with the studied RBS and the B0034 reference: AUC<sub>P, RBSx</sub>/AUC<sub>P, B0034</sub><br><br />
<sup>**</sup> The RBS efficiency for constitutive promoters expressing mRFP was computed as the ratio between S<sub>cell<sub>P, RBSx</sub></sub>/S<sub>cell<sub>P, B0034</sub></sub><br><br />
<sup>***</sup> The RBS efficiency for pTet promoter driving the expression of AiiA enzyme was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated for the measurement system pTet-RBSx-AiiA-TT. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#AiiA'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<sup>****</sup>The RBS efficiency for promoters driving the expression of LuxI was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated from the measurement systems pTet-RBSx-LuxI. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#LuxI'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<br />
<br><br />
</p><br />
<br><br><br />
The parts we used to characterize the RBSs are listed here:<br />
<ol><br />
<ul><li>mRFP expression with different promoters</li><br />
<ul><br />
<li>J23101</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> J101-RBS30-RFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> J101-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_J23101 "> BBa_J23101 </a> J101-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pTet</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pLux</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li><br />
</ul><br />
</ul><br />
<li>pTet driving the expression of different genes</li><br />
<ul><li>mRFP</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>AiiA</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> pTet-RBS34-AiiA-TT </li><br />
</ul><br />
<li>LuxI</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> pTet-RBS34-LuxI </li><br />
</ul></ul><br />
</ul></ol><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --pLux description- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<a name='pLux'></a><h2>pLux promoter</h2><br />
<p align='justify'><br />
<br />
<br />
</p><br />
<ol><ul><br />
<br />
<br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> (wiki name: E17 ) pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> (wiki name: E18 ) pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> (wiki name: E19 ) pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> (wiki name: E20 ) pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li></ul><br />
</ol><br />
<br />
<p align='justify'><br />
The estimated parameters for the Hill functions are summarized in the table below. For more details on parameter estimation, see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>model section</a>. <br />
</p><br />
<br />
<table class='data' width='100%'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Lux</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">438 [10]</td><br />
<td class="row">0.05 [>100]</td><br />
<td class="row">2 [47]</td><br />
<td class="row">1.88 [27]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">9.8 [7]</td><br />
<td class="row">0.11 [57]</td><br />
<td class="row">1.2 [29]</td><br />
<td class="row">1.5 [26]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">206 [3]</td><br />
<td class="row">0 [>>100]</td><br />
<td class="row">1.36 [10]</td><br />
<td class="row">1.87 [9]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">1105 [6]</td><br />
<td class="row">0.02 [>100]</td><br />
<td class="row">1.33 [19]</td><br />
<td class="row">2.34 [18]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br />
<br />
<p align='justify'><br />
From this table, it is evident that, whilst &alpha;<sub>pLux</sub> assumes significantly different values for different RBSs, &eta;<sub>pLux</sub> and k<sub>pLux</sub> assume very similar values. This result shows that RBS variation modulates the amplitude of Hill function, not affecting the shape of the curve. The four induction curves result to be the same Hill function modulated in amplitude by a parameter, that is the estimated RBS efficiency for this measurement system.<br><br />
These results are quite encouraging, because suggest that, given the non-modular behavior of RBS dpending on the encoded gene, the RBS has a modular behaviour respect to the promoter. <br />
<br />
</p><br />
<br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>4.28</td><td class='row'>0.20</td><td class='row'>1.08</td><td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>4.93</td><td class='row'>0.55</td><td class='row'>0.25</td><td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>9.49</td><td class='row'>0.02</td><td class='row'>0.47</td><td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>21.53</td><td class='row'>0.51</td><td class='row'>0.53</td><td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<p align='justify'><br />
<br />
These operative parameters provide useful information on the behavior of this 3OC6-HSL inducible device. RPU<sub>max</sub> assumes very different values, showing that the modulation in amplitude of the Hill can't be explained by a linear dependance on the RBS efficiency (in this case, in fact, the same RPUs should be observed for every RBS, since the standard reference used for RPUs computation)<br />
<br />
</p><br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- -pTet description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='pTet'></a><h2>pTet promoter</h2><br />
<br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> (wiki name: E21 ) pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> (wiki name: E22 ) pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT<br />
</li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT <br />
</li></ul><br />
</ul><br />
</ol><br />
<p align='justify'><br />
<br />
The protocols for the characterization of pTet promoter are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#pTet_protocol'>pTet measurement section</a>. <br><br />
This promoter is widely studied and characterized usually using the strong RBS BBa_B0034. Here we have characterized its transcriptional strength as a function of aTc induction (ng/ul) for different RBSs. Four different induction curves were obtained and are reported in figure:<br><br><br />
<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
The data collected from the mRFP measurement systems were processed as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'> data analysis section</a>. The induction curves were obtained by fitting a Hill function as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>modelling section</a> and the estimated <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>parameters</a> for pTet are reported in the pictures and in table below. </p><br />
<p align='justify'><br />
The estimated parameters of the Hill curves described in the figures are summarized in the table below:<br />
</p><br />
<br><br><br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">230.67 [3.7]</td><br />
<td class="row">0.028 [91.61]</td><br />
<td class="row">4.61 [23.73]</td><br />
<td class="row">8.75 [4.16]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">55.77 [12]</td><br />
<td class="row">1.53E-11 [>>100]</td><br />
<td class="row">4.98 [57.62]</td><br />
<td class="row">7.26 [14.98]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">120 [5.95]</td><br />
<td class="row">0.085 [40.6]</td><br />
<td class="row">24.85 [47.6]</td><br />
<td class="row">9 [5.43]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br><br><br />
<br />
<p align='justify'><br />
The measurement system pTet-B0031-mRFP-TT couldn't be assayed because its fluorescence output is under the detectability threshold of our measurement instrument. For this reason, the parameters of the corresponding Hill curve couldn't be estimated and are reported as 'Not Determined' ND. <br />
</p><br />
<br />
<p align='justify'><br />
&alpha; parameter (representing the maximum trascriptional rate in the studied range of induction) varies as expected with the RBS variation and also the &delta; and &eta; parameters are quite different among the RBS variations.<br><br />
The K<sub>pTet</sub> parameter is quite constant among the RBS variations, thus suggesting that in this case the RBS variation doesn't substantially affect the switch point of the Hill curve, even if the amplitude and the maximum slope are not quite maintained (for the &eta; parameter, maybe fitting problems). <br />
<br />
</p><br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [ng/&mu;]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [ng/&mu;]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>1.53</td><td class='row'>~0</td><td class='row'>7.95</td><td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>3.16</td><td class='row'>~0</td><td class='row'>6.7</td><td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>2.73</td><td class='row'>0.23</td><td class='row'>8.96</td><td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
From these parameters, it is evident that whilst the switch-point is almost maintained for all the RBSs, the linear boundaries are similar for RBS30 and RBS32 but for RBS34 are moved on the right of one order of magnitude. <br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --AiiA description-- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<br />
<a name='AiiA'></a><h2>AiiA gene - BBa_C0060</h2><br />
<br />
<p align='justify'><br />
The activity of AiiA enzyme has been evaluated by testing the measurement systems <a href='<a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a>. <br />
The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
</p><br />
<br />
<br />
<br><br><br />
dAiiA/dt=-&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>AiiA</sub>*AiiA<br><br><br />
d[HSL]/dt=N*K<sub>cat</sub>*AiiA*HSL/(K<sub>M, AiiA</sub>+AiiA))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
<p align='justify'><br />
<br />
Since the measurement systems are only assayed in the exponential growth phase, the third equation can be modified as follows:<br />
</p><br />
dN/dt=N*&mu;<br />
<br />
<p align='justify'><br />
because N&lt;N<sub>max</sub>. <br><br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.003152</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
<br />
<br />
<br />
<p align='justify'><br />
<br />
The parameters K<sub>cat</sub> and K<sub>M,AiiA</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of AiiA are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>cat</sub></b></td><br />
<td class='row'><b>K<sub>M, AiiA</sub></b></td><br />
</tr><br />
<tr><td class='row'></td><br />
<td class='row'></td><br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
<br />
<br />
</p><br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --LuxI description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='LuxI'></a><h2>LuxI gene - BBa_C0061</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#t9002'>modeling section</a>). <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
<br />
<br><br><br />
dLuxI/dt=&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>LuxI</sub>*LuxI<br><br><br />
d[HSL]/dt=N*V<sub>max</sub>*1/(1+(K<sub>M, LuxI</sub>/LuxI))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
<br />
<p align='justify'><br />
<br />
Since the measurement systems are only assayed in the exponential growth phase, the third equation can be modified as follows:<br />
</p><br />
dN/dt=N*&mu;<br />
<br />
<p align='justify'><br />
because N&lt;N<sub>max</sub>. <br><br />
The parameters of the first equation (estimated from the part pTet-RBSx-mRFP-TT) and N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:</p><br />
<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">215 [2]</td><br />
<td class="row">0.001 [>>100]</td><br />
<td class="row">4.3 [12]</td><br />
<td class="row">8.3 [2]</td><br />
<td class='row' rowspan='4'>1*10<sup>9</sup></td><br />
<td class='row' rowspan='4'>0.003152</td><br />
<td class='row' rowspan='4'>0</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">0.7 [>>100]</td><br />
<td class="row">0[>>100]</td><br />
<td class="row">18.84[>>100]</td><br />
<td class="row">47.51[>>100]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">45.7 [11]</td><br />
<td class="row">0.02 [>>100]</td><br />
<td class="row">83 [>>100]</td><br />
<td class="row">8 [>>100]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">125 [12]</td><br />
<td class="row">0.12 [61]</td><br />
<td class="row">71.8 [>>100]</td><br />
<td class="row">9.8 [>>100]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, K<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>pTet-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of LuxI are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>M, LuxI</sub></b></td><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>9.575*10<sup>2</sup></td><br />
<td class='row'>2.975*10<sup>-9</sup></td><br />
<td class='row'>14.08</td><br />
<td class='row'>1.67</td><br />
<td class='row'>ND</td><br />
<td class='row'>76.73</td><br />
<br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
<br />
Explain that in all these evaluations we have estimated Nmax, mu, gamma_HSL from previous experiments - see measurement and modelling sections. When do we talk about HSL stability as a function of pH?<br />
Provide parameters of HSL. How do we present these data? It would be nice also to say what's the amount of HSL produced by a liquid 5 ml culture at a given OD600 in M9 medium after tot hours starting from 1:1000 dilution of a saturated ON culture..<br />
We nee a synthetic parameter to express LuxI activity as a function of PoPS in. I suggest to report the HSL vs pH analysis here AND in the AiiA section of registry and, for what concerns our wiki, to add an appendix to the 'measurement section' to wich link when explaining.. <br />
Decide figures! <br />
</p><br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- ---pSB1C3-mRFP-- --><br />
<!-- ----------------- --><br />
<br />
<a name='K999'></a><h2>pSB1C3 plasmid with mRFP between S and P bearing pTet (easy-to-clone)</h2><br />
This vector was designed and realized in order to facilitate the cloning of coding sequences downstream of the strong promoter pTet. This vector was assembled by ligating S-P excided mRFP coding sequence from BBa_J61002 and ligating it in BBa_R0040 cut with S and P. Thus, the resulting vector contains mRFP between S and P. pTet cn be easily excided (E-P) and moved in the desired vector (E-P) and then the desired coding sequence can be easily assembled by digesting S-P the vector and X-P the coding sequence, thus obtaining a final part that is standard10-compatible. <br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<a name='Improvement'></a><h1>Existing parts: sequence debugging</h1><br />
<ol><ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516021 "> BBa_K516021 </a> (wiki name: E10 ) RBS31-AiiA-TT Rebuilt existing part from BBa_I13914 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516022 "> BBa_K516022 </a> (wiki name: E11 ) RBS32-AiiA-TT Rebuilt existing part from BBa_I13912 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516030 "> BBa_K516030 </a> (wiki name: E5 ) RBS30-mRFP-TT Rebuilt existing part from BBa_S04180 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516032 "> BBa_K516032 </a> (wiki name: E7 ) RBS32-mRFP-TT Rebuilt existing part from BBa_ J133000 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI Rebuilt existing part from BBa_S03623 (DNA available, only 2008 kit, inconsistent) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT Rebuilt existing part from BBa_ J22071 (2008 only, Bad sequencing) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT Rebuilt existing part from BBa_K077047 (Part deleted) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT Rebuilt existing part from BBa_I20252 (DNA planning) </li></ul></ol><br />
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</html><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Parts/CharacterizedTeam:UNIPV-Pavia/Parts/Characterized2011-09-17T22:25:31Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<html><br />
<a name='indice'></a><br />
<h2 class="art-postheader"><br />
Parts<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#new"><span class="tocnumber">1</span> <span class="toctext">New Parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#J101"><span class="tocnumber">1.1</span> <span class="toctext">J23101x series with different RBSs</span></a></li><br />
<li class="toclevel-2"><a href="#pTetAiiA"><span class="tocnumber">1.2</span> <span class="toctext">AiiA Expression cassette driven by aTc</span></a></li><br />
<li class="toclevel-2"><a href="#pTetLuxI"><span class="tocnumber">1.3</span> <span class="toctext">LuxI Expression cassette driven by aTc</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Existing"><span class="tocnumber">2</span> <span class="toctext">Characterization of existing parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#note"><span class="tocnumber">2.1</span> <span class="toctext">Notes on promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.2</span> <span class="toctext">RBSs from the community collection</span></a></li><br />
<li class="toclevel-2"><a href="#pLux"><span class="tocnumber">2.3</span> <span class="toctext">pLux - a 3OC6-HSL-in PoPs-out device</span></a></li><br />
<li class="toclevel-2"><a href="#pTet"><span class="tocnumber">2.4</span> <span class="toctext">pTet - BBa_R0040</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.5</span> <span class="toctext">AiiA - BBa_C0060</span></a></li><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.6</span> <span class="toctext">LuxI - BBa_C0061</span></a></li><br />
<li class="toclevel-2"><a href="#K999"><span class="tocnumber">2.7</span> <span class="toctext">pSB1C3 vector with mRFP between S and P for easy cloning</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Improvement"><span class="tocnumber">3</span> <span class="toctext">Sequence debugging</span></a></li><br />
</ul></td></tr></table><br />
</div><br />
<br />
<br />
<a name='new'></a><h1>New parts</h1><br />
<a name='J101'></a><h2>J23101x series</h2><br />
<br />
<ol type='1'><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> (wiki name: J101-E5 ) J101-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> (wiki name: J101-31 ) J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> (wiki name: J101-E7 ) J101-RBS32-mRFP-TT </li><br />
</ol><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- J23101x description --><br />
<!-- ----------------- --><br />
<p align='justify'><br />
BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in '<a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>data analysis</a>' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene. <br><br />
The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. <br>It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P.<br> <br />
This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts. <br><br />
Salis et al. [Nat Biotec, 2009] stated that<br />
<em> 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels'</em><br><br><br />
and again<br />
<em>'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.'</em> <br><br><br />
<br />
<br />
For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS.<br><br />
<br />
In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.<br />
<br />
<br><br><em><br />
<b>NB</b>: in the RPU computation, the J23101-RBS34-mRFP-TT construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency. </em><br />
<br />
<table align='center'><tr><td width='50%' ><br />
<table class='data'><br />
<tr><br />
<td class='row'><b>J23101 promoter with RBS</b><br />
</td><br />
<td class='row'><b>S<sub>cell</sub></b><br />
</td><br />
<td class='row'><b>R.P.U.s</b><br />
</td><br />
</tr><br />
<tr><td class='row'>RBS30<br />
</td><br />
<td class='row'>122 [13.23]</td><br />
<td class='row'>3.17</td><br />
</tr><br />
<tr><td class='row'>RBS31<br />
</td><br />
<td class='row'>2 [0.25]</td><br />
<td class='row'>0.05</td><br />
</tr><br />
<tr><td class='row'>RBS32<br />
</td><br />
<td class='row'>20 [1.7]</td><br />
<td class='row'>0.37</td><br />
</tr><br />
<tr><td class='row'>RBS34<br />
</td><br />
<td class='row'>50 [1.19]</td><br />
<td class='row'>1</td><br />
</tr><br />
</table></td><br />
<td width='50%' ><br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:UNIPV_RPU_J101.png" class="image"><img alt="RPUs of J23101 promoter with different RBSs" src="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="thumbimage" width="80%"></a></div></div><br />
</td><br />
</tr></table><br />
<br><br><br />
On the other hand, the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.<br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- pLuxAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetAiiA'></a><h2>AiiA expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> (wiki name: E24 ) pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> (wiki name: E25 ) pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- pTetAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetLuxI'></a><h2>LuxI expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> (wiki name: E13 ) pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> (wiki name: E14 ) pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> (wiki name: E15 ) pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<a name='Existing'></a><h1>Existing parts</h1><br />
<br />
<a name='note'></a><h2>Notes for promoter characterization</h2><br />
<p align='justify'><br />
Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection. <br />
<br><br><br />
The assembled RBSs are:<br />
<br><br><br />
<div align='center'><br />
<table class='data'><br />
<tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr><br />
<tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr><br />
<tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr><br />
<tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr><br />
<tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr><br />
</table></div><br />
<br><br />
For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.<br />
<br><br><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. <br />
RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. <br><br />
It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP). <br><br><br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element.<br />
Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.<br />
<br />
</p><br />
<br />
<p><br />
Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least sqares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs :<br />
<br><br />
<ol><ul><li><b><br />
RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity,<br />
</li><li><b><br />
RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity,<br />
</li><li><br />
<b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and represents the heart of linear region,<br />
</li><br />
<li><br />
<b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</li><br />
</ul></ol><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --RBS description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='rbs'></a><h2>RBSs</h2><br />
<p align='justify'><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of mRFP produced. <br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. <br />
The evaluation of RBS efficiency can be performed in a very intuitive fashion:<br><br />
<ul><br />
<li>1. select the RBSs you want to study, </li><br />
<li>2. assemble them in a Promoter - XX - Coding sequence circuit, </li></ul></ol><br />
<br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:Vettore_base.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d1/Vettore_base.jpg" class="thumbimage" width="50%"></a></div></div><br />
<br />
<ul><br />
<li>3. measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS. </li><br />
</ul></ol><br><br />
This simple measurement system allows the quantification of RBS efficiency depending on the whole measurement system (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in a complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments. <br><br />
To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.<br><br />
In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, pTet, pLux). Measuring the system output and evaluating the RBS efficiency. The results are summarized in the table below:<br><br><br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>pLux</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>pTet</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>J23101</sub></b><sup>**</sup></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>0.40</td><td class='row'>1.6814</td><td class='row'>3.17</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.01</td><td class='row'>ND</td><td class='row'>0.05</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.19</td><td class='row'>0.4193</td><td class='row'>0.37</td><td class='row'>0,3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<br />
<br><br />
On the other end, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (pTet-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below:<br><br><br />
<br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>mRFP</sub></b><sup>**</sup></td><br />
<td class='row'><b>eff<sub>AiiA</sub></b><sup>***</sup></td><br />
<td class='row'><b>eff<sub>LuxI</sub><sup>****</sup></b></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>1.72</td><td class='row'>0.53</td><td class='row'>0.64</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.03</td><td class='row'>0.83</td><td class='row'>0.08</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.37</td><td class='row'>0.50</td><td class='row'>N.D.</td><td class='row'>0.3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<p align='justify'><br />
<sup>*</sup> The RBS efficiency for inducible devices expressing mRFP was estimated as the ratio of the AUCs (Area under the curve) of the induction curve of the system with the studied RBS and the B0034 reference: AUC<sub>P, RBSx</sub>/AUC<sub>P, B0034</sub><br><br />
<sup>**</sup> The RBS efficiency for constitutive promoters expressing mRFP was computed as the ratio between S<sub>cell<sub>P, RBSx</sub></sub>/S<sub>cell<sub>P, B0034</sub></sub><br><br />
<sup>***</sup> The RBS efficiency for pTet promoter driving the expression of AiiA enzyme was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated for the measurement system pTet-RBSx-AiiA-TT. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#AiiA'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<sup>****</sup>The RBS efficiency for promoters driving the expression of LuxI was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated from the measurement systems pTet-RBSx-LuxI. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#LuxI'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<br />
<br><br />
</p><br />
<br><br><br />
The parts we used to characterize the RBSs are listed here:<br />
<ol><br />
<ul><li>mRFP expression with different promoters</li><br />
<ul><br />
<li>J23101</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> J101-RBS30-RFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> J101-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_J23101 "> BBa_J23101 </a> J101-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pTet</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pLux</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li><br />
</ul><br />
</ul><br />
<li>pTet driving the expression of different genes</li><br />
<ul><li>mRFP</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>AiiA</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> pTet-RBS34-AiiA-TT </li><br />
</ul><br />
<li>LuxI</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> pTet-RBS34-LuxI </li><br />
</ul></ul><br />
</ul></ol><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --pLux description- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<a name='pLux'></a><h2>pLux promoter</h2><br />
<p align='justify'><br />
<br />
<br />
</p><br />
<ol><ul><br />
<br />
<br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> (wiki name: E17 ) pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> (wiki name: E18 ) pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> (wiki name: E19 ) pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> (wiki name: E20 ) pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li></ul><br />
</ol><br />
<br />
<p align='justify'><br />
The estimated parameters for the Hill functions are summarized in the table below. For more details on parameter estimation, see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>model section</a>. <br />
</p><br />
<br />
<table class='data' width='100%'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Lux</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">438 [10]</td><br />
<td class="row">0.05 [>100]</td><br />
<td class="row">2 [47]</td><br />
<td class="row">1.88 [27]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">9.8 [7]</td><br />
<td class="row">0.11 [57]</td><br />
<td class="row">1.2 [29]</td><br />
<td class="row">1.5 [26]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">206 [3]</td><br />
<td class="row">0 [>>100]</td><br />
<td class="row">1.36 [10]</td><br />
<td class="row">1.87 [9]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">1105 [6]</td><br />
<td class="row">0.02 [>100]</td><br />
<td class="row">1.33 [19]</td><br />
<td class="row">2.34 [18]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br />
<br />
<p align='justify'><br />
From this table, it is evident that, whilst &alpha;<sub>pLux</sub> assumes significantly different values for different RBSs, &eta;<sub>pLux</sub> and k<sub>pLux</sub> assume very similar values. This result shows that RBS variation modulates the amplitude of Hill function, not affecting the shape of the curve. The four induction curves result to be the same Hill function modulated in amplitude by a parameter, that is the estimated RBS efficiency for this measurement system.<br><br />
These results are quite encouraging, because suggest that, given the non-modular behavior of RBS dpending on the encoded gene, the RBS has a modular behaviour respect to the promoter. <br />
<br />
</p><br />
<br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>4.28</td><td class='row'>0.20</td><td class='row'>1.08</td><td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>4.93</td><td class='row'>0.55</td><td class='row'>0.25</td><td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>9.49</td><td class='row'>0.02</td><td class='row'>0.47</td><td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>21.53</td><td class='row'>0.51</td><td class='row'>0.53</td><td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<p align='justify'><br />
<br />
These operative parameters provide useful information on the behavior of this 3OC6-HSL inducible device. RPU<sub>max</sub> assumes very different values, showing that the modulation in amplitude of the Hill can't be explained by a linear dependance on the RBS efficiency (in this case, in fact, the same RPUs should be observed for every RBS, since the standard reference used for RPUs computation)<br />
<br />
</p><br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- -pTet description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='pTet'></a><h2>pTet promoter</h2><br />
<br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> (wiki name: E21 ) pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> (wiki name: E22 ) pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT<br />
</li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT <br />
</li></ul><br />
</ul><br />
</ol><br />
<p align='justify'><br />
<br />
The protocols for the characterization of pTet promoter are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#pTet_protocol'>pTet measurement section</a>. <br><br />
This promoter is widely studied and characterized usually using the strong RBS BBa_B0034. Here we have characterized its transcriptional strength as a function of aTc induction (ng/ul) for different RBSs. Four different induction curves were obtained and are reported in figure:<br><br><br />
<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
The data collected from the mRFP measurement systems were processed as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'> data analysis section</a>. The induction curves were obtained by fitting a Hill function as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>modelling section</a> and the estimated <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>parameters</a> for pTet are reported in the pictures and in table below. </p><br />
<p align='justify'><br />
The estimated parameters of the Hill curves described in the figures are summarized in the table below:<br />
</p><br />
<br><br><br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">230.67 [3.7]</td><br />
<td class="row">0.028 [91.61]</td><br />
<td class="row">4.61 [23.73]</td><br />
<td class="row">8.75 [4.16]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">55.77 [12]</td><br />
<td class="row">1.53E-11 [>>100]</td><br />
<td class="row">4.98 [57.62]</td><br />
<td class="row">7.26 [14.98]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">120 [5.95]</td><br />
<td class="row">0.085 [40.6]</td><br />
<td class="row">24.85 [47.6]</td><br />
<td class="row">9 [5.43]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br><br><br />
<br />
<p align='justify'><br />
The measurement system pTet-B0031-mRFP-TT couldn't be assayed because its fluorescence output is under the detectability threshold of our measurement instrument. For this reason, the parameters of the corresponding Hill curve couldn't be estimated and are reported as 'Not Determined' ND. <br />
</p><br />
<br />
<p align='justify'><br />
&alpha; parameter (representing the maximum trascriptional rate in the studied range of induction) varies as expected with the RBS variation and also the &delta; and &eta; parameters are quite different among the RBS variations.<br><br />
The K<sub>pTet</sub> parameter is quite constant among the RBS variations, thus suggesting that in this case the RBS variation doesn't substantially affect the switch point of the Hill curve, even if the amplitude and the maximum slope are not quite maintained (for the &eta; parameter, maybe fitting problems). <br />
<br />
</p><br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [ng/&mu;]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [ng/&mu;]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>1.53</td><td class='row'>~0</td><td class='row'>7.95</td><td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>3.16</td><td class='row'>~0</td><td class='row'>6.7</td><td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>2.73</td><td class='row'>0.23</td><td class='row'>8.96</td><td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
From these parameters, it is evident that whilst the switch-point is almost maintained for all the RBSs, the linear boundaries are similar for RBS30 and RBS32 but for RBS34 are moved on the right of one order of magnitude. <br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --AiiA description-- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<br />
<a name='AiiA'></a><h2>AiiA gene - BBa_C0060</h2><br />
<br />
<p align='justify'><br />
The activity of AiiA enzyme has been evaluated by testing the measurement systems <a href='<a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a>. <br />
The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
</p><br />
<br />
<br />
<br><br><br />
dAiiA/dt=&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>AiiA</sub>*AiiA<br><br><br />
d[HSL]/dt=N*K<sub>cat</sub>*AiiA*HSL/(K<sub>M, AiiA</sub>+AiiA))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
<p align='justify'><br />
<br />
Since the measurement systems are only assayed in the exponential growth phase, the third equation can be modified as follows:<br />
</p><br />
dN/dt=N*&mu;<br />
<br />
<p align='justify'><br />
because N&lt;N<sub>max</sub>. <br><br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.003152</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
<br />
<br />
<br />
<p align='justify'><br />
<br />
The parameters K<sub>cat</sub> and K<sub>M,AiiA</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of AiiA are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>cat</sub></b></td><br />
<td class='row'><b>K<sub>M, AiiA</sub></b></td><br />
</tr><br />
<tr><td class='row'></td><br />
<td class='row'></td><br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
<br />
<br />
</p><br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --LuxI description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='LuxI'></a><h2>LuxI gene - BBa_C0061</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#t9002'>modeling section</a>). <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
<br />
<br><br><br />
dLuxI/dt=&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>LuxI</sub>*LuxI<br><br><br />
d[HSL]/dt=N*V<sub>max</sub>*1/(1+(K<sub>M, LuxI</sub>/LuxI))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
<br />
<p align='justify'><br />
<br />
Since the measurement systems are only assayed in the exponential growth phase, the third equation can be modified as follows:<br />
</p><br />
dN/dt=N*&mu;<br />
<br />
<p align='justify'><br />
because N&lt;N<sub>max</sub>. <br><br />
The parameters of the first equation (estimated from the part pTet-RBSx-mRFP-TT) and N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:</p><br />
<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">215 [2]</td><br />
<td class="row">0.001 [>>100]</td><br />
<td class="row">4.3 [12]</td><br />
<td class="row">8.3 [2]</td><br />
<td class='row' rowspan='4'>1*10<sup>9</sup></td><br />
<td class='row' rowspan='4'>0.003152</td><br />
<td class='row' rowspan='4'>0</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">0.7 [>>100]</td><br />
<td class="row">0[>>100]</td><br />
<td class="row">18.84[>>100]</td><br />
<td class="row">47.51[>>100]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">45.7 [11]</td><br />
<td class="row">0.02 [>>100]</td><br />
<td class="row">83 [>>100]</td><br />
<td class="row">8 [>>100]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">125 [12]</td><br />
<td class="row">0.12 [61]</td><br />
<td class="row">71.8 [>>100]</td><br />
<td class="row">9.8 [>>100]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, K<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>pTet-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of LuxI are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>M, LuxI</sub></b></td><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>9.575*10<sup>2</sup></td><br />
<td class='row'>2.975*10<sup>-9</sup></td><br />
<td class='row'>14.08</td><br />
<td class='row'>1.67</td><br />
<td class='row'>ND</td><br />
<td class='row'>76.73</td><br />
<br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
<br />
Explain that in all these evaluations we have estimated Nmax, mu, gamma_HSL from previous experiments - see measurement and modelling sections. When do we talk about HSL stability as a function of pH?<br />
Provide parameters of HSL. How do we present these data? It would be nice also to say what's the amount of HSL produced by a liquid 5 ml culture at a given OD600 in M9 medium after tot hours starting from 1:1000 dilution of a saturated ON culture..<br />
We nee a synthetic parameter to express LuxI activity as a function of PoPS in. I suggest to report the HSL vs pH analysis here AND in the AiiA section of registry and, for what concerns our wiki, to add an appendix to the 'measurement section' to wich link when explaining.. <br />
Decide figures! <br />
</p><br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- ---pSB1C3-mRFP-- --><br />
<!-- ----------------- --><br />
<br />
<a name='K999'></a><h2>pSB1C3 plasmid with mRFP between S and P bearing pTet (easy-to-clone)</h2><br />
This vector was designed and realized in order to facilitate the cloning of coding sequences downstream of the strong promoter pTet. This vector was assembled by ligating S-P excided mRFP coding sequence from BBa_J61002 and ligating it in BBa_R0040 cut with S and P. Thus, the resulting vector contains mRFP between S and P. pTet cn be easily excided (E-P) and moved in the desired vector (E-P) and then the desired coding sequence can be easily assembled by digesting S-P the vector and X-P the coding sequence, thus obtaining a final part that is standard10-compatible. <br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<a name='Improvement'></a><h1>Existing parts: sequence debugging</h1><br />
<ol><ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516021 "> BBa_K516021 </a> (wiki name: E10 ) RBS31-AiiA-TT Rebuilt existing part from BBa_I13914 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516022 "> BBa_K516022 </a> (wiki name: E11 ) RBS32-AiiA-TT Rebuilt existing part from BBa_I13912 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516030 "> BBa_K516030 </a> (wiki name: E5 ) RBS30-mRFP-TT Rebuilt existing part from BBa_S04180 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516032 "> BBa_K516032 </a> (wiki name: E7 ) RBS32-mRFP-TT Rebuilt existing part from BBa_ J133000 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI Rebuilt existing part from BBa_S03623 (DNA available, only 2008 kit, inconsistent) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT Rebuilt existing part from BBa_ J22071 (2008 only, Bad sequencing) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT Rebuilt existing part from BBa_K077047 (Part deleted) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT Rebuilt existing part from BBa_I20252 (DNA planning) </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
</html><br />
<br />
{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Parts/CharacterizedTeam:UNIPV-Pavia/Parts/Characterized2011-09-17T22:19:44Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<html><br />
<a name='indice'></a><br />
<h2 class="art-postheader"><br />
Parts<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#new"><span class="tocnumber">1</span> <span class="toctext">New Parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#J101"><span class="tocnumber">1.1</span> <span class="toctext">J23101x series with different RBSs</span></a></li><br />
<li class="toclevel-2"><a href="#pTetAiiA"><span class="tocnumber">1.2</span> <span class="toctext">AiiA Expression cassette driven by aTc</span></a></li><br />
<li class="toclevel-2"><a href="#pTetLuxI"><span class="tocnumber">1.3</span> <span class="toctext">LuxI Expression cassette driven by aTc</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Existing"><span class="tocnumber">2</span> <span class="toctext">Characterization of existing parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#note"><span class="tocnumber">2.1</span> <span class="toctext">Notes on promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.2</span> <span class="toctext">RBSs from the community collection</span></a></li><br />
<li class="toclevel-2"><a href="#pLux"><span class="tocnumber">2.3</span> <span class="toctext">pLux - a 3OC6-HSL-in PoPs-out device</span></a></li><br />
<li class="toclevel-2"><a href="#pTet"><span class="tocnumber">2.4</span> <span class="toctext">pTet - BBa_R0040</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.5</span> <span class="toctext">AiiA - BBa_C0060</span></a></li><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.6</span> <span class="toctext">LuxI - BBa_C0061</span></a></li><br />
<li class="toclevel-2"><a href="#K999"><span class="tocnumber">2.7</span> <span class="toctext">pSB1C3 vector with mRFP between S and P for easy cloning</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Improvement"><span class="tocnumber">3</span> <span class="toctext">Sequence debugging</span></a></li><br />
</ul></td></tr></table><br />
</div><br />
<br />
<br />
<a name='new'></a><h1>New parts</h1><br />
<a name='J101'></a><h2>J23101x series</h2><br />
<br />
<ol type='1'><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> (wiki name: J101-E5 ) J101-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> (wiki name: J101-31 ) J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> (wiki name: J101-E7 ) J101-RBS32-mRFP-TT </li><br />
</ol><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- J23101x description --><br />
<!-- ----------------- --><br />
<p align='justify'><br />
BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in '<a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>data analysis</a>' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene. <br><br />
The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. <br>It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P.<br> <br />
This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts. <br><br />
Salis et al. [Nat Biotec, 2009] stated that<br />
<em> 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels'</em><br><br><br />
and again<br />
<em>'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.'</em> <br><br><br />
<br />
<br />
For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS.<br><br />
<br />
In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.<br />
<br />
<br><br><em><br />
<b>NB</b>: in the RPU computation, the J23101-RBS34-mRFP-TT construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency. </em><br />
<br />
<table align='center'><tr><td width='50%' ><br />
<table class='data'><br />
<tr><br />
<td class='row'><b>J23101 promoter with RBS</b><br />
</td><br />
<td class='row'><b>S<sub>cell</sub></b><br />
</td><br />
<td class='row'><b>R.P.U.s</b><br />
</td><br />
</tr><br />
<tr><td class='row'>RBS30<br />
</td><br />
<td class='row'>122 [13.23]</td><br />
<td class='row'>3.17</td><br />
</tr><br />
<tr><td class='row'>RBS31<br />
</td><br />
<td class='row'>2 [0.25]</td><br />
<td class='row'>0.05</td><br />
</tr><br />
<tr><td class='row'>RBS32<br />
</td><br />
<td class='row'>20 [1.7]</td><br />
<td class='row'>0.37</td><br />
</tr><br />
<tr><td class='row'>RBS34<br />
</td><br />
<td class='row'>50 [1.19]</td><br />
<td class='row'>1</td><br />
</tr><br />
</table></td><br />
<td width='50%' ><br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:UNIPV_RPU_J101.png" class="image"><img alt="RPUs of J23101 promoter with different RBSs" src="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="thumbimage" width="80%"></a></div></div><br />
</td><br />
</tr></table><br />
<br><br><br />
On the other hand, the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.<br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- pLuxAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetAiiA'></a><h2>AiiA expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> (wiki name: E24 ) pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> (wiki name: E25 ) pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- pTetAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetLuxI'></a><h2>LuxI expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> (wiki name: E13 ) pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> (wiki name: E14 ) pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> (wiki name: E15 ) pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<a name='Existing'></a><h1>Existing parts</h1><br />
<br />
<a name='note'></a><h2>Notes for promoter characterization</h2><br />
<p align='justify'><br />
Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection. <br />
<br><br><br />
The assembled RBSs are:<br />
<br><br><br />
<div align='center'><br />
<table class='data'><br />
<tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr><br />
<tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr><br />
<tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr><br />
<tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr><br />
<tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr><br />
</table></div><br />
<br><br />
For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.<br />
<br><br><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. <br />
RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. <br><br />
It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP). <br><br><br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element.<br />
Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.<br />
<br />
</p><br />
<br />
<p><br />
Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least sqares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs :<br />
<br><br />
<ol><ul><li><b><br />
RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity,<br />
</li><li><b><br />
RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity,<br />
</li><li><br />
<b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and represents the heart of linear region,<br />
</li><br />
<li><br />
<b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</li><br />
</ul></ol><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --RBS description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='rbs'></a><h2>RBSs</h2><br />
<p align='justify'><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of mRFP produced. <br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. <br />
The evaluation of RBS efficiency can be performed in a very intuitive fashion:<br><br />
<ul><br />
<li>1. select the RBSs you want to study, </li><br />
<li>2. assemble them in a Promoter - XX - Coding sequence circuit, </li></ul></ol><br />
<br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:Vettore_base.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d1/Vettore_base.jpg" class="thumbimage" width="50%"></a></div></div><br />
<br />
<ul><br />
<li>3. measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS. </li><br />
</ul></ol><br><br />
This simple measurement system allows the quantification of RBS efficiency depending on the whole measurement system (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in a complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments. <br><br />
To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.<br><br />
In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, pTet, pLux). Measuring the system output and evaluating the RBS efficiency. The results are summarized in the table below:<br><br><br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>pLux</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>pTet</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>J23101</sub></b><sup>**</sup></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>0.40</td><td class='row'>1.6814</td><td class='row'>3.17</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.01</td><td class='row'>ND</td><td class='row'>0.05</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.19</td><td class='row'>0.4193</td><td class='row'>0.37</td><td class='row'>0,3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<br />
<br><br />
On the other end, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (pTet-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below:<br><br><br />
<br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>mRFP</sub></b><sup>**</sup></td><br />
<td class='row'><b>eff<sub>AiiA</sub></b><sup>***</sup></td><br />
<td class='row'><b>eff<sub>LuxI</sub><sup>****</sup></b></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>1.72</td><td class='row'>0.53</td><td class='row'>0.64</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.03</td><td class='row'>0.83</td><td class='row'>0.08</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.37</td><td class='row'>0.50</td><td class='row'>N.D.</td><td class='row'>0.3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<p align='justify'><br />
<sup>*</sup> The RBS efficiency for inducible devices expressing mRFP was estimated as the ratio of the AUCs (Area under the curve) of the induction curve of the system with the studied RBS and the B0034 reference: AUC<sub>P, RBSx</sub>/AUC<sub>P, B0034</sub><br><br />
<sup>**</sup> The RBS efficiency for constitutive promoters expressing mRFP was computed as the ratio between S<sub>cell<sub>P, RBSx</sub></sub>/S<sub>cell<sub>P, B0034</sub></sub><br><br />
<sup>***</sup> The RBS efficiency for pTet promoter driving the expression of AiiA enzyme was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated for the measurement system pTet-RBSx-AiiA-TT. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#AiiA'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<sup>****</sup>The RBS efficiency for promoters driving the expression of LuxI was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated from the measurement systems pTet-RBSx-LuxI. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#LuxI'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<br />
<br><br />
</p><br />
<br><br><br />
The parts we used to characterize the RBSs are listed here:<br />
<ol><br />
<ul><li>mRFP expression with different promoters</li><br />
<ul><br />
<li>J23101</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> J101-RBS30-RFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> J101-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_J23101 "> BBa_J23101 </a> J101-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pTet</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pLux</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li><br />
</ul><br />
</ul><br />
<li>pTet driving the expression of different genes</li><br />
<ul><li>mRFP</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>AiiA</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> pTet-RBS34-AiiA-TT </li><br />
</ul><br />
<li>LuxI</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> pTet-RBS34-LuxI </li><br />
</ul></ul><br />
</ul></ol><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --pLux description- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<a name='pLux'></a><h2>pLux promoter</h2><br />
<p align='justify'><br />
<br />
<br />
</p><br />
<ol><ul><br />
<br />
<br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> (wiki name: E17 ) pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> (wiki name: E18 ) pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> (wiki name: E19 ) pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> (wiki name: E20 ) pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li></ul><br />
</ol><br />
<br />
<p align='justify'><br />
The estimated parameters for the Hill functions are summarized in the table below. For more details on parameter estimation, see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>model section</a>. <br />
</p><br />
<br />
<table class='data' width='100%'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Lux</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">438 [10]</td><br />
<td class="row">0.05 [>100]</td><br />
<td class="row">2 [47]</td><br />
<td class="row">1.88 [27]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">9.8 [7]</td><br />
<td class="row">0.11 [57]</td><br />
<td class="row">1.2 [29]</td><br />
<td class="row">1.5 [26]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">206 [3]</td><br />
<td class="row">0 [>>100]</td><br />
<td class="row">1.36 [10]</td><br />
<td class="row">1.87 [9]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">1105 [6]</td><br />
<td class="row">0.02 [>100]</td><br />
<td class="row">1.33 [19]</td><br />
<td class="row">2.34 [18]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br />
<br />
<p align='justify'><br />
From this table, it is evident that, whilst &alpha;<sub>pLux</sub> assumes significantly different values for different RBSs, &eta;<sub>pLux</sub> and k<sub>pLux</sub> assume very similar values. This result shows that RBS variation modulates the amplitude of Hill function, not affecting the shape of the curve. The four induction curves result to be the same Hill function modulated in amplitude by a parameter, that is the estimated RBS efficiency for this measurement system.<br><br />
These results are quite encouraging, because suggest that, given the non-modular behavior of RBS dpending on the encoded gene, the RBS has a modular behaviour respect to the promoter. <br />
<br />
</p><br />
<br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>4.28</td><td class='row'>0.20</td><td class='row'>1.08</td><td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>4.93</td><td class='row'>0.55</td><td class='row'>0.25</td><td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>9.49</td><td class='row'>0.02</td><td class='row'>0.47</td><td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>21.53</td><td class='row'>0.51</td><td class='row'>0.53</td><td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<p align='justify'><br />
<br />
These operative parameters provide useful information on the behavior of this 3OC6-HSL inducible device. RPU<sub>max</sub> assumes very different values, showing that the modulation in amplitude of the Hill can't be explained by a linear dependance on the RBS efficiency (in this case, in fact, the same RPUs should be observed for every RBS, since the standard reference used for RPUs computation)<br />
<br />
</p><br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- -pTet description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='pTet'></a><h2>pTet promoter</h2><br />
<br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> (wiki name: E21 ) pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> (wiki name: E22 ) pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT<br />
</li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT <br />
</li></ul><br />
</ul><br />
</ol><br />
<p align='justify'><br />
<br />
The protocols for the characterization of pTet promoter are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#pTet_protocol'>pTet measurement section</a>. <br><br />
This promoter is widely studied and characterized usually using the strong RBS BBa_B0034. Here we have characterized its transcriptional strength as a function of aTc induction (ng/ul) for different RBSs. Four different induction curves were obtained and are reported in figure:<br><br><br />
<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
The data collected from the mRFP measurement systems were processed as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'> data analysis section</a>. The induction curves were obtained by fitting a Hill function as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>modelling section</a> and the estimated <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>parameters</a> for pTet are reported in the pictures and in table below. </p><br />
<p align='justify'><br />
The estimated parameters of the Hill curves described in the figures are summarized in the table below:<br />
</p><br />
<br><br><br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">230.67 [3.7]</td><br />
<td class="row">0.028 [91.61]</td><br />
<td class="row">4.61 [23.73]</td><br />
<td class="row">8.75 [4.16]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">55.77 [12]</td><br />
<td class="row">1.53E-11 [>>100]</td><br />
<td class="row">4.98 [57.62]</td><br />
<td class="row">7.26 [14.98]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">120 [5.95]</td><br />
<td class="row">0.085 [40.6]</td><br />
<td class="row">24.85 [47.6]</td><br />
<td class="row">9 [5.43]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br><br><br />
<br />
<p align='justify'><br />
The measurement system pTet-B0031-mRFP-TT couldn't be assayed because its fluorescence output is under the detectability threshold of our measurement instrument. For this reason, the parameters of the corresponding Hill curve couldn't be estimated and are reported as 'Not Determined' ND. <br />
</p><br />
<br />
<p align='justify'><br />
&alpha; parameter (representing the maximum trascriptional rate in the studied range of induction) varies as expected with the RBS variation and also the &delta; and &eta; parameters are quite different among the RBS variations.<br><br />
The K<sub>pTet</sub> parameter is quite constant among the RBS variations, thus suggesting that in this case the RBS variation doesn't substantially affect the switch point of the Hill curve, even if the amplitude and the maximum slope are not quite maintained (for the &eta; parameter, maybe fitting problems). <br />
<br />
</p><br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [ng/&mu;]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [ng/&mu;]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>1.53</td><td class='row'>~0</td><td class='row'>7.95</td><td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>3.16</td><td class='row'>~0</td><td class='row'>6.7</td><td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>2.73</td><td class='row'>0.23</td><td class='row'>8.96</td><td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
From these parameters, it is evident that whilst the switch-point is almost maintained for all the RBSs, the linear boundaries are similar for RBS30 and RBS32 but for RBS34 are moved on the right of one order of magnitude. <br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --AiiA description-- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<br />
<a name='AiiA'></a><h2>AiiA gene - BBa_C0060</h2><br />
<br />
<p align='justify'><br />
The activity of AiiA enzyme has been evaluated by testing the measurement systems <a href='<a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a>. <br />
The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
</p><br />
<br />
<br />
<br><br><br />
dAiiA/dt=&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>AiiA</sub>*AiiA<br><br><br />
d[HSL]/dt=N*K<sub>cat</sub>*AiiA*HSL/(K<sub>M, AiiA</sub>+AiiA))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
<p align='justify'><br />
<br />
Since the measurement systems are only assayed in the exponential growth phase, the third equation can be modified as follows:<br />
</p><br />
dN/dt=N*&mu;<br />
<br />
<p align='justify'><br />
because N&lt;N<sub>max</sub>. <br><br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.003152</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
<br />
<br />
<br />
<p align='justify'><br />
<br />
The parameters K<sub>cat</sub> and K<sub>M,AiiA</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of AiiA are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>cat</sub></b></td><br />
<td class='row'><b>K<sub>M, AiiA</sub></b></td><br />
</tr><br />
<tr><td class='row'></td><br />
<td class='row'></td><br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
<br />
<br />
</p><br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --LuxI description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='LuxI'></a><h2>LuxI gene - BBa_C0061</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#t9002'>modeling section</a>). <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
<br />
<br><br><br />
dLuxI/dt=&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>LuxI</sub>*LuxI<br><br><br />
d[HSL]/dt=N*V<sub>max</sub>*1/(1+(K<sub>M, LuxI</sub>/LuxI))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
<br />
<p align='justify'><br />
<br />
Since the measurement systems are only assayed in the exponential growth phase, the third equation can be modified as follows:<br />
</p><br />
dN/dt=N*&mu;<br />
<br />
<p align='justify'><br />
because N&lt;N<sub>max</sub>. <br><br />
The parameters of the first equation (estimated from the part pTet-RBSx-mRFP-TT) and N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:</p><br />
<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">215 [2]</td><br />
<td class="row">0.001 [>>100]</td><br />
<td class="row">4.3 [12]</td><br />
<td class="row">8.3 [2]</td><br />
<td class='row' rowspan='4'>1*10<sup>9</sup></td><br />
<td class='row' rowspan='4'>0.003152</td><br />
<td class='row' rowspan='4'>0</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">0.7 [>>100]</td><br />
<td class="row">0[>>100]</td><br />
<td class="row">18.84[>>100]</td><br />
<td class="row">47.51[>>100]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">45.7 [11]</td><br />
<td class="row">0.02 [>>100]</td><br />
<td class="row">83 [>>100]</td><br />
<td class="row">8 [>>100]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">125 [12]</td><br />
<td class="row">0.12 [61]</td><br />
<td class="row">71.8 [>>100]</td><br />
<td class="row">9.8 [>>100]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, K<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>pTet-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of LuxI are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>M, LuxI</sub></b></td><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>9.575*10<sup>2</sup></td><br />
<td class='row'>2.975*10<sup>-9</sup></td><br />
<td class='row'>14.08</td><br />
<td class='row'>1.67</td><br />
<td class='row'>ND</td><br />
<td class='row'>76.73</td><br />
<br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
<br />
Explain that in all these evaluations we have estimated Nmax, mu, gamma_HSL from previous experiments - see measurement and modelling sections. When do we talk about HSL stability as a function of pH?<br />
Provide parameters of HSL. How do we present these data? It would be nice also to say what's the amount of HSL produced by a liquid 5 ml culture at a given OD600 in M9 medium after tot hours starting from 1:1000 dilution of a saturated ON culture..<br />
We nee a synthetic parameter to express LuxI activity as a function of PoPS in. I suggest to report the HSL vs pH analysis here AND in the AiiA section of registry and, for what concerns our wiki, to add an appendix to the 'measurement section' to wich link when explaining.. <br />
Decide figures! <br />
</p><br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- ---pSB1C3-mRFP-- --><br />
<!-- ----------------- --><br />
<br />
<a name='K999'></a><h2>pSB1C3 plasmid with mRFP between S and P bearing pTet (easy-to-clone)</h2><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<a name='Improvement'></a><h1>Existing parts: sequence debugging</h1><br />
<ol><ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516021 "> BBa_K516021 </a> (wiki name: E10 ) RBS31-AiiA-TT Rebuilt existing part from BBa_I13914 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516022 "> BBa_K516022 </a> (wiki name: E11 ) RBS32-AiiA-TT Rebuilt existing part from BBa_I13912 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516030 "> BBa_K516030 </a> (wiki name: E5 ) RBS30-mRFP-TT Rebuilt existing part from BBa_S04180 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516032 "> BBa_K516032 </a> (wiki name: E7 ) RBS32-mRFP-TT Rebuilt existing part from BBa_ J133000 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI Rebuilt existing part from BBa_S03623 (DNA available, only 2008 kit, inconsistent) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT Rebuilt existing part from BBa_ J22071 (2008 only, Bad sequencing) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT Rebuilt existing part from BBa_K077047 (Part deleted) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT Rebuilt existing part from BBa_I20252 (DNA planning) </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
</html><br />
<br />
{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Parts/CharacterizedTeam:UNIPV-Pavia/Parts/Characterized2011-09-17T22:17:55Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<html><br />
<a name='indice'></a><br />
<h2 class="art-postheader"><br />
Parts<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#new"><span class="tocnumber">1</span> <span class="toctext">New Parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#J101"><span class="tocnumber">1.1</span> <span class="toctext">J23101x series with different RBSs</span></a></li><br />
<li class="toclevel-2"><a href="#pTetAiiA"><span class="tocnumber">1.2</span> <span class="toctext">AiiA Expression cassette driven by aTc</span></a></li><br />
<li class="toclevel-2"><a href="#pTetLuxI"><span class="tocnumber">1.3</span> <span class="toctext">LuxI Expression cassette driven by aTc</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Existing"><span class="tocnumber">2</span> <span class="toctext">Characterization of existing parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#note"><span class="tocnumber">2.1</span> <span class="toctext">Notes on promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.2</span> <span class="toctext">RBSs from the community collection</span></a></li><br />
<li class="toclevel-2"><a href="#pLux"><span class="tocnumber">2.3</span> <span class="toctext">pLux - a 3OC6-HSL-in PoPs-out device</span></a></li><br />
<li class="toclevel-2"><a href="#pTet"><span class="tocnumber">2.4</span> <span class="toctext">pTet - BBa_R0040</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.5</span> <span class="toctext">AiiA - BBa_C0060</span></a></li><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.6</span> <span class="toctext">LuxI - BBa_C0061</span></a></li><br />
<li class="toclevel-2"><a href="#K999"><span class="tocnumber">2.7</span> <span class="toctext">pSB1C3 vector with mRFP between S and P for easy cloning</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Improvement"><span class="tocnumber">3</span> <span class="toctext">Sequence debugging</span></a></li><br />
</ul></td></tr></table><br />
</div><br />
<br />
<br />
<a name='new'></a><h1>New parts</h1><br />
<a name='J101'></a><h2>J23101x series</h2><br />
<br />
<ol type='1'><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> (wiki name: J101-E5 ) J101-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> (wiki name: J101-31 ) J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> (wiki name: J101-E7 ) J101-RBS32-mRFP-TT </li><br />
</ol><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- J23101x description --><br />
<!-- ----------------- --><br />
<p align='justify'><br />
BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in '<a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>data analysis</a>' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene. <br><br />
The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. <br>It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P.<br> <br />
This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts. <br><br />
Salis et al. [Nat Biotec, 2009] stated that<br />
<em> 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels'</em><br><br><br />
and again<br />
<em>'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.'</em> <br><br><br />
<br />
<br />
For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS.<br><br />
<br />
In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.<br />
<br />
<br><br><em><br />
<b>NB</b>: in the RPU computation, the J23101-RBS34-mRFP-TT construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency. </em><br />
<br />
<table align='center'><tr><td width='50%' ><br />
<table class='data'><br />
<tr><br />
<td class='row'><b>J23101 promoter with RBS</b><br />
</td><br />
<td class='row'><b>S<sub>cell</sub></b><br />
</td><br />
<td class='row'><b>R.P.U.s</b><br />
</td><br />
</tr><br />
<tr><td class='row'>RBS30<br />
</td><br />
<td class='row'>122 [13.23]</td><br />
<td class='row'>3.17</td><br />
</tr><br />
<tr><td class='row'>RBS31<br />
</td><br />
<td class='row'>2 [0.25]</td><br />
<td class='row'>0.05</td><br />
</tr><br />
<tr><td class='row'>RBS32<br />
</td><br />
<td class='row'>20 [1.7]</td><br />
<td class='row'>0.37</td><br />
</tr><br />
<tr><td class='row'>RBS34<br />
</td><br />
<td class='row'>50 [1.19]</td><br />
<td class='row'>1</td><br />
</tr><br />
</table></td><br />
<td width='50%' ><br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:UNIPV_RPU_J101.png" class="image"><img alt="RPUs of J23101 promoter with different RBSs" src="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="thumbimage" width="80%"></a></div></div><br />
</td><br />
</tr></table><br />
<br><br><br />
On the other hand, the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.<br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- pLuxAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetAiiA'></a><h2>AiiA expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> (wiki name: E24 ) pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> (wiki name: E25 ) pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- pTetAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetLuxI'></a><h2>LuxI expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> (wiki name: E13 ) pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> (wiki name: E14 ) pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> (wiki name: E15 ) pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<a name='Existing'></a><h1>Existing parts</h1><br />
<br />
<a name='note'></a><h2>Notes for promoter characterization</h2><br />
<p align='justify'><br />
Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection. <br />
<br><br><br />
The assembled RBSs are:<br />
<br><br><br />
<div align='center'><br />
<table class='data'><br />
<tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr><br />
<tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr><br />
<tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr><br />
<tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr><br />
<tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr><br />
</table></div><br />
<br><br />
For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.<br />
<br><br><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. <br />
RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. <br><br />
It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP). <br><br><br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element.<br />
Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.<br />
<br />
</p><br />
<br />
<p><br />
Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least sqares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs :<br />
<br><br />
<ol><ul><li><b><br />
RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity,<br />
</li><li><b><br />
RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity,<br />
</li><li><br />
<b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and represents the heart of linear region,<br />
</li><br />
<li><br />
<b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</li><br />
</ul></ol><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --RBS description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='rbs'></a><h2>RBSs</h2><br />
<p align='justify'><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of mRFP produced. <br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. <br />
The evaluation of RBS efficiency can be performed in a very intuitive fashion:<br><br />
<ul><br />
<li>1. select the RBSs you want to study, </li><br />
<li>2. assemble them in a Promoter - XX - Coding sequence circuit, </li></ul></ol><br />
<br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:Vettore_base.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d1/Vettore_base.jpg" class="thumbimage" width="50%"></a></div></div><br />
<br />
<ul><br />
<li>3. measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS. </li><br />
</ul></ol><br><br />
This simple measurement system allows the quantification of RBS efficiency depending on the whole measurement system (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in a complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments. <br><br />
To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.<br><br />
In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, pTet, pLux). Measuring the system output and evaluating the RBS efficiency. The results are summarized in the table below:<br><br><br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>pLux</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>pTet</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>J23101</sub></b><sup>**</sup></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>0.40</td><td class='row'>1.6814</td><td class='row'>3.17</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.01</td><td class='row'>ND</td><td class='row'>0.05</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.19</td><td class='row'>0.4193</td><td class='row'>0.37</td><td class='row'>0,3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<br />
<br><br />
On the other end, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (pTet-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below:<br><br><br />
<br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>mRFP</sub></b><sup>**</sup></td><br />
<td class='row'><b>eff<sub>AiiA</sub></b><sup>***</sup></td><br />
<td class='row'><b>eff<sub>LuxI</sub><sup>****</sup></b></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>1.72</td><td class='row'>0.53</td><td class='row'>0.64</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.03</td><td class='row'>0.83</td><td class='row'>0.08</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.37</td><td class='row'>0.50</td><td class='row'>N.D.</td><td class='row'>0.3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<p align='justify'><br />
<sup>*</sup> The RBS efficiency for inducible devices expressing mRFP was estimated as the ratio of the AUCs (Area under the curve) of the induction curve of the system with the studied RBS and the B0034 reference: AUC<sub>P, RBSx</sub>/AUC<sub>P, B0034</sub><br><br />
<sup>**</sup> The RBS efficiency for constitutive promoters expressing mRFP was computed as the ratio between S<sub>cell<sub>P, RBSx</sub></sub>/S<sub>cell<sub>P, B0034</sub></sub><br><br />
<sup>***</sup> The RBS efficiency for pTet promoter driving the expression of AiiA enzyme was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated for the measurement system pTet-RBSx-AiiA-TT. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#AiiA'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<sup>****</sup>The RBS efficiency for promoters driving the expression of LuxI was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated from the measurement systems pTet-RBSx-LuxI. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#LuxI'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<br />
<br><br />
</p><br />
<br><br><br />
The parts we used to characterize the RBSs are listed here:<br />
<ol><br />
<ul><li>mRFP expression with different promoters</li><br />
<ul><br />
<li>J23101</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> J101-RBS30-RFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> J101-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_J23101 "> BBa_J23101 </a> J101-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pTet</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pLux</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li><br />
</ul><br />
</ul><br />
<li>pTet driving the expression of different genes</li><br />
<ul><li>mRFP</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>AiiA</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> pTet-RBS34-AiiA-TT </li><br />
</ul><br />
<li>LuxI</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> pTet-RBS34-LuxI </li><br />
</ul></ul><br />
</ul></ol><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --pLux description- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<a name='pLux'></a><h2>pLux promoter</h2><br />
<p align='justify'><br />
<br />
<br />
</p><br />
<ol><ul><br />
<br />
<br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> (wiki name: E17 ) pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> (wiki name: E18 ) pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> (wiki name: E19 ) pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> (wiki name: E20 ) pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li></ul><br />
</ol><br />
<br />
<p align='justify'><br />
The estimated parameters for the Hill functions are summarized in the table below. For more details on parameter estimation, see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>model section</a>. <br />
</p><br />
<br />
<table class='data' width='100%'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Lux</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">438 [10]</td><br />
<td class="row">0.05 [>100]</td><br />
<td class="row">2 [47]</td><br />
<td class="row">1.88 [27]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">9.8 [7]</td><br />
<td class="row">0.11 [57]</td><br />
<td class="row">1.2 [29]</td><br />
<td class="row">1.5 [26]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">206 [3]</td><br />
<td class="row">0 [>>100]</td><br />
<td class="row">1.36 [10]</td><br />
<td class="row">1.87 [9]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">1105 [6]</td><br />
<td class="row">0.02 [>100]</td><br />
<td class="row">1.33 [19]</td><br />
<td class="row">2.34 [18]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br />
<br />
<p align='justify'><br />
From this table, it is evident that, whilst &alpha;<sub>pLux</sub> assumes significantly different values for different RBSs, &eta;<sub>pLux</sub> and k<sub>pLux</sub> assume very similar values. This result shows that RBS variation modulates the amplitude of Hill function, not affecting the shape of the curve. The four induction curves result to be the same Hill function modulated in amplitude by a parameter, that is the estimated RBS efficiency for this measurement system.<br><br />
These results are quite encouraging, because suggest that, given the non-modular behavior of RBS dpending on the encoded gene, the RBS has a modular behaviour respect to the promoter. <br />
<br />
</p><br />
<br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>4.28</td><td class='row'>0.20</td><td class='row'>1.08</td><td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>4.93</td><td class='row'>0.55</td><td class='row'>0.25</td><td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>9.49</td><td class='row'>0.02</td><td class='row'>0.47</td><td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>21.53</td><td class='row'>0.51</td><td class='row'>0.53</td><td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<p align='justify'><br />
<br />
These operative parameters provide useful information on the behavior of this 3OC6-HSL inducible device. RPU<sub>max</sub> assumes very different values, showing that the modulation in amplitude of the Hill can't be explained by a linear dependance on the RBS efficiency (in this case, in fact, the same RPUs should be observed for every RBS, since the standard reference used for RPUs computation)<br />
<br />
</p><br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- -pTet description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='pTet'></a><h2>pTet promoter</h2><br />
<br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> (wiki name: E21 ) pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> (wiki name: E22 ) pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT<br />
</li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT <br />
</li></ul><br />
</ul><br />
</ol><br />
<p align='justify'><br />
<br />
The protocols for the characterization of pTet promoter are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#pTet_protocol'>pTet measurement section</a>. <br><br />
This promoter is widely studied and characterized usually using the strong RBS BBa_B0034. Here we have characterized its transcriptional strength as a function of aTc induction (ng/ul) for different RBSs. Four different induction curves were obtained and are reported in figure:<br><br><br />
<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
The data collected from the mRFP measurement systems were processed as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'> data analysis section</a>. The induction curves were obtained by fitting a Hill function as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>modelling section</a> and the estimated <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>parameters</a> for pTet are reported in the pictures and in table below. </p><br />
<p align='justify'><br />
The estimated parameters of the Hill curves described in the figures are summarized in the table below:<br />
</p><br />
<br><br><br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">230.67 [3.7]</td><br />
<td class="row">0.028 [91.61]</td><br />
<td class="row">4.61 [23.73]</td><br />
<td class="row">8.75 [4.16]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">55.77 [12]</td><br />
<td class="row">1.53E-11 [>>100]</td><br />
<td class="row">4.98 [57.62]</td><br />
<td class="row">7.26 [14.98]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">120 [5.95]</td><br />
<td class="row">0.085 [40.6]</td><br />
<td class="row">24.85 [47.6]</td><br />
<td class="row">9 [5.43]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br><br><br />
<br />
<p align='justify'><br />
The measurement system pTet-B0031-mRFP-TT couldn't be assayed because its fluorescence output is under the detectability threshold of our measurement instrument. For this reason, the parameters of the corresponding Hill curve couldn't be estimated and are reported as 'Not Determined' ND. <br />
</p><br />
<br />
<p align='justify'><br />
&alpha; parameter (representing the maximum trascriptional rate in the studied range of induction) varies as expected with the RBS variation and also the &delta; and &eta; parameters are quite different among the RBS variations.<br><br />
The K<sub>pTet</sub> parameter is quite constant among the RBS variations, thus suggesting that in this case the RBS variation doesn't substantially affect the switch point of the Hill curve, even if the amplitude and the maximum slope are not quite maintained (for the &eta; parameter, maybe fitting problems). <br />
<br />
</p><br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [ng/&mu;]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [ng/&mu;]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>1.53</td><td class='row'>~0</td><td class='row'>7.95</td><td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>3.16</td><td class='row'>~0</td><td class='row'>6.7</td><td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>2.73</td><td class='row'>0.23</td><td class='row'>8.96</td><td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
From these parameters, it is evident that whilst the switch-point is almost maintained for all the RBSs, the linear boundaries are similar for RBS30 and RBS32 but for RBS34 are moved on the right of one order of magnitude. <br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --AiiA description-- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<br />
<a name='AiiA'></a><h2>AiiA gene - BBa_C0060</h2><br />
<br />
<p align='justify'><br />
The activity of AiiA enzyme has been evaluated by testing the measurement systems <a href='<a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a>. <br />
The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
</p><br />
<br />
<br />
<br><br><br />
dAiiA/dt=&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>AiiA</sub>*AiiA<br><br><br />
d[HSL]/dt=N*K<sub>cat</sub>*AiiA*HSL/(K<sub>M, AiiA</sub>+AiiA))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
<p align='justify><br />
<br />
Since the measurement systems are only assayed in the exponential growth phase, the third equation can be modified as follows:<br />
</p><br />
dN/dt=N*&mu;<br />
<br />
<p align='justify'><br />
because N&lt;N<sub>max</sub>. <br><br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.003152</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
<br />
<br />
<br />
<p align='justify'><br />
<br />
The parameters K<sub>cat</sub> and K<sub>M,AiiA</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of AiiA are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>cat</sub></b></td><br />
<td class='row'><b>K<sub>M, AiiA</sub></b></td><br />
</tr><br />
<tr><td class='row'></td><br />
<td class='row'></td><br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
<br />
<br />
</p><br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --LuxI description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='LuxI'></a><h2>LuxI gene - BBa_C0061</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#t9002'>modeling section</a>). <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
<br />
<br><br><br />
dLuxI/dt=&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>LuxI</sub>*LuxI<br><br><br />
d[HSL]/dt=N*V<sub>max</sub>*1/(1+(K<sub>M, LuxI</sub>/LuxI))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
<br />
<p align='justify><br />
<br />
Since the measurement systems are only assayed in the exponential growth phase, the third equation can be modified as follows:<br />
</p><br />
dN/dt=N*&mu;<br />
<br />
<p align='justify'><br />
because N&lt;N<sub>max</sub>. <br><br />
The parameters of the first equation (estimated from the part pTet-RBSx-mRFP-TT) and N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:</p><br />
<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">215 [2]</td><br />
<td class="row">0.001 [>>100]</td><br />
<td class="row">4.3 [12]</td><br />
<td class="row">8.3 [2]</td><br />
<td class='row' rowspan='4'>1*10<sup>9</sup></td><br />
<td class='row' rowspan='4'>0.003152</td><br />
<td class='row' rowspan='4'>0</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">0.7 [>>100]</td><br />
<td class="row">0[>>100]</td><br />
<td class="row">18.84[>>100]</td><br />
<td class="row">47.51[>>100]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">45.7 [11]</td><br />
<td class="row">0.02 [>>100]</td><br />
<td class="row">83 [>>100]</td><br />
<td class="row">8 [>>100]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">125 [12]</td><br />
<td class="row">0.12 [61]</td><br />
<td class="row">71.8 [>>100]</td><br />
<td class="row">9.8 [>>100]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, K<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>pTet-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of LuxI are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>M, LuxI</sub></b></td><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>9.575*10<sup>2</sup></td><br />
<td class='row'>2.975*10<sup>-9</sup></td><br />
<td class='row'>14.08</td><br />
<td class='row'>1.67</td><br />
<td class='row'>ND</td><br />
<td class='row'>76.73</td><br />
<br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
<br />
Explain that in all these evaluations we have estimated Nmax, mu, gamma_HSL from previous experiments - see measurement and modelling sections. When do we talk about HSL stability as a function of pH?<br />
Provide parameters of HSL. How do we present these data? It would be nice also to say what's the amount of HSL produced by a liquid 5 ml culture at a given OD600 in M9 medium after tot hours starting from 1:1000 dilution of a saturated ON culture..<br />
We nee a synthetic parameter to express LuxI activity as a function of PoPS in. I suggest to report the HSL vs pH analysis here AND in the AiiA section of registry and, for what concerns our wiki, to add an appendix to the 'measurement section' to wich link when explaining.. <br />
Decide figures! <br />
</p><br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- ---pSB1C3-mRFP-- --><br />
<!-- ----------------- --><br />
<br />
<a name='K999'></a><h2>pSB1C3 plasmid with mRFP between S and P bearing pTet (easy-to-clone)</h2><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<a name='Improvement'></a><h1>Existing parts: sequence debugging</h1><br />
<ol><ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516021 "> BBa_K516021 </a> (wiki name: E10 ) RBS31-AiiA-TT Rebuilt existing part from BBa_I13914 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516022 "> BBa_K516022 </a> (wiki name: E11 ) RBS32-AiiA-TT Rebuilt existing part from BBa_I13912 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516030 "> BBa_K516030 </a> (wiki name: E5 ) RBS30-mRFP-TT Rebuilt existing part from BBa_S04180 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516032 "> BBa_K516032 </a> (wiki name: E7 ) RBS32-mRFP-TT Rebuilt existing part from BBa_ J133000 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI Rebuilt existing part from BBa_S03623 (DNA available, only 2008 kit, inconsistent) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT Rebuilt existing part from BBa_ J22071 (2008 only, Bad sequencing) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT Rebuilt existing part from BBa_K077047 (Part deleted) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT Rebuilt existing part from BBa_I20252 (DNA planning) </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
</html><br />
<br />
{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Parts/CharacterizedTeam:UNIPV-Pavia/Parts/Characterized2011-09-17T22:12:46Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<html><br />
<a name='indice'></a><br />
<h2 class="art-postheader"><br />
Parts<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#new"><span class="tocnumber">1</span> <span class="toctext">New Parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#J101"><span class="tocnumber">1.1</span> <span class="toctext">J23101x series with different RBSs</span></a></li><br />
<li class="toclevel-2"><a href="#pTetAiiA"><span class="tocnumber">1.2</span> <span class="toctext">AiiA Expression cassette driven by aTc</span></a></li><br />
<li class="toclevel-2"><a href="#pTetLuxI"><span class="tocnumber">1.3</span> <span class="toctext">LuxI Expression cassette driven by aTc</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Existing"><span class="tocnumber">2</span> <span class="toctext">Characterization of existing parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#note"><span class="tocnumber">2.1</span> <span class="toctext">Notes on promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.2</span> <span class="toctext">RBSs from the community collection</span></a></li><br />
<li class="toclevel-2"><a href="#pLux"><span class="tocnumber">2.3</span> <span class="toctext">pLux - a 3OC6-HSL-in PoPs-out device</span></a></li><br />
<li class="toclevel-2"><a href="#pTet"><span class="tocnumber">2.4</span> <span class="toctext">pTet - BBa_R0040</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.5</span> <span class="toctext">AiiA - BBa_C0060</span></a></li><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.6</span> <span class="toctext">LuxI - BBa_C0061</span></a></li><br />
<li class="toclevel-2"><a href="#K999"><span class="tocnumber">2.7</span> <span class="toctext">pSB1C3 vector with mRFP between S and P for easy cloning</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Improvement"><span class="tocnumber">3</span> <span class="toctext">Sequence debugging</span></a></li><br />
</ul></td></tr></table><br />
</div><br />
<br />
<br />
<a name='new'></a><h1>New parts</h1><br />
<a name='J101'></a><h2>J23101x series</h2><br />
<br />
<ol type='1'><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> (wiki name: J101-E5 ) J101-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> (wiki name: J101-31 ) J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> (wiki name: J101-E7 ) J101-RBS32-mRFP-TT </li><br />
</ol><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- J23101x description --><br />
<!-- ----------------- --><br />
<p align='justify'><br />
BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in '<a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>data analysis</a>' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene. <br><br />
The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. <br>It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P.<br> <br />
This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts. <br><br />
Salis et al. [Nat Biotec, 2009] stated that<br />
<em> 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels'</em><br><br><br />
and again<br />
<em>'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.'</em> <br><br><br />
<br />
<br />
For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS.<br><br />
<br />
In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.<br />
<br />
<br><br><em><br />
<b>NB</b>: in the RPU computation, the J23101-RBS34-mRFP-TT construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency. </em><br />
<br />
<table align='center'><tr><td width='50%' ><br />
<table class='data'><br />
<tr><br />
<td class='row'><b>J23101 promoter with RBS</b><br />
</td><br />
<td class='row'><b>S<sub>cell</sub></b><br />
</td><br />
<td class='row'><b>R.P.U.s</b><br />
</td><br />
</tr><br />
<tr><td class='row'>RBS30<br />
</td><br />
<td class='row'>122 [13.23]</td><br />
<td class='row'>3.17</td><br />
</tr><br />
<tr><td class='row'>RBS31<br />
</td><br />
<td class='row'>2 [0.25]</td><br />
<td class='row'>0.05</td><br />
</tr><br />
<tr><td class='row'>RBS32<br />
</td><br />
<td class='row'>20 [1.7]</td><br />
<td class='row'>0.37</td><br />
</tr><br />
<tr><td class='row'>RBS34<br />
</td><br />
<td class='row'>50 [1.19]</td><br />
<td class='row'>1</td><br />
</tr><br />
</table></td><br />
<td width='50%' ><br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:UNIPV_RPU_J101.png" class="image"><img alt="RPUs of J23101 promoter with different RBSs" src="https://static.igem.org/mediawiki/2011/b/bb/UNIPV_RPU_J101.png" class="thumbimage" width="80%"></a></div></div><br />
</td><br />
</tr></table><br />
<br><br><br />
On the other hand, the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.<br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- pLuxAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetAiiA'></a><h2>AiiA expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> (wiki name: E24 ) pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> (wiki name: E25 ) pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- pTetAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetLuxI'></a><h2>LuxI expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> (wiki name: E13 ) pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> (wiki name: E14 ) pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> (wiki name: E15 ) pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<a name='Existing'></a><h1>Existing parts</h1><br />
<br />
<a name='note'></a><h2>Notes for promoter characterization</h2><br />
<p align='justify'><br />
Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection. <br />
<br><br><br />
The assembled RBSs are:<br />
<br><br><br />
<div align='center'><br />
<table class='data'><br />
<tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr><br />
<tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr><br />
<tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr><br />
<tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr><br />
<tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr><br />
</table></div><br />
<br><br />
For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.<br />
<br><br><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. <br />
RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. <br><br />
It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP). <br><br><br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element.<br />
Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.<br />
<br />
</p><br />
<br />
<p><br />
Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least sqares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs :<br />
<br><br />
<ol><ul><li><b><br />
RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity,<br />
</li><li><b><br />
RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity,<br />
</li><li><br />
<b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and represents the heart of linear region,<br />
</li><br />
<li><br />
<b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</li><br />
</ul></ol><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --RBS description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='rbs'></a><h2>RBSs</h2><br />
<p align='justify'><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of mRFP produced. <br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. <br />
The evaluation of RBS efficiency can be performed in a very intuitive fashion:<br><br />
<ul><br />
<li>1. select the RBSs you want to study, </li><br />
<li>2. assemble them in a Promoter - XX - Coding sequence circuit, </li></ul></ol><br />
<br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:Vettore_base.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d1/Vettore_base.jpg" class="thumbimage" width="50%"></a></div></div><br />
<br />
<ul><br />
<li>3. measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS. </li><br />
</ul></ol><br><br />
This simple measurement system allows the quantification of RBS efficiency depending on the whole measurement system (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in a complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments. <br><br />
To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.<br><br />
In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, pTet, pLux). Measuring the system output and evaluating the RBS efficiency. The results are summarized in the table below:<br><br><br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>pLux</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>pTet</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>J23101</sub></b><sup>**</sup></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>0.40</td><td class='row'>1.6814</td><td class='row'>3.17</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.01</td><td class='row'>ND</td><td class='row'>0.05</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.19</td><td class='row'>0.4193</td><td class='row'>0.37</td><td class='row'>0,3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<br />
<br><br />
On the other end, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (pTet-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below:<br><br><br />
<br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>mRFP</sub></b><sup>**</sup></td><br />
<td class='row'><b>eff<sub>AiiA</sub></b><sup>***</sup></td><br />
<td class='row'><b>eff<sub>LuxI</sub><sup>****</sup></b></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>1.72</td><td class='row'>0.53</td><td class='row'>0.64</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.03</td><td class='row'>0.83</td><td class='row'>0.08</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.37</td><td class='row'>0.50</td><td class='row'>N.D.</td><td class='row'>0.3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<p align='justify'><br />
<sup>*</sup> The RBS efficiency for inducible devices expressing mRFP was estimated as the ratio of the AUCs (Area under the curve) of the induction curve of the system with the studied RBS and the B0034 reference: AUC<sub>P, RBSx</sub>/AUC<sub>P, B0034</sub><br><br />
<sup>**</sup> The RBS efficiency for constitutive promoters expressing mRFP was computed as the ratio between S<sub>cell<sub>P, RBSx</sub></sub>/S<sub>cell<sub>P, B0034</sub></sub><br><br />
<sup>***</sup> The RBS efficiency for pTet promoter driving the expression of AiiA enzyme was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated for the measurement system pTet-RBSx-AiiA-TT. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#AiiA'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<sup>****</sup>The RBS efficiency for promoters driving the expression of LuxI was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated from the measurement systems pTet-RBSx-LuxI. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#LuxI'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<br />
<br><br />
</p><br />
<br><br><br />
The parts we used to characterize the RBSs are listed here:<br />
<ol><br />
<ul><li>mRFP expression with different promoters</li><br />
<ul><br />
<li>J23101</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> J101-RBS30-RFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> J101-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_J23101 "> BBa_J23101 </a> J101-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pTet</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pLux</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li><br />
</ul><br />
</ul><br />
<li>pTet driving the expression of different genes</li><br />
<ul><li>mRFP</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>AiiA</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> pTet-RBS34-AiiA-TT </li><br />
</ul><br />
<li>LuxI</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> pTet-RBS34-LuxI </li><br />
</ul></ul><br />
</ul></ol><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --pLux description- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<a name='pLux'></a><h2>pLux promoter</h2><br />
<p align='justify'><br />
<br />
<br />
</p><br />
<ol><ul><br />
<br />
<br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> (wiki name: E17 ) pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> (wiki name: E18 ) pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> (wiki name: E19 ) pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> (wiki name: E20 ) pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li></ul><br />
</ol><br />
<br />
<p align='justify'><br />
The estimated parameters for the Hill functions are summarized in the table below. For more details on parameter estimation, see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>model section</a>. <br />
</p><br />
<br />
<table class='data' width='100%'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Lux</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">438 [10]</td><br />
<td class="row">0.05 [>100]</td><br />
<td class="row">2 [47]</td><br />
<td class="row">1.88 [27]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">9.8 [7]</td><br />
<td class="row">0.11 [57]</td><br />
<td class="row">1.2 [29]</td><br />
<td class="row">1.5 [26]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">206 [3]</td><br />
<td class="row">0 [>>100]</td><br />
<td class="row">1.36 [10]</td><br />
<td class="row">1.87 [9]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">1105 [6]</td><br />
<td class="row">0.02 [>100]</td><br />
<td class="row">1.33 [19]</td><br />
<td class="row">2.34 [18]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br />
<br />
<p align='justify'><br />
From this table, it is evident that, whilst &alpha;<sub>pLux</sub> assumes significantly different values for different RBSs, &eta;<sub>pLux</sub> and k<sub>pLux</sub> assume very similar values. This result shows that RBS variation modulates the amplitude of Hill function, not affecting the shape of the curve. The four induction curves result to be the same Hill function modulated in amplitude by a parameter, that is the estimated RBS efficiency for this measurement system.<br><br />
These results are quite encouraging, because suggest that, given the non-modular behavior of RBS dpending on the encoded gene, the RBS has a modular behaviour respect to the promoter. <br />
<br />
</p><br />
<br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>4.28</td><td class='row'>0.20</td><td class='row'>1.08</td><td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>4.93</td><td class='row'>0.55</td><td class='row'>0.25</td><td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>9.49</td><td class='row'>0.02</td><td class='row'>0.47</td><td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>21.53</td><td class='row'>0.51</td><td class='row'>0.53</td><td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<p align='justify'><br />
<br />
These operative parameters provide useful information on the behavior of this 3OC6-HSL inducible device. RPU<sub>max</sub> assumes very different values, showing that the modulation in amplitude of the Hill can't be explained by a linear dependance on the RBS efficiency (in this case, in fact, the same RPUs should be observed for every RBS, since the standard reference used for RPUs computation)<br />
<br />
</p><br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- -pTet description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='pTet'></a><h2>pTet promoter</h2><br />
<br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> (wiki name: E21 ) pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> (wiki name: E22 ) pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT<br />
</li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT <br />
</li></ul><br />
</ul><br />
</ol><br />
<p align='justify'><br />
<br />
The protocols for the characterization of pTet promoter are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#pTet_protocol'>pTet measurement section</a>. <br><br />
This promoter is widely studied and characterized usually using the strong RBS BBa_B0034. Here we have characterized its transcriptional strength as a function of aTc induction (ng/ul) for different RBSs. Four different induction curves were obtained and are reported in figure:<br><br><br />
<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
The data collected from the mRFP measurement systems were processed as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'> data analysis section</a>. The induction curves were obtained by fitting a Hill function as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>modelling section</a> and the estimated <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>parameters</a> for pTet are reported in the pictures and in table below. </p><br />
<p align='justify'><br />
The estimated parameters of the Hill curves described in the figures are summarized in the table below:<br />
</p><br />
<br><br><br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">230.67 [3.7]</td><br />
<td class="row">0.028 [91.61]</td><br />
<td class="row">4.61 [23.73]</td><br />
<td class="row">8.75 [4.16]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">55.77 [12]</td><br />
<td class="row">1.53E-11 [>>100]</td><br />
<td class="row">4.98 [57.62]</td><br />
<td class="row">7.26 [14.98]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">120 [5.95]</td><br />
<td class="row">0.085 [40.6]</td><br />
<td class="row">24.85 [47.6]</td><br />
<td class="row">9 [5.43]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br><br><br />
<br />
<p align='justify'><br />
The measurement system pTet-B0031-mRFP-TT couldn't be assayed because its fluorescence output is under the detectability threshold of our measurement instrument. For this reason, the parameters of the corresponding Hill curve couldn't be estimated and are reported as 'Not Determined' ND. <br />
</p><br />
<br />
<p align='justify'><br />
&alpha; parameter (representing the maximum trascriptional rate in the studied range of induction) varies as expected with the RBS variation and also the &delta; and &eta; parameters are quite different among the RBS variations.<br><br />
The K<sub>pTet</sub> parameter is quite constant among the RBS variations, thus suggesting that in this case the RBS variation doesn't substantially affect the switch point of the Hill curve, even if the amplitude and the maximum slope are not quite maintained (for the &eta; parameter, maybe fitting problems). <br />
<br />
</p><br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [ng/&mu;]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [ng/&mu;]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>1.53</td><td class='row'>~0</td><td class='row'>7.95</td><td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>3.16</td><td class='row'>~0</td><td class='row'>6.7</td><td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>2.73</td><td class='row'>0.23</td><td class='row'>8.96</td><td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
From these parameters, it is evident that whilst the switch-point is almost maintained for all the RBSs, the linear boundaries are similar for RBS30 and RBS32 but for RBS34 are moved on the right of one order of magnitude. <br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --AiiA description-- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<br />
<a name='AiiA'></a><h2>AiiA gene - BBa_C0060</h2><br />
<br />
<p align='justify'><br />
The activity of AiiA enzyme has been evaluated by testing the measurement systems <a href='<a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a>. <br />
The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
</p><br />
<br />
<br />
<br><br><br />
dAiiA/dt=&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>AiiA</sub>*AiiA<br><br><br />
d[HSL]/dt=N*K<sub>cat</sub>*AiiA*HSL/(K<sub>M, AiiA</sub>+AiiA))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
<p align='justify><br />
<br />
Since the measurement systems are only assayed in the exponential growth phase, the third equation can be modified as follows:<br />
</p><br />
dN/dt=N*&mu;<br />
<br />
<p align='justify'><br />
because N<N<sub>max</sub>. <br><br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.003152</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
<br />
<br />
<br />
<p align='justify'><br />
<br />
The parameters K<sub>cat</sub> and K<sub>M,AiiA</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of AiiA are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>cat</sub></b></td><br />
<td class='row'><b>K<sub>M, AiiA</sub></b></td><br />
</tr><br />
<tr><td class='row'></td><br />
<td class='row'></td><br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
<br />
<br />
</p><br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --LuxI description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='LuxI'></a><h2>LuxI gene - BBa_C0061</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#t9002'>modeling section</a>). <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
<br />
<br><br><br />
dLuxI/dt=&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>LuxI</sub>*LuxI<br><br><br />
d[HSL]/dt=N*V<sub>max</sub>*1/(1+(K<sub>M, LuxI</sub>/LuxI))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
The parameters of the first equation (estimated from the part pTet-RBSx-mRFP-TT) and N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:<br />
<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">215 [2]</td><br />
<td class="row">0.001 [>>100]</td><br />
<td class="row">4.3 [12]</td><br />
<td class="row">8.3 [2]</td><br />
<td class='row' rowspan='4'>1*10<sup>9</sup></td><br />
<td class='row' rowspan='4'>0.003152</td><br />
<td class='row' rowspan='4'>0</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">0.7 [>>100]</td><br />
<td class="row">0[>>100]</td><br />
<td class="row">18.84[>>100]</td><br />
<td class="row">47.51[>>100]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">45.7 [11]</td><br />
<td class="row">0.02 [>>100]</td><br />
<td class="row">83 [>>100]</td><br />
<td class="row">8 [>>100]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">125 [12]</td><br />
<td class="row">0.12 [61]</td><br />
<td class="row">71.8 [>>100]</td><br />
<td class="row">9.8 [>>100]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, K<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>pTet-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of LuxI are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>M, LuxI</sub></b></td><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>9.575*10<sup>2</sup></td><br />
<td class='row'>2.975*10<sup>-9</sup></td><br />
<td class='row'>14.08</td><br />
<td class='row'>1.67</td><br />
<td class='row'>ND</td><br />
<td class='row'>76.73</td><br />
<br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
<br />
Explain that in all these evaluations we have estimated Nmax, mu, gamma_HSL from previous experiments - see measurement and modelling sections. When do we talk about HSL stability as a function of pH?<br />
Provide parameters of HSL. How do we present these data? It would be nice also to say what's the amount of HSL produced by a liquid 5 ml culture at a given OD600 in M9 medium after tot hours starting from 1:1000 dilution of a saturated ON culture..<br />
We nee a synthetic parameter to express LuxI activity as a function of PoPS in. I suggest to report the HSL vs pH analysis here AND in the AiiA section of registry and, for what concerns our wiki, to add an appendix to the 'measurement section' to wich link when explaining.. <br />
Decide figures! <br />
</p><br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- ---pSB1C3-mRFP-- --><br />
<!-- ----------------- --><br />
<br />
<a name='K999'></a><h2>pSB1C3 plasmid with mRFP between S and P bearing pTet (easy-to-clone)</h2><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<a name='Improvement'></a><h1>Existing parts: sequence debugging</h1><br />
<ol><ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516021 "> BBa_K516021 </a> (wiki name: E10 ) RBS31-AiiA-TT Rebuilt existing part from BBa_I13914 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516022 "> BBa_K516022 </a> (wiki name: E11 ) RBS32-AiiA-TT Rebuilt existing part from BBa_I13912 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516030 "> BBa_K516030 </a> (wiki name: E5 ) RBS30-mRFP-TT Rebuilt existing part from BBa_S04180 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516032 "> BBa_K516032 </a> (wiki name: E7 ) RBS32-mRFP-TT Rebuilt existing part from BBa_ J133000 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI Rebuilt existing part from BBa_S03623 (DNA available, only 2008 kit, inconsistent) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT Rebuilt existing part from BBa_ J22071 (2008 only, Bad sequencing) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT Rebuilt existing part from BBa_K077047 (Part deleted) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT Rebuilt existing part from BBa_I20252 (DNA planning) </li></ul></ol><br />
<br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Parts/CharacterizedTeam:UNIPV-Pavia/Parts/Characterized2011-09-17T22:11:40Z<p>Nickpv: </p>
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Parts<br />
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#new"><span class="tocnumber">1</span> <span class="toctext">New Parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#J101"><span class="tocnumber">1.1</span> <span class="toctext">J23101x series with different RBSs</span></a></li><br />
<li class="toclevel-2"><a href="#pTetAiiA"><span class="tocnumber">1.2</span> <span class="toctext">AiiA Expression cassette driven by aTc</span></a></li><br />
<li class="toclevel-2"><a href="#pTetLuxI"><span class="tocnumber">1.3</span> <span class="toctext">LuxI Expression cassette driven by aTc</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Existing"><span class="tocnumber">2</span> <span class="toctext">Characterization of existing parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#note"><span class="tocnumber">2.1</span> <span class="toctext">Notes on promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.2</span> <span class="toctext">RBSs from the community collection</span></a></li><br />
<li class="toclevel-2"><a href="#pLux"><span class="tocnumber">2.3</span> <span class="toctext">pLux - a 3OC6-HSL-in PoPs-out device</span></a></li><br />
<li class="toclevel-2"><a href="#pTet"><span class="tocnumber">2.4</span> <span class="toctext">pTet - BBa_R0040</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.5</span> <span class="toctext">AiiA - BBa_C0060</span></a></li><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.6</span> <span class="toctext">LuxI - BBa_C0061</span></a></li><br />
<li class="toclevel-2"><a href="#K999"><span class="tocnumber">2.7</span> <span class="toctext">pSB1C3 vector with mRFP between S and P for easy cloning</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Improvement"><span class="tocnumber">3</span> <span class="toctext">Sequence debugging</span></a></li><br />
</ul></td></tr></table><br />
</div><br />
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<a name='new'></a><h1>New parts</h1><br />
<a name='J101'></a><h2>J23101x series</h2><br />
<br />
<ol type='1'><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> (wiki name: J101-E5 ) J101-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> (wiki name: J101-31 ) J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> (wiki name: J101-E7 ) J101-RBS32-mRFP-TT </li><br />
</ol><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- J23101x description --><br />
<!-- ----------------- --><br />
<br />
<p align='justify'><br />
BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in '<a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>data analysis</a>' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene. <br><br />
The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. <br>It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P.<br> <br />
This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts. <br><br />
Salis et al. [Nat Biotec, 2009] stated that<br />
<em> 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels'</em><br><br><br />
and again<br />
<em>'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.'</em> <br><br><br />
<br />
<br />
For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS.<br><br />
<br />
In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.<br />
<br />
<br><br><em><br />
<b>NB</b>: in the RPU computation, the J23101-RBS34-mRFP-TT construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency. </em><br />
<br />
<table align='center'><tr><td width='50%' ><br />
<table class='data'><br />
<tr><br />
<td class='row'><b>J23101 promoter with RBS</b><br />
</td><br />
<td class='row'><b>S<sub>cell</sub></b><br />
</td><br />
<td class='row'><b>R.P.U.s</b><br />
</td><br />
</tr><br />
<tr><td class='row'>RBS30<br />
</td><br />
<td class='row'>150.97 [15.34]</td><br />
<td class='row'>3.17</td><br />
</tr><br />
<tr><td class='row'>RBS31<br />
</td><br />
<td class='row'>2.30 [0.37]</td><br />
<td class='row'>0.05</td><br />
</tr><br />
<tr><td class='row'>RBS32<br />
</td><br />
<td class='row'>17.60 [5.85]</td><br />
<td class='row'>0.37</td><br />
</tr><br />
<tr><td class='row'>RBS34<br />
</td><br />
<td class='row'>47.57 [0.82]</td><br />
<td class='row'>1</td><br />
</tr><br />
</table></td><br />
<td width='50%' ><br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:RPU_J101.png" class="image"><img alt="RPUs of J23101 promoter with different RBSs" src="https://static.igem.org/mediawiki/2011/1/12/RPU_J101.png" class="thumbimage" width="80%"></a></div></div><br />
</td><br />
</tr></table><br />
<br><br><br />
On the other hand, the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.<br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!-- ----------------- --><br />
<!-- pLuxAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetAiiA'></a><h2>AiiA expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> (wiki name: E24 ) pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> (wiki name: E25 ) pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!-- ----------------- --><br />
<!-- pTetAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetLuxI'></a><h2>LuxI expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> (wiki name: E13 ) pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> (wiki name: E14 ) pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> (wiki name: E15 ) pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI </li></ul></ol><br />
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<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<a name='Existing'></a><h1>Existing parts</h1><br />
<br />
<a name='note'></a><h2>Notes for promoter characterization</h2><br />
<p align='justify'><br />
Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection. <br />
<br><br><br />
The assembled RBSs are:<br />
<br><br><br />
<div align='center'><br />
<table class='data'><br />
<tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr><br />
<tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr><br />
<tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr><br />
<tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr><br />
<tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr><br />
</table></div><br />
<br><br />
For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.<br />
<br><br><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. <br />
RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. <br><br />
It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP). <br><br><br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element.<br />
Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.<br />
<br />
</p><br />
<br />
<p><br />
Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least sqares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs :<br />
<br><br />
<ol><ul><li><b><br />
RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity,<br />
</li><li><b><br />
RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity,<br />
</li><li><br />
<b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and represents the heart of linear region,<br />
</li><br />
<li><br />
<b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</li><br />
</ul></ol><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!-- ----------------- --><br />
<!-- --RBS description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='rbs'></a><h2>RBSs</h2><br />
<p align='justify'><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of mRFP produced. <br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. <br />
The evaluation of RBS efficiency can be performed in a very intuitive fashion:<br><br />
<ul><br />
<li>1. select the RBSs you want to study, </li><br />
<li>2. assemble them in a Promoter - XX - Coding sequence circuit, </li></ul></ol><br />
<br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:Vettore_base.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d1/Vettore_base.jpg" class="thumbimage" width="50%"></a></div></div><br />
<br />
<ul><br />
<li>3. measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS. </li><br />
</ul></ol><br><br />
This simple measurement system allows the quantification of RBS efficiency depending on the whole measurement system (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in a complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments. <br><br />
To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.<br><br />
In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, pTet, pLux). Measuring the system output and evaluating the RBS efficiency. The results are summarized in the table below:<br><br><br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>pLux</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>pTet</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>J23101</sub></b><sup>**</sup></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>0.40</td><td class='row'>1.6814</td><td class='row'>3.17</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.01</td><td class='row'>ND</td><td class='row'>0.05</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.19</td><td class='row'>0.4193</td><td class='row'>0.37</td><td class='row'>0,3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<br />
<br><br />
On the other end, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (pTet-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below:<br><br><br />
<br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>mRFP</sub></b><sup>**</sup></td><br />
<td class='row'><b>eff<sub>AiiA</sub></b><sup>***</sup></td><br />
<td class='row'><b>eff<sub>LuxI</sub><sup>****</sup></b></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>1.72</td><td class='row'>0.53</td><td class='row'>0.64</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.03</td><td class='row'>0.83</td><td class='row'>0.08</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.37</td><td class='row'>0.50</td><td class='row'>N.D.</td><td class='row'>0.3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<p align='justify'><br />
<sup>*</sup> The RBS efficiency for inducible devices expressing mRFP was estimated as the ratio of the AUCs (Area under the curve) of the induction curve of the system with the studied RBS and the B0034 reference: AUC<sub>P, RBSx</sub>/AUC<sub>P, B0034</sub><br><br />
<sup>**</sup> The RBS efficiency for constitutive promoters expressing mRFP was computed as the ratio between S<sub>cell<sub>P, RBSx</sub></sub>/S<sub>cell<sub>P, B0034</sub></sub><br><br />
<sup>***</sup> The RBS efficiency for pTet promoter driving the expression of AiiA enzyme was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated for the measurement system pTet-RBSx-AiiA-TT. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#AiiA'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<sup>****</sup>The RBS efficiency for promoters driving the expression of LuxI was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated from the measurement systems pTet-RBSx-LuxI. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#LuxI'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<br />
<br><br />
</p><br />
<br><br><br />
The parts we used to characterize the RBSs are listed here:<br />
<ol><br />
<ul><li>mRFP expression with different promoters</li><br />
<ul><br />
<li>J23101</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> J101-RBS30-RFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> J101-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_J23101 "> BBa_J23101 </a> J101-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pTet</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pLux</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li><br />
</ul><br />
</ul><br />
<li>pTet driving the expression of different genes</li><br />
<ul><li>mRFP</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>AiiA</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> pTet-RBS34-AiiA-TT </li><br />
</ul><br />
<li>LuxI</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> pTet-RBS34-LuxI </li><br />
</ul></ul><br />
</ul></ol><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --pLux description- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<a name='pLux'></a><h2>pLux promoter</h2><br />
<p align='justify'><br />
<br />
<br />
</p><br />
<ol><ul><br />
<br />
<br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> (wiki name: E17 ) pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> (wiki name: E18 ) pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> (wiki name: E19 ) pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> (wiki name: E20 ) pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li></ul><br />
</ol><br />
<br />
<p align='justify'><br />
The estimated parameters for the Hill functions are summarized in the table below. For more details on parameter estimation, see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>model section</a>. <br />
</p><br />
<br />
<table class='data' width='100%'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Lux</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">438 [10]</td><br />
<td class="row">0.05 [>100]</td><br />
<td class="row">2 [47]</td><br />
<td class="row">1.88 [27]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">9.8 [7]</td><br />
<td class="row">0.11 [57]</td><br />
<td class="row">1.2 [29]</td><br />
<td class="row">1.5 [26]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">206 [3]</td><br />
<td class="row">0 [>>100]</td><br />
<td class="row">1.36 [10]</td><br />
<td class="row">1.87 [9]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">1105 [6]</td><br />
<td class="row">0.02 [>100]</td><br />
<td class="row">1.33 [19]</td><br />
<td class="row">2.34 [18]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br />
<br />
<p align='justify'><br />
From this table, it is evident that, whilst &alpha;<sub>pLux</sub> assumes significantly different values for different RBSs, &eta;<sub>pLux</sub> and k<sub>pLux</sub> assume very similar values. This result shows that RBS variation modulates the amplitude of Hill function, not affecting the shape of the curve. The four induction curves result to be the same Hill function modulated in amplitude by a parameter, that is the estimated RBS efficiency for this measurement system.<br><br />
These results are quite encouraging, because suggest that, given the non-modular behavior of RBS dpending on the encoded gene, the RBS has a modular behaviour respect to the promoter. <br />
<br />
</p><br />
<br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>4.28</td><td class='row'>0.20</td><td class='row'>1.08</td><td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>4.93</td><td class='row'>0.55</td><td class='row'>0.25</td><td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>9.49</td><td class='row'>0.02</td><td class='row'>0.47</td><td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>21.53</td><td class='row'>0.51</td><td class='row'>0.53</td><td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<p align='justify'><br />
<br />
These operative parameters provide useful information on the behavior of this 3OC6-HSL inducible device. RPU<sub>max</sub> assumes very different values, showing that the modulation in amplitude of the Hill can't be explained by a linear dependance on the RBS efficiency (in this case, in fact, the same RPUs should be observed for every RBS, since the standard reference used for RPUs computation)<br />
<br />
</p><br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- -pTet description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='pTet'></a><h2>pTet promoter</h2><br />
<br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> (wiki name: E21 ) pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> (wiki name: E22 ) pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT<br />
</li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT <br />
</li></ul><br />
</ul><br />
</ol><br />
<p align='justify'><br />
<br />
The protocols for the characterization of pTet promoter are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#pTet_protocol'>pTet measurement section</a>. <br><br />
This promoter is widely studied and characterized usually using the strong RBS BBa_B0034. Here we have characterized its transcriptional strength as a function of aTc induction (ng/ul) for different RBSs. Four different induction curves were obtained and are reported in figure:<br><br><br />
<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
The data collected from the mRFP measurement systems were processed as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'> data analysis section</a>. The induction curves were obtained by fitting a Hill function as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>modelling section</a> and the estimated <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>parameters</a> for pTet are reported in the pictures and in table below. </p><br />
<p align='justify'><br />
The estimated parameters of the Hill curves described in the figures are summarized in the table below:<br />
</p><br />
<br><br><br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">230.67 [3.7]</td><br />
<td class="row">0.028 [91.61]</td><br />
<td class="row">4.61 [23.73]</td><br />
<td class="row">8.75 [4.16]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">55.77 [12]</td><br />
<td class="row">1.53E-11 [>>100]</td><br />
<td class="row">4.98 [57.62]</td><br />
<td class="row">7.26 [14.98]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">120 [5.95]</td><br />
<td class="row">0.085 [40.6]</td><br />
<td class="row">24.85 [47.6]</td><br />
<td class="row">9 [5.43]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br><br><br />
<br />
<p align='justify'><br />
The measurement system pTet-B0031-mRFP-TT couldn't be assayed because its fluorescence output is under the detectability threshold of our measurement instrument. For this reason, the parameters of the corresponding Hill curve couldn't be estimated and are reported as 'Not Determined' ND. <br />
</p><br />
<br />
<p align='justify'><br />
&alpha; parameter (representing the maximum trascriptional rate in the studied range of induction) varies as expected with the RBS variation and also the &delta; and &eta; parameters are quite different among the RBS variations.<br><br />
The K<sub>pTet</sub> parameter is quite constant among the RBS variations, thus suggesting that in this case the RBS variation doesn't substantially affect the switch point of the Hill curve, even if the amplitude and the maximum slope are not quite maintained (for the &eta; parameter, maybe fitting problems). <br />
<br />
</p><br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [ng/&mu;]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [ng/&mu;]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>1.53</td><td class='row'>~0</td><td class='row'>7.95</td><td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>3.16</td><td class='row'>~0</td><td class='row'>6.7</td><td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>2.73</td><td class='row'>0.23</td><td class='row'>8.96</td><td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
From these parameters, it is evident that whilst the switch-point is almost maintained for all the RBSs, the linear boundaries are similar for RBS30 and RBS32 but for RBS34 are moved on the right of one order of magnitude. <br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --AiiA description-- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<br />
<a name='AiiA'></a><h2>AiiA gene - BBa_C0060</h2><br />
<br />
<p align='justify'><br />
The activity of AiiA enzyme has been evaluated by testing the measurement systems <a href='<a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a>. <br />
The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
</p><br />
<br />
<br />
<br><br><br />
dAiiA/dt=&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>AiiA</sub>*AiiA<br><br><br />
d[HSL]/dt=N*K<sub>cat</sub>*AiiA*HSL/(K<sub>M, AiiA</sub>+AiiA))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
<p align='justify><br />
<br />
Since the measurement systems are only assayed in the exponential growth phase, the third equation can be modified as follows:<br />
</p><br />
dN/dt=N*&mu;<br />
<br />
<p align='justify'><br />
because N<N<sub>max</sub>. <br><br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.003152</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
<br />
<br />
<br />
<p align='justify'><br />
<br />
The parameters K<sub>cat</sub> and K<sub>M,AiiA</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of AiiA are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>cat</sub></b></td><br />
<td class='row'><b>K<sub>M, AiiA</sub></b></td><br />
</tr><br />
<tr><td class='row'></td><br />
<td class='row'></td><br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
<br />
<br />
</p><br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --LuxI description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='LuxI'></a><h2>LuxI gene - BBa_C0061</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#t9002'>modeling section</a>). <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
<br />
<br><br><br />
dLuxI/dt=&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>LuxI</sub>*LuxI<br><br><br />
d[HSL]/dt=N*V<sub>max</sub>*1/(1+(K<sub>M, LuxI</sub>/LuxI))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
The parameters of the first equation (estimated from the part pTet-RBSx-mRFP-TT) and N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:<br />
<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">215 [2]</td><br />
<td class="row">0.001 [>>100]</td><br />
<td class="row">4.3 [12]</td><br />
<td class="row">8.3 [2]</td><br />
<td class='row' rowspan='4'>1*10<sup>9</sup></td><br />
<td class='row' rowspan='4'>0.003152</td><br />
<td class='row' rowspan='4'>0</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">0.7 [>>100]</td><br />
<td class="row">0[>>100]</td><br />
<td class="row">18.84[>>100]</td><br />
<td class="row">47.51[>>100]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">45.7 [11]</td><br />
<td class="row">0.02 [>>100]</td><br />
<td class="row">83 [>>100]</td><br />
<td class="row">8 [>>100]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">125 [12]</td><br />
<td class="row">0.12 [61]</td><br />
<td class="row">71.8 [>>100]</td><br />
<td class="row">9.8 [>>100]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, K<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>pTet-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of LuxI are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>M, LuxI</sub></b></td><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>9.575*10<sup>2</sup></td><br />
<td class='row'>2.975*10<sup>-9</sup></td><br />
<td class='row'>14.08</td><br />
<td class='row'>1.67</td><br />
<td class='row'>ND</td><br />
<td class='row'>76.73</td><br />
<br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
<br />
Explain that in all these evaluations we have estimated Nmax, mu, gamma_HSL from previous experiments - see measurement and modelling sections. When do we talk about HSL stability as a function of pH?<br />
Provide parameters of HSL. How do we present these data? It would be nice also to say what's the amount of HSL produced by a liquid 5 ml culture at a given OD600 in M9 medium after tot hours starting from 1:1000 dilution of a saturated ON culture..<br />
We nee a synthetic parameter to express LuxI activity as a function of PoPS in. I suggest to report the HSL vs pH analysis here AND in the AiiA section of registry and, for what concerns our wiki, to add an appendix to the 'measurement section' to wich link when explaining.. <br />
Decide figures! <br />
</p><br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- ---pSB1C3-mRFP-- --><br />
<!-- ----------------- --><br />
<br />
<a name='K999'></a><h2>pSB1C3 plasmid with mRFP between S and P bearing pTet (easy-to-clone)</h2><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<a name='Improvement'></a><h1>Existing parts: sequence debugging</h1><br />
<ol><ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516021 "> BBa_K516021 </a> (wiki name: E10 ) RBS31-AiiA-TT Rebuilt existing part from BBa_I13914 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516022 "> BBa_K516022 </a> (wiki name: E11 ) RBS32-AiiA-TT Rebuilt existing part from BBa_I13912 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516030 "> BBa_K516030 </a> (wiki name: E5 ) RBS30-mRFP-TT Rebuilt existing part from BBa_S04180 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516032 "> BBa_K516032 </a> (wiki name: E7 ) RBS32-mRFP-TT Rebuilt existing part from BBa_ J133000 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI Rebuilt existing part from BBa_S03623 (DNA available, only 2008 kit, inconsistent) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT Rebuilt existing part from BBa_ J22071 (2008 only, Bad sequencing) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT Rebuilt existing part from BBa_K077047 (Part deleted) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT Rebuilt existing part from BBa_I20252 (DNA planning) </li></ul></ol><br />
<br />
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<br />
</html><br />
<br />
{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/Parts/CharacterizedTeam:UNIPV-Pavia/Parts/Characterized2011-09-17T22:01:47Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<html><br />
<a name='indice'></a><br />
<h2 class="art-postheader"><br />
Parts<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div> <br />
<ul> <br />
<li class="toclevel-1"><a href="#new"><span class="tocnumber">1</span> <span class="toctext">New Parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#J101"><span class="tocnumber">1.1</span> <span class="toctext">J23101x series with different RBSs</span></a></li><br />
<li class="toclevel-2"><a href="#pTetAiiA"><span class="tocnumber">1.2</span> <span class="toctext">AiiA Expression cassette driven by aTc</span></a></li><br />
<li class="toclevel-2"><a href="#pTetLuxI"><span class="tocnumber">1.3</span> <span class="toctext">LuxI Expression cassette driven by aTc</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Existing"><span class="tocnumber">2</span> <span class="toctext">Characterization of existing parts</span></a> <ul><br />
<li class="toclevel-2"><a href="#note"><span class="tocnumber">2.1</span> <span class="toctext">Notes on promoter characterization</span></a></li><br />
<li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.2</span> <span class="toctext">RBSs from the community collection</span></a></li><br />
<li class="toclevel-2"><a href="#pLux"><span class="tocnumber">2.3</span> <span class="toctext">pLux - a 3OC6-HSL-in PoPs-out device</span></a></li><br />
<li class="toclevel-2"><a href="#pTet"><span class="tocnumber">2.4</span> <span class="toctext">pTet - BBa_R0040</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.5</span> <span class="toctext">AiiA - BBa_C0060</span></a></li><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.6</span> <span class="toctext">LuxI - BBa_C0061</span></a></li><br />
<li class="toclevel-2"><a href="#K999"><span class="tocnumber">2.7</span> <span class="toctext">pSB1C3 vector with mRFP between S and P for easy cloning</span></a></li></ul><br />
<li class="toclevel-1"><a href="#Improvement"><span class="tocnumber">3</span> <span class="toctext">Sequence debugging</span></a></li><br />
</ul></td></tr></table><br />
</div><br />
<br />
<br />
<a name='new'></a><h1>New parts</h1><br />
<a name='J101'></a><h2>J23101x series</h2><br />
<br />
<ol type='1'><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> (wiki name: J101-E5 ) J101-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> (wiki name: J101-31 ) J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> (wiki name: J101-E7 ) J101-RBS32-mRFP-TT </li><br />
</ol><br />
<br />
<br />
<br />
<!-- ----------------- --><br />
<!-- J23101x description --><br />
<!-- ----------------- --><br />
<br />
<p align='justify'><br />
BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in '<a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>data analysis</a>' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene. <br><br />
The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. <br>It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P.<br> <br />
This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts. <br><br />
Salis et al. [Nat Biotec, 2009] stated that<br />
<em> 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels'</em><br><br><br />
and again<br />
<em>'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.'</em> <br><br><br />
<br />
<br />
For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS.<br><br />
<br />
In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.<br />
<br />
<br><br><em><br />
<b>NB</b>: in the RPU computation, the J23101-RBS34-mRFP-TT construct has been considered as the reference standard. With this assumption, RPUs are identical to the estimated RBS efficiency. </em><br />
<br />
<table align='center'><tr><td width='50%' ><br />
<table class='data'><br />
<tr><br />
<td class='row'><b>J23101 promoter with RBS</b><br />
</td><br />
<td class='row'><b>S<sub>cell</sub></b><br />
</td><br />
<td class='row'><b>R.P.U.s</b><br />
</td><br />
</tr><br />
<tr><td class='row'>RBS30<br />
</td><br />
<td class='row'>150.97 [15.34]</td><br />
<td class='row'>3.17</td><br />
</tr><br />
<tr><td class='row'>RBS31<br />
</td><br />
<td class='row'>2.30 [0.37]</td><br />
<td class='row'>0.05</td><br />
</tr><br />
<tr><td class='row'>RBS32<br />
</td><br />
<td class='row'>17.60 [5.85]</td><br />
<td class='row'>0.37</td><br />
</tr><br />
<tr><td class='row'>RBS34<br />
</td><br />
<td class='row'>47.57 [0.82]</td><br />
<td class='row'>1</td><br />
</tr><br />
</table></td><br />
<td width='50%' ><br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:RPU_J101.png" class="image"><img alt="RPUs of J23101 promoter with different RBSs" src="https://static.igem.org/mediawiki/2011/1/12/RPU_J101.png" class="thumbimage" width="80%"></a></div></div><br />
</td><br />
</tr></table><br />
<br><br><br />
On the other hand, the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.<br />
<br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- pLuxAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetAiiA'></a><h2>AiiA expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> (wiki name: E24 ) pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> (wiki name: E25 ) pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- pTetAiiA description --><br />
<!-- ----------------- --><br />
<br />
<a name='pTetLuxI'></a><h2>LuxI expression cassette driven by aTc-inducible pTet promoter</h2><br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> (wiki name: E13 ) pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> (wiki name: E14 ) pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> (wiki name: E15 ) pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI </li></ul></ol><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<a name='Existing'></a><h1>Existing parts</h1><br />
<br />
<a name='note'></a><h2>Notes for promoter characterization</h2><br />
<p align='justify'><br />
Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection. <br />
<br><br><br />
The assembled RBSs are:<br />
<br><br><br />
<div align='center'><br />
<table class='data'><br />
<tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr><br />
<tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr><br />
<tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr><br />
<tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr><br />
<tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr><br />
</table></div><br />
<br><br />
For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.<br />
<br><br><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. <br />
RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. <br><br />
It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP). <br><br><br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element.<br />
Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.<br />
<br />
</p><br />
<br />
<p><br />
Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least sqares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs :<br />
<br><br />
<ol><ul><li><b><br />
RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity,<br />
</li><li><b><br />
RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity,<br />
</li><li><br />
<b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and represents the heart of linear region,<br />
</li><br />
<li><br />
<b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</li><br />
</ul></ol><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --RBS description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='rbs'></a><h2>RBSs</h2><br />
<p align='justify'><br />
The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. In addition, it is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount of mRFP produced. <br />
For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. <br />
The evaluation of RBS efficiency can be performed in a very intuitive fashion:<br><br />
<ul><br />
<li>1. select the RBSs you want to study, </li><br />
<li>2. assemble them in a Promoter - XX - Coding sequence circuit, </li></ul></ol><br />
<br />
<div style="text-align:justify"><div class="thumbinner" width='80%'><a href="File:Vettore_base.jpg" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d1/Vettore_base.jpg" class="thumbimage" width="50%"></a></div></div><br />
<br />
<ul><br />
<li>3. measure the output of the circuits and calculate the RBS efficiency as the ratio of the output relative to the output of the circuit with the standard RBS. </li><br />
</ul></ol><br><br />
This simple measurement system allows the quantification of RBS efficiency depending on the whole measurement system (i.e.: promoter and encoded gene). Today it has not still been completely validated the hypothesis that every functional module in a genetic circuit maintains its behavior when assembled in a complex circuits, even if many researchers implicitly accept this hypothesis when performing characterization experiments. <br><br />
To rationally assess the impact that this hypothesis has on the genetic circuit design and fine tuning, several measurement systems were built to evaluate the dependance of RBS modularity from the promoter or the coding sequence separately.<br><br />
In particular, in order to investigate if RBS efficiency depends on the promoter, the same coding devices (RBSx-RFP-TT) were assembled downstream of different promoters (J23101, pTet, pLux). Measuring the system output and evaluating the RBS efficiency. The results are summarized in the table below:<br><br><br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>pLux</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>pTet</sub></b><sup>*</sup></td><br />
<td class='row'><b>eff<sub>J23101</sub></b><sup>**</sup></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>0.40</td><td class='row'>1.6814</td><td class='row'>3.17</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.01</td><td class='row'>ND</td><td class='row'>0.05</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.19</td><td class='row'>0.4193</td><td class='row'>0.37</td><td class='row'>0,3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<br />
<br><br />
On the other end, to investigate the dependance of RBS modularity on the coding sequence, the same regulatory elements (pTet-RBSx) were assembled upstream of different encoded gene (mRFP, AiiA and LuxI). RBS efficiency was assessed and the results are summarized in the table below:<br><br><br />
<br />
<table class='data' width='70%'><tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>eff<sub>mRFP</sub></b><sup>**</sup></td><br />
<td class='row'><b>eff<sub>AiiA</sub></b><sup>***</sup></td><br />
<td class='row'><b>eff<sub>LuxI</sub><sup>****</sup></b></td><br />
<td class='row'><b>Declared efficiency</b></td><br />
</tr><tr><br />
<td class='row'>B0030</td><td class='row'>1.72</td><td class='row'>0.53</td><td class='row'>0.64</td><td class='row'>0,6</td><br />
</tr><tr><br />
<td class='row'>B0031</td><td class='row'>0.03</td><td class='row'>0.83</td><td class='row'>0.08</td><td class='row'>0,07</td><br />
</tr><tr><br />
<td class='row'>B0032</td><td class='row'>0.37</td><td class='row'>0.50</td><td class='row'>N.D.</td><td class='row'>0.3</td><br />
</tr><tr><br />
<td class='row'>B0034</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><td class='row'>1</td><br />
</tr></table><br />
<p align='justify'><br />
<sup>*</sup> The RBS efficiency for inducible devices expressing mRFP was estimated as the ratio of the AUCs (Area under the curve) of the induction curve of the system with the studied RBS and the B0034 reference: AUC<sub>P, RBSx</sub>/AUC<sub>P, B0034</sub><br><br />
<sup>**</sup> The RBS efficiency for constitutive promoters expressing mRFP was computed as the ratio between S<sub>cell<sub>P, RBSx</sub></sub>/S<sub>cell<sub>P, B0034</sub></sub><br><br />
<sup>***</sup> The RBS efficiency for pTet promoter driving the expression of AiiA enzyme was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated for the measurement system pTet-RBSx-AiiA-TT. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#AiiA'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<sup>****</sup>The RBS efficiency for promoters driving the expression of LuxI was computed as the ratio &alpha;<sub>pTet, RBSx</sub>/&alpha;<sub>pTet, B0034</sub> estimated from the measurement systems pTet-RBSx-LuxI. &alpha;<sub>pTet</sub> was estimated as described <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#LuxI'>here</a>. pTet was tested at full induction (100 ng/ml).<br><br />
<br />
<br><br />
</p><br />
<br><br><br />
The parts we used to characterize the RBSs are listed here:<br />
<ol><br />
<ul><li>mRFP expression with different promoters</li><br />
<ul><br />
<li>J23101</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516130 "> BBa_K516130 </a> J101-RBS30-RFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516131 "> BBa_K516131 </a> J101-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516132 "> BBa_K516132 </a> J101-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_J23101 "> BBa_J23101 </a> J101-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pTet</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>pLux</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li><br />
</ul><br />
</ul><br />
<li>pTet driving the expression of different genes</li><br />
<ul><li>mRFP</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> pTet-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT </li><br />
</ul><br />
<li>AiiA</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516220 "> BBa_K516220 </a> pTet-RBS30-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516221 "> BBa_K516221 </a> pTet-RBS31-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> pTet-RBS32-AiiA-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> pTet-RBS34-AiiA-TT </li><br />
</ul><br />
<li>LuxI</li><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516210 "> BBa_K516210 </a> pTet-RBS30-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516211 "> BBa_K516211 </a> pTet-RBS31-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516212 "> BBa_K516212 </a> pTet-RBS32-LuxI </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> pTet-RBS34-LuxI </li><br />
</ul></ul><br />
</ul></ol><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --pLux description- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<a name='pLux'></a><h2>pLux promoter</h2><br />
<p align='justify'><br />
<br />
<br />
</p><br />
<ol><ul><br />
<br />
<br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516330 "> BBa_K516330 </a> (wiki name: E17 ) pLambda-RBS30-LuxR-T-pLux-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516331 "> BBa_K516331 </a> (wiki name: E18 ) pLambda-RBS30-LuxR-T-pLux-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516332 "> BBa_K516332 </a> (wiki name: E19 ) pLambda-RBS30-LuxR-T-pLux-RBS32-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516334 "> BBa_K516334 </a> (wiki name: E20 ) pLambda-RBS30-LuxR-T-pLux-RBS34-mRFP-TT </li></ul><br />
</ol><br />
<br />
<p align='justify'><br />
The estimated parameters for the Hill functions are summarized in the table below. For more details on parameter estimation, see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>model section</a>. <br />
</p><br />
<br />
<table class='data' width='100%'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Lux</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Lux</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">438 [10]</td><br />
<td class="row">0.05 [>100]</td><br />
<td class="row">2 [47]</td><br />
<td class="row">1.88 [27]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">9.8 [7]</td><br />
<td class="row">0.11 [57]</td><br />
<td class="row">1.2 [29]</td><br />
<td class="row">1.5 [26]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">206 [3]</td><br />
<td class="row">0 [>>100]</td><br />
<td class="row">1.36 [10]</td><br />
<td class="row">1.87 [9]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">1105 [6]</td><br />
<td class="row">0.02 [>100]</td><br />
<td class="row">1.33 [19]</td><br />
<td class="row">2.34 [18]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br />
<br />
<p align='justify'><br />
From this table, it is evident that, whilst &alpha;<sub>pLux</sub> assumes significantly different values for different RBSs, &eta;<sub>pLux</sub> and k<sub>pLux</sub> assume very similar values. This result shows that RBS variation modulates the amplitude of Hill function, not affecting the shape of the curve. The four induction curves result to be the same Hill function modulated in amplitude by a parameter, that is the estimated RBS efficiency for this measurement system.<br><br />
These results are quite encouraging, because suggest that, given the non-modular behavior of RBS dpending on the encoded gene, the RBS has a modular behaviour respect to the promoter. <br />
<br />
</p><br />
<br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [nM]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>4.28</td><td class='row'>0.20</td><td class='row'>1.08</td><td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>4.93</td><td class='row'>0.55</td><td class='row'>0.25</td><td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>9.49</td><td class='row'>0.02</td><td class='row'>0.47</td><td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>21.53</td><td class='row'>0.51</td><td class='row'>0.53</td><td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<p align='justify'><br />
<br />
These operative parameters provide useful information on the behavior of this 3OC6-HSL inducible device. RPU<sub>max</sub> assumes very different values, showing that the modulation in amplitude of the Hill can't be explained by a linear dependance on the RBS efficiency (in this case, in fact, the same RPUs should be observed for every RBS, since the standard reference used for RPUs computation)<br />
<br />
</p><br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- -pTet description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='pTet'></a><h2>pTet promoter</h2><br />
<br />
<ol><br />
<ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516230 "> BBa_K516230 </a> (wiki name: E21 ) pTet-RBS30-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516231 "> BBa_K516231 </a> (wiki name: E22 ) pTet-RBS31-mRFP-TT </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT<br />
</li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_I13521 "> BBa_I13521 </a> pTet-RBS34-mRFP-TT <br />
</li></ul><br />
</ul><br />
</ol><br />
<p align='justify'><br />
<br />
The protocols for the characterization of pTet promoter are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#pTet_protocol'>pTet measurement section</a>. <br><br />
This promoter is widely studied and characterized usually using the strong RBS BBa_B0034. Here we have characterized its transcriptional strength as a function of aTc induction (ng/ul) for different RBSs. Four different induction curves were obtained and are reported in figure:<br><br><br />
<br />
<br />
<br />
<table width='100%'><tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f6/PTet_-_mRFP_with_RBS_B0030_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d0/PTet_-_mRFP_with_RBS_B0031_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
<tr><td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/6/64/PTet_-_mRFP_with_RBS_B0032_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td><br />
<td width='50%'><br />
<div style="text-align:justify"><div class="thumbinner" width='100%'><a href="File:PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/1/12/PTet_-_mRFP_with_RBS_B0034_RFP_8_0.png" class="thumbimage" width="100%"></a></div></div><br />
</td></tr><br />
</table><br />
<br />
The data collected from the mRFP measurement systems were processed as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'> data analysis section</a>. The induction curves were obtained by fitting a Hill function as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Ptet_&_Plux'>modelling section</a> and the estimated <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Table_of_parameters'>parameters</a> for pTet are reported in the pictures and in table below. </p><br />
<p align='justify'><br />
The estimated parameters of the Hill curves described in the figures are summarized in the table below:<br />
</p><br />
<br><br><br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">230.67 [3.7]</td><br />
<td class="row">0.028 [91.61]</td><br />
<td class="row">4.61 [23.73]</td><br />
<td class="row">8.75 [4.16]</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
<td class="row">ND</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">55.77 [12]</td><br />
<td class="row">1.53E-11 [>>100]</td><br />
<td class="row">4.98 [57.62]</td><br />
<td class="row">7.26 [14.98]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">120 [5.95]</td><br />
<td class="row">0.085 [40.6]</td><br />
<td class="row">24.85 [47.6]</td><br />
<td class="row">9 [5.43]</td><br />
</tr><br />
</table><br />
Data are provided as average [CV%]<br />
<br><br><br />
<br />
<p align='justify'><br />
The measurement system pTet-B0031-mRFP-TT couldn't be assayed because its fluorescence output is under the detectability threshold of our measurement instrument. For this reason, the parameters of the corresponding Hill curve couldn't be estimated and are reported as 'Not Determined' ND. <br />
</p><br />
<br />
<p align='justify'><br />
&alpha; parameter (representing the maximum trascriptional rate in the studied range of induction) varies as expected with the RBS variation and also the &delta; and &eta; parameters are quite different among the RBS variations.<br><br />
The K<sub>pTet</sub> parameter is quite constant among the RBS variations, thus suggesting that in this case the RBS variation doesn't substantially affect the switch point of the Hill curve, even if the amplitude and the maximum slope are not quite maintained (for the &eta; parameter, maybe fitting problems). <br />
<br />
</p><br />
<p align='justify'><br />
The operative parameters are summarized in the table below:<br />
</p><br />
<br />
<table align='center' class='data' width='100%'><br />
<tr><br />
<td class='row'><b>RBS</b></td><br />
<td class='row'><b>RPU<sub>max</sub></b></td><br />
<td class='row'><b>RPU<sub>min</sub></b></td><br />
<td class='row'><b>Switch point [ng/&mu;]</b></td><br />
<td class='row'><b>Linear boundaries [MIN; MAX] [ng/&mu;]</b></td><br />
</tr><br />
<tr><br />
<td class='row'>B0030</td><td class='row'>1.53</td><td class='row'>~0</td><td class='row'>7.95</td><td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><td class='row'>ND</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><td class='row'>3.16</td><td class='row'>~0</td><td class='row'>6.7</td><td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><td class='row'>2.73</td><td class='row'>0.23</td><td class='row'>8.96</td><td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
From these parameters, it is evident that whilst the switch-point is almost maintained for all the RBSs, the linear boundaries are similar for RBS30 and RBS32 but for RBS34 are moved on the right of one order of magnitude. <br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<!-- ----------------- --><br />
<!-- --AiiA description-- --><br />
<!-- ----------------- --><br />
<br />
<br />
<br />
<br />
<a name='AiiA'></a><h2>AiiA gene - BBa_C0060</h2><br />
<br />
<p align='justify'><br />
The activity of AiiA enzyme has been evaluated by testing the measurement systems <a href='<a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a>. <br />
The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
</p><br />
<br />
<br />
<br><br><br />
dAiiA/dt=&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>AiiA</sub>*AiiA<br><br><br />
d[HSL]/dt=N*K<sub>cat</sub>*AiiA*HSL/(K<sub>M, AiiA</sub>+AiiA))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
<p align='justify><br />
<br />
Since the measurement systems are only assayed in the exponential growth phase, the third equation can be modified as follows:<br />
</p><br />
dN/dt=N*&mu;<br />
<br />
<p align='justify'><br />
because N<N<sub>max</sub>. <br><br />
The parameters N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><br />
<td class='row' >1*10<sup>9</sup></td><br />
<td class='row' >0.003152</td><br />
<td class='row' >0</td><br />
</table><br />
</p><br />
<br />
<br />
<br />
<p align='justify'><br />
<br />
The parameters K<sub>cat</sub> and K<sub>M,AiiA</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#AiiA'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetAiiA'>pTet-RBSx-AiiA-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of AiiA are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>cat</sub></b></td><br />
<td class='row'><b>K<sub>M, AiiA</sub></b></td><br />
</tr><br />
<tr><td class='row'></td><br />
<td class='row'></td><br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
<br />
<br />
</p><br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- --LuxI description-- --><br />
<!-- ----------------- --><br />
<br />
<a name='LuxI'></a><h2>LuxI gene - BBa_C0061</h2><br />
<br />
<br />
<p align='justify'><br />
<br />
LuxI has been characterized through the Biosensor BBa_T9002 (see <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#t9002'>modeling section</a>). <br />
<br>The HSL synthesis rate has been evaluated according to the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>model equations</a>, properly adjusted. In particular, the ODE system is reported here:<br />
<br />
<br><br><br />
dLuxI/dt=&alpha;<sub>pTet</sub>*(&delta;<sub>pTet</sub>+(1-&delta;<sub>pTet</sub>)/(1+(K<sub>pTet</sub>/aTc)<sup>&eta;</sup>))-&gamma;<sub>LuxI</sub>*LuxI<br><br><br />
d[HSL]/dt=N*V<sub>max</sub>*1/(1+(K<sub>M, LuxI</sub>/LuxI))<br />
<br><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br />
<br><br><br />
<br />
The parameters of the first equation (estimated from the part pTet-RBSx-mRFP-TT) and N<sub>max</sub>, &mu; and &gamma;<sub>HSL</sub> (see the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Results'>Results section</a> for more details) are known. <br> These parameters are summarized in the table below:<br />
<br />
<br />
<table class='data' width='100%' title='parameter value'><br />
<tr><br />
<td class="row"><b>RBS</b></td><br />
<td class="row"><b>&alpha;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&delta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>&eta;<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>k<sub>p<sub>Tet</sub></sub></b></td><br />
<td class="row"><b>N<sub>max</sub></sub></b></td><br />
<td class="row"><b>&mu;</b></td><br />
<td class="row"><b>&gamma;<sub>HSL</sub></b></td><br />
</tr><br />
<tr><td class="row">BBa_B0030</td><br />
<td class="row">215 [2]</td><br />
<td class="row">0.001 [>>100]</td><br />
<td class="row">4.3 [12]</td><br />
<td class="row">8.3 [2]</td><br />
<td class='row' rowspan='4'>1*10<sup>9</sup></td><br />
<td class='row' rowspan='4'>0.003152</td><br />
<td class='row' rowspan='4'>0</td><br />
</tr><br />
<tr><td class="row">BBa_B0031</td><br />
<td class="row">0.7 [>>100]</td><br />
<td class="row">0[>>100]</td><br />
<td class="row">18.84[>>100]</td><br />
<td class="row">47.51[>>100]</td><br />
</tr><br />
<tr><td class="row">BBa_B0032</td><br />
<td class="row">45.7 [11]</td><br />
<td class="row">0.02 [>>100]</td><br />
<td class="row">83 [>>100]</td><br />
<td class="row">8 [>>100]</td><br />
</tr><br />
<tr><td class="row">BBa_B0034</td><br />
<td class="row">125 [12]</td><br />
<td class="row">0.12 [61]</td><br />
<td class="row">71.8 [>>100]</td><br />
<td class="row">9.8 [>>100]</td><br />
</tr><br />
</table><br />
<br />
<p align='justify'><br />
The parameters V<sub>max</sub>, K<sub>M,LuxI</sub> and &alpha;<sub>RBSx</sub> were estimated with a simultaneous fitting of the data collected as described in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#LuxI'>measurement section</a> for the four measurement parts <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#pTetLuxI'>pTet-RBSx-LuxI-TT</a> assayed by <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements#T9002'>BBa_T9002 biosensor</a> section. <br />
<br />
The estimated parameters for the enzymatic activity of LuxI are reported in the table below:<br />
<br />
<table align='center' width='50%'><br />
<tr><td width='100%'><br />
<table class='data' width='100%'><br />
<tr><br />
<td class='row'><b>K<sub>M, LuxI</sub></b></td><br />
<td class='row'><b>V<sub>max</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0030</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0031</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0032</sub></b></td><br />
<td class='row'><b>&alpha;<sub>B0034</sub></b></td><br />
</tr><br />
<tr><td class='row'>9.575*10<sup>2</sup></td><br />
<td class='row'>2.975*10<sup>-9</sup></td><br />
<td class='row'>14.08</td><br />
<td class='row'>1.67</td><br />
<td class='row'>ND</td><br />
<td class='row'>76.73</td><br />
<br />
</tr></table></td></tr></table><br />
<br />
<br><br><br />
<br />
Explain that in all these evaluations we have estimated Nmax, mu, gamma_HSL from previous experiments - see measurement and modelling sections. When do we talk about HSL stability as a function of pH?<br />
Provide parameters of HSL. How do we present these data? It would be nice also to say what's the amount of HSL produced by a liquid 5 ml culture at a given OD600 in M9 medium after tot hours starting from 1:1000 dilution of a saturated ON culture..<br />
We nee a synthetic parameter to express LuxI activity as a function of PoPS in. I suggest to report the HSL vs pH analysis here AND in the AiiA section of registry and, for what concerns our wiki, to add an appendix to the 'measurement section' to wich link when explaining.. <br />
Decide figures! <br />
</p><br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!-- ----------------- --><br />
<!-- ---pSB1C3-mRFP-- --><br />
<!-- ----------------- --><br />
<br />
<a name='K999'></a><h2>pSB1C3 plasmid with mRFP between S and P bearing pTet (easy-to-clone)</h2><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<a name='Improvement'></a><h1>Existing parts: sequence debugging</h1><br />
<ol><ul><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516021 "> BBa_K516021 </a> (wiki name: E10 ) RBS31-AiiA-TT Rebuilt existing part from BBa_I13914 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516022 "> BBa_K516022 </a> (wiki name: E11 ) RBS32-AiiA-TT Rebuilt existing part from BBa_I13912 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516030 "> BBa_K516030 </a> (wiki name: E5 ) RBS30-mRFP-TT Rebuilt existing part from BBa_S04180 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516032 "> BBa_K516032 </a> (wiki name: E7 ) RBS32-mRFP-TT Rebuilt existing part from BBa_ J133000 (DNA planning) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516214 "> BBa_K516214 </a> (wiki name: E16 ) pTet-RBS34-LuxI Rebuilt existing part from BBa_S03623 (DNA available, only 2008 kit, inconsistent) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516222 "> BBa_K516222 </a> (wiki name: E26 ) pTet-RBS32-AiiA-TT Rebuilt existing part from BBa_ J22071 (2008 only, Bad sequencing) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516224 "> BBa_K516224 </a> (wiki name: E27 ) pTet-RBS34-AiiA-TT Rebuilt existing part from BBa_K077047 (Part deleted) </li><br />
<li> <A HREF="http://partsregistry.org/wiki/index.php/Part: BBa_K516232 "> BBa_K516232 </a> (wiki name: E23 ) pTet-RBS32-mRFP-TT Rebuilt existing part from BBa_I20252 (DNA planning) </li></ul></ol><br />
<br />
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<br />
<br />
<br />
</html><br />
<br />
{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/MeasurementsTeam:UNIPV-Pavia/Measurements2011-09-17T17:13:57Z<p>Nickpv: </p>
<hr />
<div>{{main}}<br />
<br />
<html><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<a name="top_page"></a><br />
<br />
<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div><br />
<ul><br />
<li class="toclevel-1"><a href="#Promoters"><span class="tocnumber">1</span> <span class="toctext">Measuring promoters transcriptional strength</span></a></li><br />
<br />
<ul><br />
<li class="toclevel-1"><a href="#pTet_protocol"><span class="tocnumber">1.1</span> <span class="toctext">pTet transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#pLux_protocol"><span class="tocnumber">1.2</span> <span class="toctext">pLux transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#j101_protocol"><span class="tocnumber">1.3</span> <span class="toctext">Constitutive BBa_J23101x promoters transcriptional strength</span></a><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#Enzyme"><span class="tocnumber">2</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</span></a><br />
<br />
<ul><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.1</span> <span class="toctext">LuxI enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.2</span> <span class="toctext">AiiA enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#Deg"><span class="tocnumber">2.3</span> <span class="toctext">3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH</span></a></li><br />
<li class="toclevel-2"><a href="#T9002"><span class="tocnumber">2.4</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL concentration with BBa_T9002</span></a></li><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#data_analysis"><span class="tocnumber">3</span> <span class="toctext">Data analysis</span></a><br />
<ul><br />
<li class="toclevel-2"><a href="#preprocessing"><span class="tocnumber">3.1</span> <span class="toctext">Data pre-processing</span></a></li><br />
<li class="toclevel-2"><a href="#doubtime"><span class="tocnumber">3.2</span> <span class="toctext">Doubling time evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#scell"><span class="tocnumber">3.3</span> <span class="toctext">Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#RPU"><span class="tocnumber">3.4</span> <span class="toctext">R.P.U. evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#HSL"><span class="tocnumber">3.5</span> <span class="toctext">Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration</span></a></li><br />
</ul><br />
<br />
</td></tr></table><br />
</div><br />
<br />
<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C. <br />
</em><br />
<br />
<a name="Promoters"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring promoters transcriptional strength</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pTet_protocol"></a> <h2 class="art-postheader"><br />
Measuring pTet transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for three hours.<br />
<li> Induce cultures in falcon tube with anhydrotetracycline (aTc); final concentrations:<br />
<ul><br />
<li> 0 ng/ml<br />
<li> 1 ng/ml<br />
<li> 2 ng/ml<br />
<li> 3 ng/ml<br />
<li> 4 ng/ml<br />
<li> 5 ng/ml<br />
<li> 8 ng/ml<br />
<li> 10 ng/ml<br />
<li> 50 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow at 37°C, 220 rpm for three hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pLux_protocol"></a> <h2 class="art-postheader"><br />
Measuring pLux transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours at 37°C, 220 rpm.<br />
<li> Induce cultures in falcon tube with 3OC<sub><small>6</small></sub>-HSL; final concentrations: <br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
</ul><br />
<li> Let the cultures grow for three hours at 37°C, 220 rpm.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="j101_protocol"></a> <h2 class="art-postheader"><br />
Constitutive BBa_J23101x promoters transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for six hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Enzyme"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="LuxI"></a> <h2 class="art-postheader"><br />
LuxI enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and grow them for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of induction, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="AiiA"></a> <h2 class="art-postheader"><br />
AiiA enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow for one more hour at 37°C, 220 rpm.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Deg"></a> <h2 class="art-postheader"><br />
3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks not expressing lactonases in 1 ml of M9 with the proper antibiotic. Use M9 at different pHs, for example pH = 6.0 and pH = 7.0. Let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 with the proper antibiotic in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Prepare falcon tubes with M9 at pH = 6.0 and pH = 7.0.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL to each falcon tube.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="T9002"></a> <h2 class="art-postheader"><br />
Measuring 3OC<sub><small>6</small></sub>-HSL concentration withBBa_T9002<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> in 1 ml M9 with the proper antibiotic (Ampicillin when you use <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> or Ampicillin + Chloramphenicol 12.5 mg/ml if you use T9002-ENTERO, see <a href="https://2011.igem.org/Team:UNIPV-Pavia/Freezer">Freezer Management</a>) together with a non-fluorescent culture; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in M9 with the proper antibiotic; let the cultures grow for two hours at 37°C, 220 rpm.<br />
<li> Induce <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with the previously collected supernatants, diluting them 1:20: aliquot 190&mu;l of inducible cultures and 10 &mu;l of supernatants in the wells of the microplate.<br />
<li> Don't forget to build a calibration curve, by inducing <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with known 3OC<sub><small>6</small></sub>-HSL concentrations:<br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.2 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
<li> 1 &mu;M<br />
</ul><br />
<li>Use Tecan Infinite F200 to read O.D. at 600 nm and green fluorescence, setting the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50<br />
<li> O.D. filter: 600 nm<br />
<li> GFP filters: 485 nm (excitation) / 540 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
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<a name="data_analysis"></a> <h2 class="art-postheader"><br />
<font size = "5">Data analysis</font><br />
</h2><br />
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<br />
<a name="preprocessing"></a> <h2 class="art-postheader"><br />
Data pre-processing<br />
</h2><br />
<p><br />
Data from TECAN Infinite F200 need to be pre-processed in order to remove spurious effects affecting measurements. Before evaluating the parameters of interest (for example the synthesis rate per cell, S<sub><small>cell</small></sub>, or Relative Promoter Unit, R.P.U.) blanking is needed:<br />
<ol><ul><br />
<li> each time sample of O.D. at 600 nm, (O.D.<sub><small>600</small></sub>) was subtracted the reference value (i.e. the measure of O.D. at 600 nm of the broth in which cultures grow)<br />
<li> each time sample of red and green fluorescence was subtracted the reference value (i.e. the red or green fluorescence of a non-fluorescent culture, also known as auto-fluorescence)<br />
</ul></ol><br />
</p><br />
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<a name="doubtime"></a> <h2 class="art-postheader"><br />
Doubling time evaluation<br />
</h2><br />
<p><br />
After blanking O.D.<sub><small>600</small></sub> data you can compute the doubling time of cultures growth, i.e. the time needed to double O.D.<sub><small>600</small></sub>.<br />
<br><br />
Identifying the exponential phase of growth curve can be done by visual inspection, plotting the natural logarithm of O.D.<sub><small>600</small></sub> over time. Linear regression on logarithmic data is performed to estimate the growth rate, named &mu;. Finally doubling time can be evaluated as:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_DoubTime.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/7d/UNIPV_DoubTime.PNG"class="thumbimage" width="80%"></a></div></div><br />
<br />
<br />
<p><br />
When measuring multiple growth for a strain this value was evaluated for each reaplicate and then average was computed.<br />
</p><br />
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<br />
<a name="scell"></a><h2 class="art-postheader"><br />
Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation<br />
</h2><br />
<p><br />
Another interesting parameter, which describes the activity of a promoter of interest (&phi;), is the S<sub><small>cell</small></sub>; it needs to be measured using reporter genes as RFP or GFP.<br />
<br><br />
Compute the average of the time derivative of the red or green fluorescence, divided by the O.D.<sub><small>600</small></sub>, in the time interval corresponding to the exponential growth phase which boundaries can be identified by visual inspection of the logarithmic growth curve:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 225px;"><a href="/File:UNIPV_Scell.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c5/UNIPV_Scell.PNG"class="thumbimage" width="80%"></a></div></div><br />
<br />
<p><br />
The S<sub><small>cell</small></sub> is basic to measure the Relative Promoter Unit, a robust parameter expressing the transcriptional activity of a promoter.<br />
</p><br />
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<br />
<a name="RPU"></a> <h2 class="art-postheader"><br />
R.P.U. evaluation<br />
</h2><br />
<p><br />
As described in Kelly J. et al., 2009, Relative Promoter Units express the activity of a promoter of interest (&phi;), reported to the one of a reference promoter, <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a> (from Anderson promoters collection). The R.P.U. of a promoter is expressed as the ratio between its S<sub><small>cell</small></sub> and the one of <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a>:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 250px;"><a href="/File:UNIPV_RPU.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/70/UNIPV_RPU.PNG"class="thumbimage" width="80%"></a></div></div><br />
<br />
<p><br />
This procedure has the advantage of being robust to variations in experimental conditions (for example the instrument used to measure absorbance and fluorescence) and proportional to PoPS (Polymerase Per Second) if the subsequent specifications are satisfied:<br />
<ol><ul><br />
<li> strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in BBa_J23101 reference standard<br />
<li> the reporter protein must have a half life higher than the experiment duration<br />
</ul></ol><br />
Moreover the S<sub><small>cell</small></sub> signal of both the promoter of interest and of BBa_J23101 has to be constant on the time interval considered for R.P.U. evaluation.<br />
</p><br />
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<br />
<a name="HSL"></a> <h2 class="art-postheader"><br />
Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration<br />
</h2><br />
<p><br />
The procedure described below is useful in order to quantify the concentration of 3OC<small><sub>6</sub></small>-HSL in cultures expressing LuxI and AiiA enzyme.<br />
<br><br />
Once collected (according to the <a href = "#T9002">protocol above</a>) and pre-processed data, it is necessary to compute the S<sub><small>cell</small></sub> of every culture.<br />
<br><br />
BBa_T9002 induced with known 3OC<small><sub>6</sub></small>-HSL concentration is useful to estimate the parameters of the activation Hill function of pLux promoter:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 350px;"><a href="/File:UNIPV_activation_pLux.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f2/UNIPV_activation_pLux.png"class="thumbimage" width="80%"></a></div></div><br />
<br />
<p><br />
Provided that the S<sub><small>cell</small></sub> measured for BBa_T9002 induced with supernatants of cultures producing or degrading 3OC<small><sub>6</sub></small>-HSL has a value included in the linear zone of the biosensor (tuning of the dilution factor is necessary), you can easily extrapolate the auto-inducer concentration as follows:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 650px;"><a href="/File:UNIPV_HSL.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e2/UNIPV_HSL.PNG"class="thumbimage" width="40%"></a></div></div><br />
<br />
<p><br />
Finally, don't forget to multiply the value obtained for the dilution factor (in our experiments it was 20).<br />
</p><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/MeasurementsTeam:UNIPV-Pavia/Measurements2011-09-17T17:09:18Z<p>Nickpv: </p>
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div><br />
<ul><br />
<li class="toclevel-1"><a href="#Promoters"><span class="tocnumber">1</span> <span class="toctext">Measuring promoters transcriptional strength</span></a></li><br />
<br />
<ul><br />
<li class="toclevel-1"><a href="#pTet_protocol"><span class="tocnumber">1.1</span> <span class="toctext">pTet transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#pLux_protocol"><span class="tocnumber">1.2</span> <span class="toctext">pLux transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#j101_protocol"><span class="tocnumber">1.3</span> <span class="toctext">Constitutive BBa_J23101x promoters transcriptional strength</span></a><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#Enzyme"><span class="tocnumber">2</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</span></a><br />
<br />
<ul><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.1</span> <span class="toctext">LuxI enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.2</span> <span class="toctext">AiiA enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#Deg"><span class="tocnumber">2.3</span> <span class="toctext">3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH</span></a></li><br />
<li class="toclevel-2"><a href="#T9002"><span class="tocnumber">2.4</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL concentration with BBa_T9002</span></a></li><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#data_analysis"><span class="tocnumber">3</span> <span class="toctext">Data analysis</span></a><br />
<ul><br />
<li class="toclevel-2"><a href="#preprocessing"><span class="tocnumber">3.1</span> <span class="toctext">Data pre-processing</span></a></li><br />
<li class="toclevel-2"><a href="#doubtime"><span class="tocnumber">3.2</span> <span class="toctext">Doubling time evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#scell"><span class="tocnumber">3.3</span> <span class="toctext">Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#RPU"><span class="tocnumber">3.4</span> <span class="toctext">R.P.U. evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#HSL"><span class="tocnumber">3.5</span> <span class="toctext">Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration</span></a></li><br />
</ul><br />
<br />
</td></tr></table><br />
</div><br />
<br />
<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C. <br />
</em><br />
<br />
<a name="Promoters"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring promoters transcriptional strength</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pTet_protocol"></a> <h2 class="art-postheader"><br />
Measuring pTet transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for three hours.<br />
<li> Induce cultures in falcon tube with anhydrotetracycline (aTc); final concentrations:<br />
<ul><br />
<li> 0 ng/ml<br />
<li> 1 ng/ml<br />
<li> 2 ng/ml<br />
<li> 3 ng/ml<br />
<li> 4 ng/ml<br />
<li> 5 ng/ml<br />
<li> 8 ng/ml<br />
<li> 10 ng/ml<br />
<li> 50 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow at 37°C, 220 rpm for three hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
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<br />
<a name="pLux_protocol"></a> <h2 class="art-postheader"><br />
Measuring pLux transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours at 37°C, 220 rpm.<br />
<li> Induce cultures in falcon tube with 3OC<sub><small>6</small></sub>-HSL; final concentrations: <br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
</ul><br />
<li> Let the cultures grow for three hours at 37°C, 220 rpm.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="j101_protocol"></a> <h2 class="art-postheader"><br />
Constitutive BBa_J23101x promoters transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for six hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Enzyme"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="LuxI"></a> <h2 class="art-postheader"><br />
LuxI enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and grow them for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of induction, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="AiiA"></a> <h2 class="art-postheader"><br />
AiiA enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow for one more hour at 37°C, 220 rpm.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Deg"></a> <h2 class="art-postheader"><br />
3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks not expressing lactonases in 1 ml of M9 with the proper antibiotic. Use M9 at different pHs, for example pH = 6.0 and pH = 7.0. Let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 with the proper antibiotic in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Prepare falcon tubes with M9 at pH = 6.0 and pH = 7.0.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL to each falcon tube.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="T9002"></a> <h2 class="art-postheader"><br />
Measuring 3OC<sub><small>6</small></sub>-HSL concentration withBBa_T9002<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> in 1 ml M9 with the proper antibiotic (Ampicillin when you use <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> or Ampicillin + Chloramphenicol 12.5 mg/ml if you use T9002-ENTERO, see <a href="https://2011.igem.org/Team:UNIPV-Pavia/Freezer">Freezer Management</a>) together with a non-fluorescent culture; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in M9 with the proper antibiotic; let the cultures grow for two hours at 37°C, 220 rpm.<br />
<li> Induce <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with the previously collected supernatants, diluting them 1:20: aliquot 190&mu;l of inducible cultures and 10 &mu;l of supernatants in the wells of the microplate.<br />
<li> Don't forget to build a calibration curve, by inducing <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with known 3OC<sub><small>6</small></sub>-HSL concentrations:<br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.2 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
<li> 1 &mu;M<br />
</ul><br />
<li>Use Tecan Infinite F200 to read O.D. at 600 nm and green fluorescence, setting the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50<br />
<li> O.D. filter: 600 nm<br />
<li> GFP filters: 485 nm (excitation) / 540 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
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<br />
<a name="data_analysis"></a> <h2 class="art-postheader"><br />
<font size = "5">Data analysis</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="preprocessing"></a> <h2 class="art-postheader"><br />
Data pre-processing<br />
</h2><br />
<p><br />
Data from TECAN Infinite F200 need to be pre-processed in order to remove spurious effects affecting measurements. Before evaluating the parameters of interest (for example the synthesis rate per cell, S<sub><small>cell</small></sub>, or Relative Promoter Unit, R.P.U.) blanking is needed:<br />
<ol><ul><br />
<li> each time sample of O.D. at 600 nm, (O.D.<sub><small>600</small></sub>) was subtracted the reference value (i.e. the measure of O.D. at 600 nm of the broth in which cultures grow)<br />
<li> each time sample of red and green fluorescence was subtracted the reference value (i.e. the red or green fluorescence of a non-fluorescent culture)<br />
</ul></ol><br />
</p><br />
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<a name="doubtime"></a> <h2 class="art-postheader"><br />
Doubling time evaluation<br />
</h2><br />
<p><br />
After blanking O.D.<sub><small>600</small></sub> data you can compute the doubling time of cultures growth, i.e. the time needed to double O.D.<sub><small>600</small></sub>.<br />
<br><br />
Identifying the exponential phase of growth curve can be done by visual inspection, plotting the natural logarithm of O.D.<sub><small>600</small></sub> over time. Linear regression on logarithmic data is performed to estimate the growth rate, named &mu;. Finally doubling time can be evaluated as:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_DoubTime.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/7d/UNIPV_DoubTime.PNG"class="thumbimage" width="80%"></a></div></div><br />
<br />
<br />
<p><br />
When measuring multiple growth for a strain this value was evaluated for each reaplicate and then average was computed.<br />
</p><br />
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<br />
<a name="scell"></a><h2 class="art-postheader"><br />
Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation<br />
</h2><br />
<p><br />
Another interesting parameter, which describes the activity of a promoter of interest (&phi;), is the S<sub><small>cell</small></sub>; it needs to be measured using reporter genes as RFP or GFP.<br />
<br><br />
Compute the average of the time derivative of the red or green fluorescence, divided by the O.D.<sub><small>600</small></sub>, in the time interval corresponding to the exponential growth phase which boundaries can be identified by visual inspection of the logarithmic growth curve:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 225px;"><a href="/File:UNIPV_Scell.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c5/UNIPV_Scell.PNG"class="thumbimage" width="80%"></a></div></div><br />
<br />
<p><br />
The S<sub><small>cell</small></sub> is basic to measure the Relative Promoter Unit, a robust parameter expressing the transcriptional activity of a promoter.<br />
</p><br />
<br />
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<br />
<a name="RPU"></a> <h2 class="art-postheader"><br />
R.P.U. evaluation<br />
</h2><br />
<p><br />
As described in Kelly J. et al., 2009, Relative Promoter Units express the activity of a promoter of interest (&phi;), reported to the one of a reference promoter, <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a> (from Anderson promoters collection). The R.P.U. of a promoter is expressed as the ratio between its S<sub><small>cell</small></sub> and the one of <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a>:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 250px;"><a href="/File:UNIPV_RPU.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/70/UNIPV_RPU.PNG"class="thumbimage" width="80%"></a></div></div><br />
<br />
<p><br />
This procedure has the advantage of being robust to variations in experimental conditions and proportional to PoPS (Polymerase Per Second) if the subsequent specifications are satisfied:<br />
<ol><ul><br />
<li> strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in BBa_J23101 reference standard<br />
<li> the reporter protein must have a half life higher than the experiment duration<br />
</ul></ol><br />
Moreover the S<sub><small>cell</small></sub> signal of both the promoter of interest and of BBa_J23101 has to be constant on the time interval considered for R.P.U. evaluation.<br />
</p><br />
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<br />
<a name="HSL"></a> <h2 class="art-postheader"><br />
Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration<br />
</h2><br />
<p><br />
The procedure described below is useful in order to quantify the concentration of 3OC<small><sub>6</sub></small>-HSL in cultures expressing LuxI and AiiA enzyme.<br />
<br><br />
Once collected (according to the <a href = "#T9002">protocol above</a>) and pre-processed data, it is necessary to compute the S<sub><small>cell</small></sub> of every culture.<br />
<br><br />
BBa_T9002 induced with known 3OC<small><sub>6</sub></small>-HSL concentration is useful to estimate the parameters of the activation Hill function of pLux promoter:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 350px;"><a href="/File:UNIPV_activation_pLux.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f2/UNIPV_activation_pLux.png"class="thumbimage" width="80%"></a></div></div><br />
<br />
<p><br />
Provided that the S<sub><small>cell</small></sub> measured for BBa_T9002 induced with supernatants of cultures producing or degrading 3OC<small><sub>6</sub></small>-HSL has a value included in the linear zone of the biosensor (tuning of the dilution factor is necessary), you can easily extrapolate the auto-inducer concentration as follows:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 650px;"><a href="/File:UNIPV_HSL.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e2/UNIPV_HSL.PNG"class="thumbimage" width="40%"></a></div></div><br />
<br />
<p><br />
Finally, don't forget to multiply this value for the dilution factor (in our experiments it was 20).<br />
</p><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/MeasurementsTeam:UNIPV-Pavia/Measurements2011-09-17T17:07:11Z<p>Nickpv: </p>
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div><br />
<ul><br />
<li class="toclevel-1"><a href="#Promoters"><span class="tocnumber">1</span> <span class="toctext">Measuring promoters transcriptional strength</span></a></li><br />
<br />
<ul><br />
<li class="toclevel-1"><a href="#pTet_protocol"><span class="tocnumber">1.1</span> <span class="toctext">pTet transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#pLux_protocol"><span class="tocnumber">1.2</span> <span class="toctext">pLux transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#j101_protocol"><span class="tocnumber">1.3</span> <span class="toctext">Constitutive BBa_J23101x promoters transcriptional strength</span></a><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#Enzyme"><span class="tocnumber">2</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</span></a><br />
<br />
<ul><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.1</span> <span class="toctext">LuxI enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.2</span> <span class="toctext">AiiA enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#Deg"><span class="tocnumber">2.3</span> <span class="toctext">3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH</span></a></li><br />
<li class="toclevel-2"><a href="#T9002"><span class="tocnumber">2.4</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL concentration with BBa_T9002</span></a></li><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#data_analysis"><span class="tocnumber">3</span> <span class="toctext">Data analysis</span></a><br />
<ul><br />
<li class="toclevel-2"><a href="#preprocessing"><span class="tocnumber">3.1</span> <span class="toctext">Data pre-processing</span></a></li><br />
<li class="toclevel-2"><a href="#doubtime"><span class="tocnumber">3.2</span> <span class="toctext">Doubling time evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#scell"><span class="tocnumber">3.3</span> <span class="toctext">Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#RPU"><span class="tocnumber">3.4</span> <span class="toctext">R.P.U. evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#HSL"><span class="tocnumber">3.5</span> <span class="toctext">Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration</span></a></li><br />
</ul><br />
<br />
</td></tr></table><br />
</div><br />
<br />
<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C. <br />
</em><br />
<br />
<a name="Promoters"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring promoters transcriptional strength</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pTet_protocol"></a> <h2 class="art-postheader"><br />
Measuring pTet transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for three hours.<br />
<li> Induce cultures in falcon tube with anhydrotetracycline (aTc); final concentrations:<br />
<ul><br />
<li> 0 ng/ml<br />
<li> 1 ng/ml<br />
<li> 2 ng/ml<br />
<li> 3 ng/ml<br />
<li> 4 ng/ml<br />
<li> 5 ng/ml<br />
<li> 8 ng/ml<br />
<li> 10 ng/ml<br />
<li> 50 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow at 37°C, 220 rpm for three hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pLux_protocol"></a> <h2 class="art-postheader"><br />
Measuring pLux transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours at 37°C, 220 rpm.<br />
<li> Induce cultures in falcon tube with 3OC<sub><small>6</small></sub>-HSL; final concentrations: <br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
</ul><br />
<li> Let the cultures grow for three hours at 37°C, 220 rpm.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="j101_protocol"></a> <h2 class="art-postheader"><br />
Constitutive BBa_J23101x promoters transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for six hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Enzyme"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="LuxI"></a> <h2 class="art-postheader"><br />
LuxI enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and grow them for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of induction, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
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<br />
<a name="AiiA"></a> <h2 class="art-postheader"><br />
AiiA enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow for one more hour at 37°C, 220 rpm.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Deg"></a> <h2 class="art-postheader"><br />
3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks not expressing lactonases in 1 ml of M9 with the proper antibiotic. Use M9 at different pHs, for example pH = 6.0 and pH = 7.0. Let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 with the proper antibiotic in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Prepare falcon tubes with M9 at pH = 6.0 and pH = 7.0.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL to each falcon tube.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
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<br />
<a name="T9002"></a> <h2 class="art-postheader"><br />
Measuring 3OC<sub><small>6</small></sub>-HSL concentration withBBa_T9002<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> in 1 ml M9 with the proper antibiotic (Ampicillin when you use <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> or Ampicillin + Chloramphenicol 12.5 mg/ml if you use T9002-ENTERO, see <a href="https://2011.igem.org/Team:UNIPV-Pavia/Freezer">Freezer Management</a>) together with a non-fluorescent culture; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in M9 with the proper antibiotic; let the cultures grow for two hours at 37°C, 220 rpm.<br />
<li> Induce <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with the previously collected supernatants, diluting them 1:20: aliquot 190&mu;l of inducible cultures and 10 &mu;l of supernatants in the wells of the microplate.<br />
<li> Don't forget to build a calibration curve, by inducing <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with known 3OC<sub><small>6</small></sub>-HSL concentrations:<br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.2 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
<li> 1 &mu;M<br />
</ul><br />
<li>Use Tecan Infinite F200 to read O.D. at 600 nm and green fluorescence, setting the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50<br />
<li> O.D. filter: 600 nm<br />
<li> GFP filters: 485 nm (excitation) / 540 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
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<br />
<a name="data_analysis"></a> <h2 class="art-postheader"><br />
<font size = "5">Data analysis</font><br />
</h2><br />
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<a name="preprocessing"></a> <h2 class="art-postheader"><br />
Data pre-processing<br />
</h2><br />
<p><br />
Data from TECAN Infinite F200 need to be pre-processed in order to remove spurious effects affecting measurements. Before evaluating the parameters of interest (for example the synthesis rate per cell, S<sub><small>cell</small></sub>, or Relative Promoter Unit, R.P.U.) blanking is needed:<br />
<ol><ul><br />
<li> each time sample of O.D. at 600 nm, (O.D.<sub><small>600</small></sub>) was subtracted the reference value (i.e. the measure of O.D. at 600 nm of the broth in which cultures grow)<br />
<li> each time sample of red and green fluorescence was subtracted the reference value (i.e. the red or green fluorescence of a non-fluorescent culture)<br />
</ul></ol><br />
</p><br />
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<a name="doubtime"></a> <h2 class="art-postheader"><br />
Doubling time evaluation<br />
</h2><br />
<p><br />
After blanking O.D.<sub><small>600</small></sub> data you can compute the doubling time of cultures growth, i.e. the time needed to double O.D.<sub><small>600</small></sub>.<br />
<br><br />
Identifying the exponential phase of growth curve can be done by visual inspection, plotting the natural logarithm of O.D.<sub><small>600</small></sub> over time. Linear regression on logarithmic data is performed to estimate the growth rate, named &mu;. Finally doubling time can be evaluated as:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_DoubTime.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/7d/UNIPV_DoubTime.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<br />
<p><br />
When measuring multiple growth for a strain this value was evaluated for each reaplicate and then average was computed.<br />
</p><br />
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<a name="scell"></a><h2 class="art-postheader"><br />
Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation<br />
</h2><br />
<p><br />
Another interesting parameter, which describes the activity of a promoter of interest (&phi;), is the S<sub><small>cell</small></sub>; it needs to be measured using reporter genes as RFP or GFP.<br />
<br><br />
Compute the average of the time derivative of the red or green fluorescence, divided by the O.D.<sub><small>600</small></sub>, in the time interval corresponding to the exponential growth phase which boundaries can be identified by visual inspection of the logarithmic growth curve:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_Scell.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c5/UNIPV_Scell.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
The S<sub><small>cell</small></sub> is basic to measure the Relative Promoter Unit, a robust parameter expressing the transcriptional activity of a promoter.<br />
</p><br />
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<br />
<a name="RPU"></a> <h2 class="art-postheader"><br />
R.P.U. evaluation<br />
</h2><br />
<p><br />
As described in Kelly J. et al., 2009, Relative Promoter Units express the activity of a promoter of interest (&phi;), reported to the one of a reference promoter, <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a> (from Anderson promoters collection). The R.P.U. of a promoter is expressed as the ratio between its S<sub><small>cell</small></sub> and the one of <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a>:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_RPU.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/70/UNIPV_RPU.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
This procedure has the advantage of being robust to variations in experimental conditions and proportional to PoPS (Polymerase Per Second) if the subsequent specifications are satisfied:<br />
<ol><ul><br />
<li> strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in BBa_J23101 reference standard<br />
<li> the reporter protein must have a half life higher than the experiment duration<br />
</ul></ol><br />
Moreover the S<sub><small>cell</small></sub> signal of both the promoter of interest and of BBa_J23101 has to be constant on the time interval considered for R.P.U. evaluation.<br />
</p><br />
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<a name="HSL"></a> <h2 class="art-postheader"><br />
Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration<br />
</h2><br />
<p><br />
The procedure described below is useful in order to quantify the concentration of 3OC<small><sub>6</sub></small>-HSL in cultures expressing LuxI and AiiA enzyme.<br />
<br><br />
Once collected (according to the <a href = "#T9002">protocol above</a>) and pre-processed data, it is necessary to compute the S<sub><small>cell</small></sub> of every culture.<br />
<br><br />
BBa_T9002 induced with known 3OC<small><sub>6</sub></small>-HSL concentration is useful to estimate the parameters of the activation Hill function of pLux promoter:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_activation_pLux.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f2/UNIPV_activation_pLux.png"class="thumbimage" width="80%"></a></div></div><br />
<br />
<p><br />
Provided that the S<sub><small>cell</small></sub> measured for BBa_T9002 induced with supernatants of cultures producing or degrading 3OC<small><sub>6</sub></small>-HSL has a value included in the linear zone of the biosensor (tuning of the dilution factor is necessary), you can easily extrapolate the auto-inducer concentration as follows:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 600px;"><a href="/File:UNIPV_HSL.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e2/UNIPV_HSL.PNG"class="thumbimage" width="40%"></a></div></div><br />
<br />
<p><br />
Finally, don't forget to multiply this value for the dilution factor (in our experiments it was 20).<br />
</p><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/MeasurementsTeam:UNIPV-Pavia/Measurements2011-09-17T17:04:28Z<p>Nickpv: </p>
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div><br />
<ul><br />
<li class="toclevel-1"><a href="#Promoters"><span class="tocnumber">1</span> <span class="toctext">Measuring promoters transcriptional strength</span></a></li><br />
<br />
<ul><br />
<li class="toclevel-1"><a href="#pTet_protocol"><span class="tocnumber">1.1</span> <span class="toctext">pTet transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#pLux_protocol"><span class="tocnumber">1.2</span> <span class="toctext">pLux transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#j101_protocol"><span class="tocnumber">1.3</span> <span class="toctext">Constitutive BBa_J23101x promoters transcriptional strength</span></a><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#Enzyme"><span class="tocnumber">2</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</span></a><br />
<br />
<ul><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.1</span> <span class="toctext">LuxI enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.2</span> <span class="toctext">AiiA enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#Deg"><span class="tocnumber">2.3</span> <span class="toctext">3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH</span></a></li><br />
<li class="toclevel-2"><a href="#T9002"><span class="tocnumber">2.4</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL concentration with BBa_T9002</span></a></li><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#data_analysis"><span class="tocnumber">3</span> <span class="toctext">Data analysis</span></a><br />
<ul><br />
<li class="toclevel-2"><a href="#preprocessing"><span class="tocnumber">3.1</span> <span class="toctext">Data pre-processing</span></a></li><br />
<li class="toclevel-2"><a href="#doubtime"><span class="tocnumber">3.2</span> <span class="toctext">Doubling time evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#scell"><span class="tocnumber">3.3</span> <span class="toctext">Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#RPU"><span class="tocnumber">3.4</span> <span class="toctext">R.P.U. evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#HSL"><span class="tocnumber">3.5</span> <span class="toctext">Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration</span></a></li><br />
</ul><br />
<br />
</td></tr></table><br />
</div><br />
<br />
<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C. <br />
</em><br />
<br />
<a name="Promoters"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring promoters transcriptional strength</font><br />
</h2><br />
<br />
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<br />
<a name="pTet_protocol"></a> <h2 class="art-postheader"><br />
Measuring pTet transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for three hours.<br />
<li> Induce cultures in falcon tube with anhydrotetracycline (aTc); final concentrations:<br />
<ul><br />
<li> 0 ng/ml<br />
<li> 1 ng/ml<br />
<li> 2 ng/ml<br />
<li> 3 ng/ml<br />
<li> 4 ng/ml<br />
<li> 5 ng/ml<br />
<li> 8 ng/ml<br />
<li> 10 ng/ml<br />
<li> 50 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow at 37°C, 220 rpm for three hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
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<br />
<a name="pLux_protocol"></a> <h2 class="art-postheader"><br />
Measuring pLux transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours at 37°C, 220 rpm.<br />
<li> Induce cultures in falcon tube with 3OC<sub><small>6</small></sub>-HSL; final concentrations: <br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
</ul><br />
<li> Let the cultures grow for three hours at 37°C, 220 rpm.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="j101_protocol"></a> <h2 class="art-postheader"><br />
Constitutive BBa_J23101x promoters transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for six hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Enzyme"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="LuxI"></a> <h2 class="art-postheader"><br />
LuxI enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and grow them for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of induction, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
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<br />
<a name="AiiA"></a> <h2 class="art-postheader"><br />
AiiA enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow for one more hour at 37°C, 220 rpm.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Deg"></a> <h2 class="art-postheader"><br />
3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks not expressing lactonases in 1 ml of M9 with the proper antibiotic. Use M9 at different pHs, for example pH = 6.0 and pH = 7.0. Let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 with the proper antibiotic in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Prepare falcon tubes with M9 at pH = 6.0 and pH = 7.0.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL to each falcon tube.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="T9002"></a> <h2 class="art-postheader"><br />
Measuring 3OC<sub><small>6</small></sub>-HSL concentration withBBa_T9002<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> in 1 ml M9 with the proper antibiotic (Ampicillin when you use <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> or Ampicillin + Chloramphenicol 12.5 mg/ml if you use T9002-ENTERO, see <a href="https://2011.igem.org/Team:UNIPV-Pavia/Freezer">Freezer Management</a>) together with a non-fluorescent culture; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in M9 with the proper antibiotic; let the cultures grow for two hours at 37°C, 220 rpm.<br />
<li> Induce <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with the previously collected supernatants, diluting them 1:20: aliquot 190&mu;l of inducible cultures and 10 &mu;l of supernatants in the wells of the microplate.<br />
<li> Don't forget to build a calibration curve, by inducing <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with known 3OC<sub><small>6</small></sub>-HSL concentrations:<br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.2 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
<li> 1 &mu;M<br />
</ul><br />
<li>Use Tecan Infinite F200 to read O.D. at 600 nm and green fluorescence, setting the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50<br />
<li> O.D. filter: 600 nm<br />
<li> GFP filters: 485 nm (excitation) / 540 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
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<br />
<a name="data_analysis"></a> <h2 class="art-postheader"><br />
<font size = "5">Data analysis</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="preprocessing"></a> <h2 class="art-postheader"><br />
Data pre-processing<br />
</h2><br />
<p><br />
Data from TECAN Infinite F200 need to be pre-processed in order to remove spurious effects affecting measurements. Before evaluating the parameters of interest (for example the synthesis rate per cell, S<sub><small>cell</small></sub>, or Relative Promoter Unit, R.P.U.) blanking is needed:<br />
<ol><ul><br />
<li> each time sample of O.D. at 600 nm, (O.D.<sub><small>600</small></sub>) was subtracted the reference value (i.e. the measure of O.D. at 600 nm of the broth in which cultures grow)<br />
<li> each time sample of red and green fluorescence was subtracted the reference value (i.e. the red or green fluorescence of a non-fluorescent culture)<br />
</ul></ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="doubtime"></a> <h2 class="art-postheader"><br />
Doubling time evaluation<br />
</h2><br />
<p><br />
After blanking O.D.<sub><small>600</small></sub> data you can compute the doubling time of cultures growth, i.e. the time needed to double O.D.<sub><small>600</small></sub>.<br />
<br><br />
Identifying the exponential phase of growth curve can be done by visual inspection, plotting the natural logarithm of O.D.<sub><small>600</small></sub> over time. Linear regression on logarithmic data is performed to estimate the growth rate, named &mu;. Finally doubling time can be evaluated as:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_DoubTime.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/7d/UNIPV_DoubTime.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<br />
<p><br />
When measuring multiple growth for a strain this value was evaluated for each reaplicate and then average was computed.<br />
</p><br />
<br />
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<a name="scell"></a><h2 class="art-postheader"><br />
Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation<br />
</h2><br />
<p><br />
Another interesting parameter, which describes the activity of a promoter of interest (&phi;), is the S<sub><small>cell</small></sub>; it needs to be measured using reporter genes as RFP or GFP.<br />
<br><br />
Compute the average of the time derivative of the red or green fluorescence, divided by the O.D.<sub><small>600</small></sub>, in the time interval corresponding to the exponential growth phase which boundaries can be identified by visual inspection of the logarithmic growth curve:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_Scell.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c5/UNIPV_Scell.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
The S<sub><small>cell</small></sub> is basic to measure the Relative Promoter Unit, a robust parameter expressing the transcriptional activity of a promoter.<br />
</p><br />
<br />
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<br />
<a name="RPU"></a> <h2 class="art-postheader"><br />
R.P.U. evaluation<br />
</h2><br />
<p><br />
As described in Kelly J. et al., 2009, Relative Promoter Units express the activity of a promoter of interest (&phi;), reported to the one of a reference promoter, <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a> (from Anderson promoters collection). The R.P.U. of a promoter is expressed as the ratio between its S<sub><small>cell</small></sub> and the one of <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a>:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_RPU.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/70/UNIPV_RPU.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
This procedure has the advantage of being robust to variations in experimental conditions and proportional to PoPS (Polymerase Per Second) if the subsequent specifications are satisfied:<br />
<ol><ul><br />
<li> strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in BBa_J23101 reference standard<br />
<li> the reporter protein must have a half life higher than the experiment duration<br />
</ul></ol><br />
Moreover the S<sub><small>cell</small></sub> signal of both the promoter of interest and of BBa_J23101 has to be constant.<br />
</p><br />
<br />
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<br />
<a name="HSL"></a> <h2 class="art-postheader"><br />
Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration<br />
</h2><br />
<p><br />
The procedure described below is useful in order to quantify the concentration of 3OC<small><sub>6</sub></small>-HSL in cultures expressing LuxI and AiiA enzyme.<br />
<br><br />
Once collected (according to the <a href = "#T9002">protocol above</a>) and pre-processed data, it is necessary to compute the S<sub><small>cell</small></sub> of every culture.<br />
<br><br />
BBa_T9002 induced with known 3OC<small><sub>6</sub></small>-HSL concentration is useful to estimate the parameters of the activation Hill function of pLux promoter:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_activation_pLux.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f2/UNIPV_activation_pLux.png"class="thumbimage" width="80%"></a></div></div><br />
<br />
<p><br />
Provided that the S<sub><small>cell</small></sub> measured for BBa_T9002 induced with supernatants of cultures producing or degrading 3OC<small><sub>6</sub></small>-HSL has a value included in the linear zone of the biosensor (tuning of the dilution factor is necessary), you can easily extrapolate the auto-inducer concentration as follows:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 600px;"><a href="/File:UNIPV_HSL.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e2/UNIPV_HSL.PNG"class="thumbimage" width="40%"></a></div></div><br />
<br />
<p><br />
Finally, don't forget to multiply this value for the dilution factor (in our experiments it was 20).<br />
</p><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/MeasurementsTeam:UNIPV-Pavia/Measurements2011-09-17T17:01:32Z<p>Nickpv: </p>
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div><br />
<ul><br />
<li class="toclevel-1"><a href="#Promoters"><span class="tocnumber">1</span> <span class="toctext">Measuring promoters transcriptional strength</span></a></li><br />
<br />
<ul><br />
<li class="toclevel-1"><a href="#pTet_protocol"><span class="tocnumber">1.1</span> <span class="toctext">pTet transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#pLux_protocol"><span class="tocnumber">1.2</span> <span class="toctext">pLux transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#j101_protocol"><span class="tocnumber">1.3</span> <span class="toctext">Constitutive BBa_J23101x promoters transcriptional strength</span></a><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#Enzyme"><span class="tocnumber">2</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</span></a><br />
<br />
<ul><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.1</span> <span class="toctext">LuxI enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.2</span> <span class="toctext">AiiA enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#Deg"><span class="tocnumber">2.3</span> <span class="toctext">3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH</span></a></li><br />
<li class="toclevel-2"><a href="#T9002"><span class="tocnumber">2.4</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL concentration with BBa_T9002</span></a></li><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#data_analysis"><span class="tocnumber">3</span> <span class="toctext">Data analysis</span></a><br />
<ul><br />
<li class="toclevel-2"><a href="#preprocessing"><span class="tocnumber">3.1</span> <span class="toctext">Data pre-processing</span></a></li><br />
<li class="toclevel-2"><a href="#doubtime"><span class="tocnumber">3.2</span> <span class="toctext">Doubling time evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#scell"><span class="tocnumber">3.3</span> <span class="toctext">Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#RPU"><span class="tocnumber">3.4</span> <span class="toctext">R.P.U. evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#HSL"><span class="tocnumber">3.5</span> <span class="toctext">Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration</span></a></li><br />
</ul><br />
<br />
</td></tr></table><br />
</div><br />
<br />
<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C. <br />
</em><br />
<br />
<a name="Promoters"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring promoters transcriptional strength</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pTet_protocol"></a> <h2 class="art-postheader"><br />
Measuring pTet transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for three hours.<br />
<li> Induce cultures in falcon tube with anhydrotetracycline (aTc); final concentrations:<br />
<ul><br />
<li> 0 ng/ml<br />
<li> 1 ng/ml<br />
<li> 2 ng/ml<br />
<li> 3 ng/ml<br />
<li> 4 ng/ml<br />
<li> 5 ng/ml<br />
<li> 8 ng/ml<br />
<li> 10 ng/ml<br />
<li> 50 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow at 37°C, 220 rpm for three hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pLux_protocol"></a> <h2 class="art-postheader"><br />
Measuring pLux transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours at 37°C, 220 rpm.<br />
<li> Induce cultures in falcon tube with 3OC<sub><small>6</small></sub>-HSL; final concentrations: <br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
</ul><br />
<li> Let the cultures grow for three hours at 37°C, 220 rpm.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="j101_protocol"></a> <h2 class="art-postheader"><br />
Constitutive BBa_J23101x promoters transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for six hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Enzyme"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="LuxI"></a> <h2 class="art-postheader"><br />
LuxI enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and grow them for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of induction, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="AiiA"></a> <h2 class="art-postheader"><br />
AiiA enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow for one more hour at 37°C, 220 rpm.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Deg"></a> <h2 class="art-postheader"><br />
3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks not expressing lactonases in 1 ml of M9 with the proper antibiotic. Use M9 at different pHs, for example pH = 6.0 and pH = 7.0. Let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 with the proper antibiotic in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Prepare falcon tubes with M9 at pH = 6.0 and pH = 7.0.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL to each falcon tube.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="T9002"></a> <h2 class="art-postheader"><br />
Measuring 3OC<sub><small>6</small></sub>-HSL concentration withBBa_T9002<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> in 1 ml M9 with the proper antibiotic (Ampicillin when you use <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> or Ampicillin + Chloramphenicol 12.5 mg/ml if you use T9002-ENTERO, see <a href="https://2011.igem.org/Team:UNIPV-Pavia/Freezer">Freezer Management</a>) together with a non-fluorescent culture; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in M9 with the proper antibiotic; let the cultures grow for two hours at 37°C, 220 rpm.<br />
<li> Induce <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with the previously collected supernatants, diluting them 1:20: aliquot 190&mu;l of inducible cultures and 10 &mu;l of supernatants in the wells of the microplate.<br />
<li> Don't forget to build a calibration curve, by inducing <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with known 3OC<sub><small>6</small></sub>-HSL concentrations:<br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.2 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
<li> 1 &mu;M<br />
</ul><br />
<li>Use Tecan Infinite F200 to read O.D. at 600 nm and green fluorescence, setting the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50<br />
<li> O.D. filter: 600 nm<br />
<li> GFP filters: 485 nm (excitation) / 540 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
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<a name="data_analysis"></a> <h2 class="art-postheader"><br />
<font size = "5">Data analysis</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
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<a name="preprocessing"></a> <h2 class="art-postheader"><br />
Data pre-processing<br />
</h2><br />
<p><br />
Data from TECAN Infinite F200 need to be pre-processed in order to remove spurious effects affecting measurements. Before evaluating the parameters of interest (for example the synthesis rate per cell, S<sub><small>cell</small></sub>, or Relative Promoter Unit, R.P.U.) blanking is needed:<br />
<ol><ul><br />
<li> each time sample of O.D. at 600 nm, (O.D.<sub><small>600</small></sub>) was subtracted the reference value (i.e. the measure of O.D. at 600 nm of the broth in which cultures grow)<br />
<li> each time sample of red and green fluorescence was subtracted the reference value (i.e. the red or green fluorescence of a non-fluorescent culture)<br />
</ul></ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="doubtime"></a> <h2 class="art-postheader"><br />
Doubling time evaluation<br />
</h2><br />
<p><br />
After blanking O.D.<sub><small>600</small></sub> data you can compute the doubling time of cultures growth, i.e. the time needed to double O.D.<sub><small>600</small></sub>.<br />
<br><br />
Identifying the exponential phase of growth curve can be done by visual inspection, plotting the natural logarithm of O.D.<sub><small>600</small></sub> over time. Linear regression on logarithmic data is performed to estimate the growth rate, named &mu;. Finally doubling time can be evaluated as:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_DoubTime.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/7d/UNIPV_DoubTime.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<br />
<p><br />
When measuring multiple growth for a strain this value was evaluated for each reaplicate and then average was computed.<br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
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<br />
<a name="scell"></a><h2 class="art-postheader"><br />
Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation<br />
</h2><br />
<p><br />
Another interesting parameter, which describes the activity of a promoter of interest (&phi;), is the S<sub><small>cell</small></sub>; it needs to be measured using reporter genes as RFP or GFP.<br />
<br><br />
Compute the average of the time derivative of the red or green fluorescence, divided by the O.D.<sub><small>600</small></sub>, in the time interval corresponding to the exponential growth phase which boundaries can be identified by visual inspection of the logarithmic growth curve:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_Scell.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c5/UNIPV_Scell.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
The S<sub><small>cell</small></sub> is basic to measure the Relative Promoter Unit, a robust parameter expressing the transcriptional activity of a promoter.<br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
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<a name="RPU"></a> <h2 class="art-postheader"><br />
R.P.U. evaluation<br />
</h2><br />
<p><br />
As described in Kelly J. et al., 2009, Relative Promoter Units express the activity of a promoter of interest (&phi;), reported to the one of a reference promoter, <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a> (from Anderson promoters collection):<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_RPU.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/70/UNIPV_RPU.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
This procedure has the advantage of being robust to variations in experimental conditions and proportional to PoPS (Polymerase Per Second) if the subsequent specifications are satisfied:<br />
<ol><ul><br />
<li> strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in BBa_J23101 reference standard<br />
<li> the reporter protein must have a half life higher than the experiment duration<br />
</ul></ol><br />
Moreover the S<sub><small>cell</small></sub> signal of both the promoter of interest and of BBa_J23101 has to be constant.<br />
</p><br />
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<a name="HSL"></a> <h2 class="art-postheader"><br />
Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration<br />
</h2><br />
<p><br />
The procedure described below is useful in order to quantify the concentration of 3OC<small><sub>6</sub></small>-HSL in cultures expressing LuxI and AiiA enzyme.<br />
<br><br />
Once collected (according to the <a href = "#T9002">protocol above</a>) and pre-processed data, it is necessary to compute the S<sub><small>cell</small></sub> of every culture.<br />
<br><br />
BBa_T9002 induced with known 3OC<small><sub>6</sub></small>-HSL concentration is useful to estimate the parameters of the activation Hill function of pLux promoter:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_activation_pLux.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f2/UNIPV_activation_pLux.png"class="thumbimage" width="80%"></a></div></div><br />
<br />
<p><br />
Provided that the S<sub><small>cell</small></sub> measured for BBa_T9002 induced with supernatants of cultures producing or degrading 3OC<small><sub>6</sub></small>-HSL has a value included in the linear zone of the biosensor (tuning of the dilution factor is necessary), you can easily extrapolate the auto-inducer concentration as follows:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 600px;"><a href="/File:UNIPV_HSL.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e2/UNIPV_HSL.PNG"class="thumbimage" width="40%"></a></div></div><br />
<br />
<p><br />
Finally, don't forget to multiply this value for the dilution factor (in our experiments it was 20).<br />
</p><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/MeasurementsTeam:UNIPV-Pavia/Measurements2011-09-17T17:00:20Z<p>Nickpv: </p>
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div><br />
<ul><br />
<li class="toclevel-1"><a href="#Promoters"><span class="tocnumber">1</span> <span class="toctext">Measuring promoters transcriptional strength</span></a></li><br />
<br />
<ul><br />
<li class="toclevel-1"><a href="#pTet_protocol"><span class="tocnumber">1.1</span> <span class="toctext">pTet transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#pLux_protocol"><span class="tocnumber">1.2</span> <span class="toctext">pLux transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#j101_protocol"><span class="tocnumber">1.3</span> <span class="toctext">Constitutive BBa_J23101x promoters transcriptional strength</span></a><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#Enzyme"><span class="tocnumber">2</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</span></a><br />
<br />
<ul><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.1</span> <span class="toctext">LuxI enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.2</span> <span class="toctext">AiiA enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#Deg"><span class="tocnumber">2.3</span> <span class="toctext">3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH</span></a></li><br />
<li class="toclevel-2"><a href="#T9002"><span class="tocnumber">2.4</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL concentration with BBa_T9002</span></a></li><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#data_analysis"><span class="tocnumber">3</span> <span class="toctext">Data analysis</span></a><br />
<ul><br />
<li class="toclevel-2"><a href="#preprocessing"><span class="tocnumber">3.1</span> <span class="toctext">Data pre-processing</span></a></li><br />
<li class="toclevel-2"><a href="#doubtime"><span class="tocnumber">3.2</span> <span class="toctext">Doubling time evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#scell"><span class="tocnumber">3.3</span> <span class="toctext">Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#RPU"><span class="tocnumber">3.4</span> <span class="toctext">R.P.U. evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#HSL"><span class="tocnumber">3.5</span> <span class="toctext">Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration</span></a></li><br />
</ul><br />
<br />
</td></tr></table><br />
</div><br />
<br />
<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C. <br />
</em><br />
<br />
<a name="Promoters"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring promoters transcriptional strength</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pTet_protocol"></a> <h2 class="art-postheader"><br />
Measuring pTet transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for three hours.<br />
<li> Induce cultures in falcon tube with anhydrotetracycline (aTc); final concentrations:<br />
<ul><br />
<li> 0 ng/ml<br />
<li> 1 ng/ml<br />
<li> 2 ng/ml<br />
<li> 3 ng/ml<br />
<li> 4 ng/ml<br />
<li> 5 ng/ml<br />
<li> 8 ng/ml<br />
<li> 10 ng/ml<br />
<li> 50 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow at 37°C, 220 rpm for three hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
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<br />
<a name="pLux_protocol"></a> <h2 class="art-postheader"><br />
Measuring pLux transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours at 37°C, 220 rpm.<br />
<li> Induce cultures in falcon tube with 3OC<sub><small>6</small></sub>-HSL; final concentrations: <br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
</ul><br />
<li> Let the cultures grow for three hours at 37°C, 220 rpm.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="j101_protocol"></a> <h2 class="art-postheader"><br />
Constitutive BBa_J23101x promoters transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for six hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Enzyme"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="LuxI"></a> <h2 class="art-postheader"><br />
LuxI enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and grow them for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of induction, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="AiiA"></a> <h2 class="art-postheader"><br />
AiiA enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow for one more hour at 37°C, 220 rpm.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Deg"></a> <h2 class="art-postheader"><br />
3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks not expressing lactonases in 1 ml of M9 with the proper antibiotic. Use M9 at different pHs, for example pH = 6.0 and pH = 7.0. Let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 with the proper antibiotic in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Prepare falcon tubes with M9 at pH = 6.0 and pH = 7.0.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL to each falcon tube.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="T9002"></a> <h2 class="art-postheader"><br />
Measuring 3OC<sub><small>6</small></sub>-HSL concentration withBBa_T9002<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> in 1 ml M9 with the proper antibiotic (Ampicillin when you use <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> or Ampicillin + Chloramphenicol 12.5 mg/ml if you use T9002-ENTERO, see <a href="https://2011.igem.org/Team:UNIPV-Pavia/Freezer">Freezer Management</a>) together with a non-fluorescent culture; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in M9 with the proper antibiotic; let the cultures grow for two hours at 37°C, 220 rpm.<br />
<li> Induce <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with the previously collected supernatants, diluting them 1:20: aliquot 190&mu;l of inducible cultures and 10 &mu;l of supernatants in the wells of the microplate.<br />
<li> Don't forget to build a calibration curve, by inducing <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with known 3OC<sub><small>6</small></sub>-HSL concentrations:<br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.2 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
<li> 1 &mu;M<br />
</ul><br />
<li>Use Tecan Infinite F200 to read O.D. at 600 nm and green fluorescence, setting the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50<br />
<li> O.D. filter: 600 nm<br />
<li> GFP filters: 485 nm (excitation) / 540 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
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<a name="data_analysis"></a> <h2 class="art-postheader"><br />
<font size = "5">Data analysis</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
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<a name="preprocessing"></a> <h2 class="art-postheader"><br />
Data pre-processing<br />
</h2><br />
<p><br />
Data from TECAN Infinite F200 need to be pre-processed in order to remove spurious effects affecting measurements. Before evaluating the parameters of interest (for example the synthesis rate per cell, S<sub><small>cell</small></sub>, or Relative Promoter Unit, R.P.U.) blanking is needed:<br />
<ol><ul><br />
<li> each time sample of O.D. at 600 nm, (O.D.<sub><small>600</small></sub>) was subtracted the reference value (i.e. the measure of O.D. at 600 nm of the broth in which cultures grow)<br />
<li> each time sample of red and green fluorescence was subtracted the reference value (i.e. the red or green fluorescence of a non-fluorescent culture)<br />
</ul></ol><br />
</p><br />
<br />
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<br />
<a name="doubtime"></a> <h2 class="art-postheader"><br />
Doubling time evaluation<br />
</h2><br />
<p><br />
After blanking O.D.<sub><small>600</small></sub> data you can compute the doubling time of cultures growth, i.e. the time needed to double O.D.<sub><small>600</small></sub>.<br />
<br><br />
Identifying the exponential phase of growth curve can be done by visual inspection, plotting the natural logarithm of O.D.<sub><small>600</small></sub> over time. Linear regression on logarithmic data is performed to estimate the growth rate, named &mu;. Finally doubling time can be evaluated as:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_DoubTime.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/7d/UNIPV_DoubTime.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
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<br />
<p><br />
When measuring multiple growth for a strain this value was evaluated for each reaplicate and then average was computed.<br />
</p><br />
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<a name="scell"></a><h2 class="art-postheader"><br />
Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation<br />
</h2><br />
<p><br />
Another interesting parameter, which describes the activity of a promoter of interest (&phi;), is the S<sub><small>cell</small></sub>; it needs to be measured using reporter genes as RFP or GFP.<br />
<br><br />
Compute the average of the time derivative of the red or green fluorescence, divided by the O.D.<sub><small>600</small></sub>, in the time interval corresponding to the exponential growth phase which boundaries can be identified by visual inspection of the logarithmic growth curve:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_Scell.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c5/UNIPV_Scell.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
The S<sub><small>cell</small></sub> is basic to measure the Relative Promoter Unit, a robust parameter expressing the transcriptional activity of a promoter.<br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="RPU"></a> <h2 class="art-postheader"><br />
R.P.U. evaluation<br />
</h2><br />
<p><br />
As described in Kelly J. et al., 2009, Relative Promoter Units express the activity of a promoter of interest (&phi;), reported to the one of a reference promoter, <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a> (from Anderson promoters collection):<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_RPU.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/70/UNIPV_RPU.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
This procedure has the advantage of being robust to variations in experimental conditions and proportional to PoPS (Polymerase Per Second) if the subsequent specifications are satisfied:<br />
<ol><ul><br />
<li> strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in BBa_J23101 reference standard<br />
<li> the reporter protein must have a half life higher than the experiment duration<br />
</ul></ol><br />
Moreover the S<sub><small>cell</small></sub> signal of both the promoter of interest and of BBa_J23101 has to be constant.<br />
</p><br />
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<a name="HSL"></a> <h2 class="art-postheader"><br />
Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration<br />
</h2><br />
<p><br />
The procedure described below is useful in order to quantify the concentration of 3OC<small><sub>6</sub></small>-HSL in cultures expressing LuxI and AiiA enzyme.<br />
<br><br />
Once collected (according to the <a href = "#T9002">protocol above</a>) and pre-processed data, it is necessary to compute the S<sub><small>cell</small></sub> of every culture.<br />
<br><br />
BBa_T9002 induced with known 3OC<small><sub>6</sub></small>-HSL concentration is useful to estimate the parameters of the activation Hill function of pLux promoter:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_activation_pLux.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f2/UNIPV_activation_pLux.png"class="thumbimage" width="80%"></a></div></div><br />
<br />
<p><br />
Provided that the S<sub><small>cell</small></sub> measured for BBa_T9002 induced with supernatants of cultures producing or degrading 3OC<small><sub>6</sub></small>-HSL has a value included in the linear zone of the biosensor (tuning of the dilution factor is necessary), you can easily extrapolate the auto-inducer concentration as follows:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 550px;"><a href="/File:UNIPV_HSL.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e2/UNIPV_HSL.PNG"class="thumbimage" width="40%"></a></div></div><br />
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<p><br />
Finally, don't forget to multiply this value for the dilution factor (in our experiments it was 20).<br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/MeasurementsTeam:UNIPV-Pavia/Measurements2011-09-17T17:00:05Z<p>Nickpv: </p>
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div><br />
<ul><br />
<li class="toclevel-1"><a href="#Promoters"><span class="tocnumber">1</span> <span class="toctext">Measuring promoters transcriptional strength</span></a></li><br />
<br />
<ul><br />
<li class="toclevel-1"><a href="#pTet_protocol"><span class="tocnumber">1.1</span> <span class="toctext">pTet transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#pLux_protocol"><span class="tocnumber">1.2</span> <span class="toctext">pLux transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#j101_protocol"><span class="tocnumber">1.3</span> <span class="toctext">Constitutive BBa_J23101x promoters transcriptional strength</span></a><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#Enzyme"><span class="tocnumber">2</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</span></a><br />
<br />
<ul><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.1</span> <span class="toctext">LuxI enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.2</span> <span class="toctext">AiiA enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#Deg"><span class="tocnumber">2.3</span> <span class="toctext">3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH</span></a></li><br />
<li class="toclevel-2"><a href="#T9002"><span class="tocnumber">2.4</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL concentration with BBa_T9002</span></a></li><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#data_analysis"><span class="tocnumber">3</span> <span class="toctext">Data analysis</span></a><br />
<ul><br />
<li class="toclevel-2"><a href="#preprocessing"><span class="tocnumber">3.1</span> <span class="toctext">Data pre-processing</span></a></li><br />
<li class="toclevel-2"><a href="#doubtime"><span class="tocnumber">3.2</span> <span class="toctext">Doubling time evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#scell"><span class="tocnumber">3.3</span> <span class="toctext">Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#RPU"><span class="tocnumber">3.4</span> <span class="toctext">R.P.U. evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#HSL"><span class="tocnumber">3.5</span> <span class="toctext">Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration</span></a></li><br />
</ul><br />
<br />
</td></tr></table><br />
</div><br />
<br />
<em><br />
NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C. <br />
</em><br />
<br />
<a name="Promoters"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring promoters transcriptional strength</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pTet_protocol"></a> <h2 class="art-postheader"><br />
Measuring pTet transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for three hours.<br />
<li> Induce cultures in falcon tube with anhydrotetracycline (aTc); final concentrations:<br />
<ul><br />
<li> 0 ng/ml<br />
<li> 1 ng/ml<br />
<li> 2 ng/ml<br />
<li> 3 ng/ml<br />
<li> 4 ng/ml<br />
<li> 5 ng/ml<br />
<li> 8 ng/ml<br />
<li> 10 ng/ml<br />
<li> 50 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow at 37°C, 220 rpm for three hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
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<br />
<a name="pLux_protocol"></a> <h2 class="art-postheader"><br />
Measuring pLux transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours at 37°C, 220 rpm.<br />
<li> Induce cultures in falcon tube with 3OC<sub><small>6</small></sub>-HSL; final concentrations: <br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
</ul><br />
<li> Let the cultures grow for three hours at 37°C, 220 rpm.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="j101_protocol"></a> <h2 class="art-postheader"><br />
Constitutive BBa_J23101x promoters transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for six hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Enzyme"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="LuxI"></a> <h2 class="art-postheader"><br />
LuxI enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and grow them for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of induction, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="AiiA"></a> <h2 class="art-postheader"><br />
AiiA enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow for one more hour at 37°C, 220 rpm.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Deg"></a> <h2 class="art-postheader"><br />
3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks not expressing lactonases in 1 ml of M9 with the proper antibiotic. Use M9 at different pHs, for example pH = 6.0 and pH = 7.0. Let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 with the proper antibiotic in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Prepare falcon tubes with M9 at pH = 6.0 and pH = 7.0.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL to each falcon tube.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="T9002"></a> <h2 class="art-postheader"><br />
Measuring 3OC<sub><small>6</small></sub>-HSL concentration withBBa_T9002<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> in 1 ml M9 with the proper antibiotic (Ampicillin when you use <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> or Ampicillin + Chloramphenicol 12.5 mg/ml if you use T9002-ENTERO, see <a href="https://2011.igem.org/Team:UNIPV-Pavia/Freezer">Freezer Management</a>) together with a non-fluorescent culture; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in M9 with the proper antibiotic; let the cultures grow for two hours at 37°C, 220 rpm.<br />
<li> Induce <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with the previously collected supernatants, diluting them 1:20: aliquot 190&mu;l of inducible cultures and 10 &mu;l of supernatants in the wells of the microplate.<br />
<li> Don't forget to build a calibration curve, by inducing <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with known 3OC<sub><small>6</small></sub>-HSL concentrations:<br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.2 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
<li> 1 &mu;M<br />
</ul><br />
<li>Use Tecan Infinite F200 to read O.D. at 600 nm and green fluorescence, setting the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50<br />
<li> O.D. filter: 600 nm<br />
<li> GFP filters: 485 nm (excitation) / 540 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
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<a name="data_analysis"></a> <h2 class="art-postheader"><br />
<font size = "5">Data analysis</font><br />
</h2><br />
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<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
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<a name="preprocessing"></a> <h2 class="art-postheader"><br />
Data pre-processing<br />
</h2><br />
<p><br />
Data from TECAN Infinite F200 need to be pre-processed in order to remove spurious effects affecting measurements. Before evaluating the parameters of interest (for example the synthesis rate per cell, S<sub><small>cell</small></sub>, or Relative Promoter Unit, R.P.U.) blanking is needed:<br />
<ol><ul><br />
<li> each time sample of O.D. at 600 nm, (O.D.<sub><small>600</small></sub>) was subtracted the reference value (i.e. the measure of O.D. at 600 nm of the broth in which cultures grow)<br />
<li> each time sample of red and green fluorescence was subtracted the reference value (i.e. the red or green fluorescence of a non-fluorescent culture)<br />
</ul></ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="doubtime"></a> <h2 class="art-postheader"><br />
Doubling time evaluation<br />
</h2><br />
<p><br />
After blanking O.D.<sub><small>600</small></sub> data you can compute the doubling time of cultures growth, i.e. the time needed to double O.D.<sub><small>600</small></sub>.<br />
<br><br />
Identifying the exponential phase of growth curve can be done by visual inspection, plotting the natural logarithm of O.D.<sub><small>600</small></sub> over time. Linear regression on logarithmic data is performed to estimate the growth rate, named &mu;. Finally doubling time can be evaluated as:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_DoubTime.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/7d/UNIPV_DoubTime.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
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<br />
<p><br />
When measuring multiple growth for a strain this value was evaluated for each reaplicate and then average was computed.<br />
</p><br />
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<a name="scell"></a><h2 class="art-postheader"><br />
Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation<br />
</h2><br />
<p><br />
Another interesting parameter, which describes the activity of a promoter of interest (&phi;), is the S<sub><small>cell</small></sub>; it needs to be measured using reporter genes as RFP or GFP.<br />
<br><br />
Compute the average of the time derivative of the red or green fluorescence, divided by the O.D.<sub><small>600</small></sub>, in the time interval corresponding to the exponential growth phase which boundaries can be identified by visual inspection of the logarithmic growth curve:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_Scell.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c5/UNIPV_Scell.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
The S<sub><small>cell</small></sub> is basic to measure the Relative Promoter Unit, a robust parameter expressing the transcriptional activity of a promoter.<br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
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<a name="RPU"></a> <h2 class="art-postheader"><br />
R.P.U. evaluation<br />
</h2><br />
<p><br />
As described in Kelly J. et al., 2009, Relative Promoter Units express the activity of a promoter of interest (&phi;), reported to the one of a reference promoter, <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a> (from Anderson promoters collection):<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_RPU.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/70/UNIPV_RPU.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
This procedure has the advantage of being robust to variations in experimental conditions and proportional to PoPS (Polymerase Per Second) if the subsequent specifications are satisfied:<br />
<ol><ul><br />
<li> strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in BBa_J23101 reference standard<br />
<li> the reporter protein must have a half life higher than the experiment duration<br />
</ul></ol><br />
Moreover the S<sub><small>cell</small></sub> signal of both the promoter of interest and of BBa_J23101 has to be constant.<br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="HSL"></a> <h2 class="art-postheader"><br />
Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration<br />
</h2><br />
<p><br />
The procedure described below is useful in order to quantify the concentration of 3OC<small><sub>6</sub></small>-HSL in cultures expressing LuxI and AiiA enzyme.<br />
<br><br />
Once collected (according to the <a href = "#T9002">protocol above</a>) and pre-processed data, it is necessary to compute the S<sub><small>cell</small></sub> of every culture.<br />
<br><br />
BBa_T9002 induced with known 3OC<small><sub>6</sub></small>-HSL concentration is useful to estimate the parameters of the activation Hill function of pLux promoter:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_activation_pLux.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f2/UNIPV_activation_pLux.png"class="thumbimage" width="80%"></a></div></div><br />
<br />
<p><br />
Provided that the S<sub><small>cell</small></sub> measured for BBa_T9002 induced with supernatants of cultures producing or degrading 3OC<small><sub>6</sub></small>-HSL has a value included in the linear zone of the biosensor (tuning of the dilution factor is necessary), you can easily extrapolate the auto-inducer concentration as follows:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 450px;"><a href="/File:UNIPV_HSL.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e2/UNIPV_HSL.PNG"class="thumbimage" width="40%"></a></div></div><br />
<br />
<p><br />
Finally, don't forget to multiply this value for the dilution factor (in our experiments it was 20).<br />
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{{end}}</div>Nickpvhttp://2011.igem.org/File:UNIPV_HSL.PNGFile:UNIPV HSL.PNG2011-09-17T16:59:00Z<p>Nickpv: uploaded a new version of &quot;File:UNIPV HSL.PNG&quot;: Reverted to version as of 16:56, 17 September 2011</p>
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<div></div>Nickpvhttp://2011.igem.org/File:UNIPV_HSL.PNGFile:UNIPV HSL.PNG2011-09-17T16:57:39Z<p>Nickpv: uploaded a new version of &quot;File:UNIPV HSL.PNG&quot;</p>
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<div></div>Nickpvhttp://2011.igem.org/File:UNIPV_HSL.PNGFile:UNIPV HSL.PNG2011-09-17T16:56:02Z<p>Nickpv: uploaded a new version of &quot;File:UNIPV HSL.PNG&quot;</p>
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<div></div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/MeasurementsTeam:UNIPV-Pavia/Measurements2011-09-17T13:47:17Z<p>Nickpv: </p>
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div><br />
<ul><br />
<li class="toclevel-1"><a href="#Promoters"><span class="tocnumber">1</span> <span class="toctext">Measuring promoters transcriptional strength</span></a></li><br />
<br />
<ul><br />
<li class="toclevel-1"><a href="#pTet_protocol"><span class="tocnumber">1.1</span> <span class="toctext">pTet transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#pLux_protocol"><span class="tocnumber">1.2</span> <span class="toctext">pLux transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#j101_protocol"><span class="tocnumber">1.3</span> <span class="toctext">Constitutive BBa_J23101x promoters transcriptional strength</span></a><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#Enzyme"><span class="tocnumber">2</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</span></a><br />
<br />
<ul><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.1</span> <span class="toctext">LuxI enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.2</span> <span class="toctext">AiiA enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#Deg"><span class="tocnumber">2.3</span> <span class="toctext">3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH</span></a></li><br />
<li class="toclevel-2"><a href="#T9002"><span class="tocnumber">2.4</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL concentration with BBa_T9002</span></a></li><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#data_analysis"><span class="tocnumber">3</span> <span class="toctext">Data analysis</span></a><br />
<ul><br />
<li class="toclevel-2"><a href="#preprocessing"><span class="tocnumber">3.1</span> <span class="toctext">Data pre-processing</span></a></li><br />
<li class="toclevel-2"><a href="#doubtime"><span class="tocnumber">3.2</span> <span class="toctext">Doubling time evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#scell"><span class="tocnumber">3.3</span> <span class="toctext">Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#RPU"><span class="tocnumber">3.4</span> <span class="toctext">R.P.U. evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#HSL"><span class="tocnumber">3.5</span> <span class="toctext">Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration</span></a></li><br />
</ul><br />
<br />
</td></tr></table><br />
</div><br />
<br />
<a name="Promoters"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring promoters transcriptional strength</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pTet_protocol"></a> <h2 class="art-postheader"><br />
Measuring pTet transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for three hours.<br />
<li> Induce cultures in falcon tube with anhydrotetracycline (aTc); final concentrations:<br />
<ul><br />
<li> 0 ng/ml<br />
<li> 1 ng/ml<br />
<li> 2 ng/ml<br />
<li> 3 ng/ml<br />
<li> 4 ng/ml<br />
<li> 5 ng/ml<br />
<li> 8 ng/ml<br />
<li> 10 ng/ml<br />
<li> 50 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow at 37°C, 220 rpm for three hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pLux_protocol"></a> <h2 class="art-postheader"><br />
Measuring pLux transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours at 37°C, 220 rpm.<br />
<li> Induce cultures in falcon tube with 3OC<sub><small>6</small></sub>-HSL; final concentrations: <br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
</ul><br />
<li> Let the cultures grow for three hours at 37°C, 220 rpm.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="j101_protocol"></a> <h2 class="art-postheader"><br />
Constitutive BBa_J23101x promoters transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for six hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Enzyme"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="LuxI"></a> <h2 class="art-postheader"><br />
LuxI enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and grow them for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of induction, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="AiiA"></a> <h2 class="art-postheader"><br />
AiiA enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow for one more hour at 37°C, 220 rpm.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Deg"></a> <h2 class="art-postheader"><br />
3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks not expressing lactonases in 1 ml of M9 with the proper antibiotic. Use M9 at different pHs, for example pH = 6.0 and pH = 7.0. Let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 with the proper antibiotic in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Prepare falcon tubes with M9 at pH = 6.0 and pH = 7.0.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL to each falcon tube.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="T9002"></a> <h2 class="art-postheader"><br />
Measuring 3OC<sub><small>6</small></sub>-HSL concentration withBBa_T9002<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> in 1 ml M9 with the proper antibiotic (Ampicillin when you use <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> or Ampicillin + Chloramphenicol 12.5 mg/ml if you use T9002-ENTERO, see <a href="https://2011.igem.org/Team:UNIPV-Pavia/Freezer">Freezer Management</a>) together with a non-fluorescent culture; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in M9 with the proper antibiotic; let the cultures grow for two hours at 37°C, 220 rpm.<br />
<li> Induce <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with the previously collected supernatants, diluting them 1:20: aliquot 190&mu;l of inducible cultures and 10 &mu;l of supernatants in the wells of the microplate.<br />
<li> Don't forget to build a calibration curve, by inducing <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with known 3OC<sub><small>6</small></sub>-HSL concentrations:<br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.2 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
<li> 1 &mu;M<br />
</ul><br />
<li>Use Tecan Infinite F200 to read O.D. at 600 nm and green fluorescence, setting the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50<br />
<li> O.D. filter: 600 nm<br />
<li> GFP filters: 485 nm (excitation) / 540 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="data_analysis"></a> <h2 class="art-postheader"><br />
<font size = "5">Data analysis</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="preprocessing"></a> <h2 class="art-postheader"><br />
Data pre-processing<br />
</h2><br />
<p><br />
Data from TECAN Infinite F200 need to be pre-processed in order to remove spurious effects affecting measurements. Before evaluating the parameters of interest (for example the synthesis rate per cell, S<sub><small>cell</small></sub>, or Relative Promoter Unit, R.P.U.) blanking is needed:<br />
<ol><ul><br />
<li> each time sample of O.D. at 600 nm, (O.D.<sub><small>600</small></sub>) was subtracted the reference value (i.e. the measure of O.D. at 600 nm of the broth in which cultures grow)<br />
<li> each time sample of red and green fluorescence was subtracted the reference value (i.e. the red or green fluorescence of a non-fluorescent culture)<br />
</ul></ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="doubtime"></a> <h2 class="art-postheader"><br />
Doubling time evaluation<br />
</h2><br />
<p><br />
After blanking O.D.<sub><small>600</small></sub> data you can compute the doubling time of cultures growth, i.e. the time needed to double O.D.<sub><small>600</small></sub>.<br />
<br><br />
Identifying the exponential phase of growth curve can be done by visual inspection, plotting the natural logarithm of O.D.<sub><small>600</small></sub> over time. Linear regression on logarithmic data is performed to estimate the growth rate, named &mu;. Finally doubling time can be evaluated as:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_DoubTime.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/7d/UNIPV_DoubTime.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<br />
<p><br />
When measuring multiple growth for a strain this value was evaluated for each reaplicate and then average was computed.<br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
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<br />
<a name="scell"></a><h2 class="art-postheader"><br />
Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation<br />
</h2><br />
<p><br />
Another interesting parameter, which describes the activity of a promoter of interest (&phi;), is the S<sub><small>cell</small></sub>; it needs to be measured using reporter genes as RFP or GFP.<br />
<br><br />
Compute the average of the time derivative of the red or green fluorescence, divided by the O.D.<sub><small>600</small></sub>, in the time interval corresponding to the exponential growth phase which boundaries can be identified by visual inspection of the logarithmic growth curve:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_Scell.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c5/UNIPV_Scell.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
The S<sub><small>cell</small></sub> is basic to measure the Relative Promoter Unit, a robust parameter expressing the transcriptional activity of a promoter.<br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="RPU"></a> <h2 class="art-postheader"><br />
R.P.U. evaluation<br />
</h2><br />
<p><br />
As described in Kelly J. et al., 2009, Relative Promoter Units express the activity of a promoter of interest (&phi;), reported to the one of a reference promoter, <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a> (from Anderson promoters collection):<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_RPU.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/70/UNIPV_RPU.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
This procedure has the advantage of being robust to variations in experimental conditions and proportional to PoPS (Polymerase Per Second) if the subsequent specifications are satisfied:<br />
<ol><ul><br />
<li> strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in BBa_J23101 reference standard<br />
<li> the reporter protein must have a half life higher than the experiment duration<br />
</ul></ol><br />
Moreover the S<sub><small>cell</small></sub> signal of both the promoter of interest and of BBa_J23101 has to be constant.<br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="HSL"></a> <h2 class="art-postheader"><br />
Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration<br />
</h2><br />
<p><br />
The procedure described below is useful in order to quantify the concentration of 3OC<small><sub>6</sub></small>-HSL in cultures expressing LuxI and AiiA enzyme.<br />
<br><br />
Once collected (according to the <a href = "#T9002">protocol above</a>) and pre-processed data, it is necessary to compute the S<sub><small>cell</small></sub> of every culture.<br />
<br><br />
BBa_T9002 induced with known 3OC<small><sub>6</sub></small>-HSL concentration is useful to estimate the parameters of the activation Hill function of pLux promoter:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_activation_pLux.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f2/UNIPV_activation_pLux.png"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
Provided that the S<sub><small>cell</small></sub> measured for BBa_T9002 induced with supernatants of cultures producing or degrading 3OC<small><sub>6</sub></small>-HSL has a value included in the linear zone of the biosensor (tuning of the dilution factor is necessary), you can easily extrapolate the auto-inducer concentration as follows:<br />
</p><br />
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<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_HSL.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e2/UNIPV_HSL.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
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Finally, don't forget to multiply this value for the dilution factor (in our experiments it was 20).<br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/MeasurementsTeam:UNIPV-Pavia/Measurements2011-09-17T13:45:41Z<p>Nickpv: </p>
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div><br />
<ul><br />
<li class="toclevel-1"><a href="#Promoters"><span class="tocnumber">1</span> <span class="toctext">Measuring promoters transcriptional strength</span></a></li><br />
<br />
<ul><br />
<li class="toclevel-1"><a href="#pTet_protocol"><span class="tocnumber">1.1</span> <span class="toctext">pTet transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#pLux_protocol"><span class="tocnumber">1.2</span> <span class="toctext">pLux transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#j101_protocol"><span class="tocnumber">1.3</span> <span class="toctext">Constitutive BBa_J23101x promoters transcriptional strength</span></a><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#Enzyme"><span class="tocnumber">2</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</span></a><br />
<br />
<ul><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.1</span> <span class="toctext">LuxI enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.2</span> <span class="toctext">AiiA enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#Deg"><span class="tocnumber">2.3</span> <span class="toctext">3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH</span></a></li><br />
<li class="toclevel-2"><a href="#T9002"><span class="tocnumber">2.4</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL concentration with BBa_T9002</span></a></li><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#data_analysis"><span class="tocnumber">3</span> <span class="toctext">Data analysis</span></a><br />
<ul><br />
<li class="toclevel-2"><a href="#preprocessing"><span class="tocnumber">3.1</span> <span class="toctext">Data pre-processing</span></a></li><br />
<li class="toclevel-2"><a href="#doubtime"><span class="tocnumber">3.2</span> <span class="toctext">Doubling time evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#scell"><span class="tocnumber">3.3</span> <span class="toctext">Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#RPU"><span class="tocnumber">3.4</span> <span class="toctext">R.P.U. evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#HSL"><span class="tocnumber">3.5</span> <span class="toctext">Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration</span></a></li><br />
</ul><br />
<br />
</td></tr></table><br />
</div><br />
<br />
<a name="Promoters"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring promoters transcriptional strength</font><br />
</h2><br />
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<br />
<a name="pTet_protocol"></a> <h2 class="art-postheader"><br />
Measuring pTet transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for three hours.<br />
<li> Induce cultures in falcon tube with anhydrotetracycline (aTc); final concentrations:<br />
<ul><br />
<li> 0 ng/ml<br />
<li> 1 ng/ml<br />
<li> 2 ng/ml<br />
<li> 3 ng/ml<br />
<li> 4 ng/ml<br />
<li> 5 ng/ml<br />
<li> 8 ng/ml<br />
<li> 10 ng/ml<br />
<li> 50 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow at 37°C, 220 rpm for three hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
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<br />
<a name="pLux_protocol"></a> <h2 class="art-postheader"><br />
Measuring pLux transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours at 37°C, 220 rpm.<br />
<li> Induce cultures in falcon tube with 3OC<sub><small>6</small></sub>-HSL; final concentrations: <br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
</ul><br />
<li> Let the cultures grow for three hours at 37°C, 220 rpm.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
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<br />
<a name="j101_protocol"></a> <h2 class="art-postheader"><br />
Constitutive BBa_J23101x promoters transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for six hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
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<a name="Enzyme"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</font><br />
</h2><br />
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<br />
<a name="LuxI"></a> <h2 class="art-postheader"><br />
LuxI enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and grow them for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of induction, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
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<a name="AiiA"></a> <h2 class="art-postheader"><br />
AiiA enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow for one more hour at 37°C, 220 rpm.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
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<a name="Deg"></a> <h2 class="art-postheader"><br />
3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks not expressing lactonases in 1 ml of M9 with the proper antibiotic. Use M9 at different pHs, for example pH = 6.0 and pH = 7.0. Let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 with the proper antibiotic in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Prepare falcon tubes with M9 at pH = 6.0 and pH = 7.0.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL to each falcon tube.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
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<a name="T9002"></a> <h2 class="art-postheader"><br />
Measuring 3OC<sub><small>6</small></sub>-HSL concentration withBBa_T9002<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> in 1 ml M9 with the proper antibiotic (Ampicillin when you use <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> or Ampicillin + Chloramphenicol 12.5 mg/ml if you use T9002-ENTERO, see <a href="https://2011.igem.org/Team:UNIPV-Pavia/Freezer">Freezer Management</a>) together with a non-fluorescent culture; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in M9 with the proper antibiotic; let the cultures grow for two hours at 37°C, 220 rpm.<br />
<li> Induce <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with the previously collected supernatants, diluting them 1:20: aliquot 190&mu;l of inducible cultures and 10 &mu;l of supernatants in the wells of the microplate.<br />
<li> Don't forget to build a calibration curve, by inducing <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with known 3OC<sub><small>6</small></sub>-HSL concentrations:<br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.2 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
<li> 1 &mu;M<br />
</ul><br />
<li>Use Tecan Infinite F200 to read O.D. at 600 nm and green fluorescence, setting the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50<br />
<li> O.D. filter: 600 nm<br />
<li> GFP filters: 485 nm (excitation) / 540 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
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<a name="data_analysis"></a> <h2 class="art-postheader"><br />
<font size = "5">Data analysis</font><br />
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<a name="preprocessing"></a> <h2 class="art-postheader"><br />
Data pre-processing<br />
</h2><br />
<p><br />
Data from TECAN Infinite F200 need to be pre-processed in order to remove spurious effects affecting measurements. Before evaluating the parameters of interest (for example the synthesis rate per cell, S<sub><small>cell</small></sub>, or Relative Promoter Unit, R.P.U.) blanking is needed:<br />
<ol><ul><br />
<li> each time sample of O.D. at 600 nm, (O.D.<sub><small>600</small></sub>) was subtracted the reference value (i.e. the measure of O.D. at 600 nm of the broth in which cultures grow)<br />
<li> each time sample of red and green fluorescence was subtracted the reference value (i.e. the red or green fluorescence of a non-fluorescent culture)<br />
</ul></ol><br />
</p><br />
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<a name="doubtime"></a> <h2 class="art-postheader"><br />
Doubling time evaluation<br />
</h2><br />
<p><br />
After blanking O.D.<sub><small>600</small></sub> data you can compute the doubling time of cultures growth, i.e. the time needed to double O.D.<sub><small>600</small></sub>.<br />
<br><br />
Identifying the exponential phase of growth curve can be done by visual inspection, plotting the natural logarithm of O.D.<sub><small>600</small></sub> over time. Linear regression on logarithmic data is performed to estimate the growth rate, named &mu;. Finally doubling time can be evaluated as:<br />
</p><br />
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<div class="center"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_DoubTime.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/7d/UNIPV_DoubTime.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
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<p><br />
When measuring multiple growth for a strain this value was evaluated for each reaplicate and then average was computed.<br />
</p><br />
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<a name="scell"></a><h2 class="art-postheader"><br />
Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation<br />
</h2><br />
<p><br />
Another interesting parameter, which describes the activity of a promoter of interest (&phi;), is the S<sub><small>cell</small></sub>; it needs to be measured using reporter genes as RFP or GFP.<br />
<br><br />
Compute the average of the time derivative of the red or green fluorescence, divided by the O.D.<sub><small>600</small></sub>, in the time interval corresponding to the exponential growth phase which boundaries can be identified by visual inspection of the logarithmic growth curve:<br />
</p><br />
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<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_Scell.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c5/UNIPV_Scell.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
The S<sub><small>cell</small></sub> is basic to measure the Relative Promoter Unit, a robust parameter expressing the transcriptional activity of a promoter.<br />
</p><br />
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<a name="RPU"></a> <h2 class="art-postheader"><br />
R.P.U. evaluation<br />
</h2><br />
<p><br />
As described in Kelly J. et al., 2009, Relative Promoter Units express the activity of a promoter of interest (&phi;), reported to the one of a reference promoter, <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a> (from Anderson promoters collection):<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_RPU.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/70/UNIPV_RPU.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
This procedure has the advantage of being robust to variations in experimental conditions and proportional to PoPS (Polymerase Per Second) if the subsequent specifications are satisfied:<br />
<ol><ul><br />
<li> strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in BBa_J23101 reference standard<br />
<li> the reporter protein must have a half life higher than the experiment duration<br />
</ul></ol><br />
Moreover the S<sub><small>cell</small></sub> signal of both the promoter of interest and of BBa_J23101 has to be at the steady state.<br />
</p><br />
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<a name="HSL"></a> <h2 class="art-postheader"><br />
Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration<br />
</h2><br />
<p><br />
The procedure described below is useful in order to quantify the concentration of 3OC<small><sub>6</sub></small>-HSL in cultures expressing LuxI and AiiA enzyme.<br />
<br><br />
Once collected (according to the <a href = "#T9002">protocol above</a>) and pre-processed data, it is necessary to compute the S<sub><small>cell</small></sub> of every culture.<br />
<br><br />
BBa_T9002 induced with known 3OC<small><sub>6</sub></small>-HSL concentration is useful to estimate the parameters of the activation Hill function of pLux promoter:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_activation_pLux.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f2/UNIPV_activation_pLux.png"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
Provided that the S<sub><small>cell</small></sub> measured for BBa_T9002 induced with supernatants of cultures producing or degrading 3OC<small><sub>6</sub></small>-HSL has a value included in the linear zone of the biosensor (tuning of the dilution factor is necessary), you can easily extrapolate the auto-inducer concentration as follows:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_HSL.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e2/UNIPV_HSL.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
Finally, don't forget to multiply this value for the dilution factor (in our experiments it was 20).<br />
</p><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/MeasurementsTeam:UNIPV-Pavia/Measurements2011-09-17T13:42:33Z<p>Nickpv: </p>
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div><br />
<ul><br />
<li class="toclevel-1"><a href="#Promoters"><span class="tocnumber">1</span> <span class="toctext">Measuring promoters transcriptional strength</span></a></li><br />
<br />
<ul><br />
<li class="toclevel-1"><a href="#pTet_protocol"><span class="tocnumber">1.1</span> <span class="toctext">pTet transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#pLux_protocol"><span class="tocnumber">1.2</span> <span class="toctext">pLux transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#j101_protocol"><span class="tocnumber">1.3</span> <span class="toctext">Constitutive BBa_J23101x promoters transcriptional strength</span></a><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#Enzyme"><span class="tocnumber">2</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</span></a><br />
<br />
<ul><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.1</span> <span class="toctext">LuxI enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.2</span> <span class="toctext">AiiA enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#Deg"><span class="tocnumber">2.3</span> <span class="toctext">3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH</span></a></li><br />
<li class="toclevel-2"><a href="#T9002"><span class="tocnumber">2.4</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL concentration with BBa_T9002</span></a></li><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#data_analysis"><span class="tocnumber">3</span> <span class="toctext">Data analysis</span></a><br />
<ul><br />
<li class="toclevel-2"><a href="#preprocessing"><span class="tocnumber">3.1</span> <span class="toctext">Data pre-processing</span></a></li><br />
<li class="toclevel-2"><a href="#doubtime"><span class="tocnumber">3.2</span> <span class="toctext">Doubling time evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#scell"><span class="tocnumber">3.3</span> <span class="toctext">Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#RPU"><span class="tocnumber">3.4</span> <span class="toctext">R.P.U. evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#HSL"><span class="tocnumber">3.5</span> <span class="toctext">Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration</span></a></li><br />
</ul><br />
<br />
</td></tr></table><br />
</div><br />
<br />
<a name="Promoters"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring promoters transcriptional strength</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pTet_protocol"></a> <h2 class="art-postheader"><br />
Measuring pTet transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for three hours.<br />
<li> Induce cultures in falcon tube with anhydrotetracycline (aTc); final concentrations:<br />
<ul><br />
<li> 0 ng/ml<br />
<li> 1 ng/ml<br />
<li> 2 ng/ml<br />
<li> 3 ng/ml<br />
<li> 4 ng/ml<br />
<li> 5 ng/ml<br />
<li> 8 ng/ml<br />
<li> 10 ng/ml<br />
<li> 50 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow at 37°C, 220 rpm for three hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pLux_protocol"></a> <h2 class="art-postheader"><br />
Measuring pLux transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours at 37°C, 220 rpm.<br />
<li> Induce cultures in falcon tube with 3OC<sub><small>6</small></sub>-HSL; final concentrations: <br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
</ul><br />
<li> Let the cultures grow for three hours at 37°C, 220 rpm.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="j101_protocol"></a> <h2 class="art-postheader"><br />
Constitutive BBa_J23101x promoters transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for six hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Enzyme"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="LuxI"></a> <h2 class="art-postheader"><br />
LuxI enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and grow them for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of induction, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="AiiA"></a> <h2 class="art-postheader"><br />
AiiA enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow for one more hour at 37°C, 220 rpm.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Deg"></a> <h2 class="art-postheader"><br />
3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks not expressing lactonases in 1 ml of M9 with the proper antibiotic. Use M9 at different pHs, for example pH = 6.0 and pH = 7.0. Let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 with the proper antibiotic in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Prepare falcon tubes with M9 at pH = 6.0 and pH = 7.0.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL to each falcon tube.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="T9002"></a> <h2 class="art-postheader"><br />
Measuring 3OC<sub><small>6</small></sub>-HSL concentration withBBa_T9002<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> in 1 ml M9 with the proper antibiotic (Ampicillin when you use <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> or Ampicillin + Chloramphenicol 12.5 mg/ml if you use T9002-ENTERO, see <a href="https://2011.igem.org/Team:UNIPV-Pavia/Freezer">Freezer Management</a>) together with a non-fluorescent culture; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in M9 with the proper antibiotic; let the cultures grow for two hours at 37°C, 220 rpm.<br />
<li> Induce <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with the previously collected supernatants, diluting them 1:20: aliquot 190&mu;l of inducible cultures and 10 &mu;l of supernatants in the wells of the microplate.<br />
<li> Don't forget to build a calibration curve, by inducing <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with known 3OC<sub><small>6</small></sub>-HSL concentrations:<br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.2 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
<li> 1 &mu;M<br />
</ul><br />
<li>Use Tecan Infinite F200 to read O.D. at 600 nm and green fluorescence, setting the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50<br />
<li> O.D. filter: 600 nm<br />
<li> GFP filters: 485 nm (excitation) / 540 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
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<br />
<a name="data_analysis"></a> <h2 class="art-postheader"><br />
<font size = "5">Data analysis</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="preprocessing"></a> <h2 class="art-postheader"><br />
Data pre-processing<br />
</h2><br />
<p><br />
Data from TECAN Infinite F200 need to be pre-processed in order to remove spurious effects affecting measurements. Before evaluating the parameters of interest (for example the synthesis rate per cell, S<sub><small>cell</small></sub>, or Relative Promoter Unit, R.P.U.) blanking is needed:<br />
<ol><ul><br />
<li> each time sample of O.D. at 600 nm, (O.D.<sub><small>600</small></sub>) was subtracted the reference value (i.e. the measure of O.D. at 600 nm of the broth in which cultures grow)<br />
<li> each time sample of red and green fluorescence was subtracted the reference value (i.e. the red or green fluorescence of a non-fluorescent culture)<br />
</ul></ol><br />
</p><br />
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<br />
<a name="doubtime"></a> <h2 class="art-postheader"><br />
Doubling time evaluation<br />
</h2><br />
<p><br />
After blanking O.D.<sub><small>600</small></sub> data you can compute the doubling time of cultures growth, i.e. the time needed to double O.D.<sub><small>600</small></sub>.<br />
<br><br />
Identifying the exponential phase of growth curve can be done by visual inspection, plotting the natural logarithm of O.D.<sub><small>600</small></sub> over time. Linear regression on logarithmic data is performed to estimate the growth rate, named &mu;. Finally doubling time can be evaluated as:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_DoubTime.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/7d/UNIPV_DoubTime.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<br />
<p><br />
When measuring multiple growth for a strain this value was evaluated for each reaplicate and then average was computed.<br />
</p><br />
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<br />
<a name="scell"></a><h2 class="art-postheader"><br />
Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation<br />
</h2><br />
<p><br />
Another interesting parameter, which describes the activity of a promoter of interest (&phi;), is the S<sub><small>cell</small></sub>; it needs to be measured using reporter genes as RFP or GFP.<br />
<br><br />
Compute the average of the time derivative of the red or green fluorescence, divided by the O.D.<sub><small>600</small></sub>, in the time interval corresponding to the exponential growth phase which boundaries can be identified by visual inspection of the logarithmic growth curve:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_Scell.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c5/UNIPV_Scell.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
The S<sub><small>cell</small></sub> is basic to measure the Relative Promoter Unit, a robust parameter expressing the transcriptional activity of a promoter.<br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="RPU"></a> <h2 class="art-postheader"><br />
R.P.U. evaluation<br />
</h2><br />
<p><br />
As described in Kelly J. et al., 2009, Relative Promoter Units express the activity of a promoter of interest (&phi;), reported to the one of a reference promoter, <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a> (from Anderson promoters collection):<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_RPU.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/70/UNIPV_RPU.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
This procedure has the advantage of being robust to variations in experimental conditions and proportional to PoPS (Polymerase Per Second) if the subsequent specifications are satisfied:<br />
<ol><ul><br />
<li> strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in BBa_J23101 reference standard<br />
<li> the reporter protein must have a half life higher than the experiment duration<br />
</ul></ol><br />
Moreover the S<sub><small>cell</small></sub> signal of both the promoter of interest and of BBa_J23101 has tp be at the steady state.<br />
</p><br />
<br />
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<br />
<a name="HSL"></a> <h2 class="art-postheader"><br />
Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration<br />
</h2><br />
<p><br />
The procedure described below is useful in order to quantify the concentration of 3OC<small><sub>6</sub></small>-HSL in cultures expressing LuxI and AiiA enzyme.<br />
<br><br />
Once collected (according to the <a href = "#T9002">protocol above</a>) and pre-processed data, it is necessary to compute the S<sub><small>cell</small></sub> of every culture.<br />
<br><br />
BBa_T9002 induced with known 3OC<small><sub>6</sub></small>-HSL concentration is useful to estimate the parameters of the activation Hill function of pLux promoter:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_activation_pLux.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f2/UNIPV_activation_pLux.png"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
Provided that the S<sub><small>cell</small></sub> measured for BBa_T9002 induced with supernatants of cultures producing or degrading 3OC<small><sub>6</sub></small>-HSL has a value included in the linear zone of the biosensor (tuning of the dilution factor is necessary), you can easily extrapolate the auto-inducer concentration as follows:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_HSL.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e2/UNIPV_HSL.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
Finally, don't forget to multiply this value for the dilution factor (in our experiments it was 20).<br />
</p><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/MeasurementsTeam:UNIPV-Pavia/Measurements2011-09-17T13:31:25Z<p>Nickpv: </p>
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div><br />
<ul><br />
<li class="toclevel-1"><a href="#Promoters"><span class="tocnumber">1</span> <span class="toctext">Measuring promoters transcriptional strength</span></a></li><br />
<br />
<ul><br />
<li class="toclevel-1"><a href="#pTet_protocol"><span class="tocnumber">1.1</span> <span class="toctext">pTet transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#pLux_protocol"><span class="tocnumber">1.2</span> <span class="toctext">pLux transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#j101_protocol"><span class="tocnumber">1.3</span> <span class="toctext">Constitutive BBa_J23101x promoters transcriptional strength</span></a><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#Enzyme"><span class="tocnumber">2</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</span></a><br />
<br />
<ul><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.1</span> <span class="toctext">LuxI enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.2</span> <span class="toctext">AiiA enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#Deg"><span class="tocnumber">2.3</span> <span class="toctext">3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH</span></a></li><br />
<li class="toclevel-2"><a href="#T9002"><span class="tocnumber">2.4</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL concentration with BBa_T9002</span></a></li><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#data_analysis"><span class="tocnumber">3</span> <span class="toctext">Data analysis</span></a><br />
<ul><br />
<li class="toclevel-2"><a href="#preprocessing"><span class="tocnumber">3.1</span> <span class="toctext">Data pre-processing</span></a></li><br />
<li class="toclevel-2"><a href="#doubtime"><span class="tocnumber">3.2</span> <span class="toctext">Doubling time evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#scell"><span class="tocnumber">3.3</span> <span class="toctext">Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#RPU"><span class="tocnumber">3.4</span> <span class="toctext">R.P.U. evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#HSL"><span class="tocnumber">3.5</span> <span class="toctext">Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration</span></a></li><br />
</ul><br />
<br />
</td></tr></table><br />
</div><br />
<br />
<a name="Promoters"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring promoters transcriptional strength</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pTet_protocol"></a> <h2 class="art-postheader"><br />
Measuring pTet transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for three hours.<br />
<li> Induce cultures in falcon tube with anhydrotetracycline (aTc); final concentrations:<br />
<ul><br />
<li> 0 ng/ml<br />
<li> 1 ng/ml<br />
<li> 2 ng/ml<br />
<li> 3 ng/ml<br />
<li> 4 ng/ml<br />
<li> 5 ng/ml<br />
<li> 8 ng/ml<br />
<li> 10 ng/ml<br />
<li> 50 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow at 37°C, 220 rpm for three hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pLux_protocol"></a> <h2 class="art-postheader"><br />
Measuring pLux transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours at 37°C, 220 rpm.<br />
<li> Induce cultures in falcon tube with 3OC<sub><small>6</small></sub>-HSL; final concentrations: <br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
</ul><br />
<li> Let the cultures grow for three hours at 37°C, 220 rpm.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="j101_protocol"></a> <h2 class="art-postheader"><br />
Constitutive BBa_J23101x promoters transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for six hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Enzyme"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="LuxI"></a> <h2 class="art-postheader"><br />
LuxI enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and grow them for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of induction, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="AiiA"></a> <h2 class="art-postheader"><br />
AiiA enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow for one more hour at 37°C, 220 rpm.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Deg"></a> <h2 class="art-postheader"><br />
3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks not expressing lactonases in 1 ml of M9 with the proper antibiotic. Use M9 at different pHs, for example pH = 6.0 and pH = 7.0. Let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 with the proper antibiotic in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Prepare falcon tubes with M9 at pH = 6.0 and pH = 7.0.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL to each falcon tube.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="T9002"></a> <h2 class="art-postheader"><br />
Measuring 3OC<sub><small>6</small></sub>-HSL concentration withBBa_T9002<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> in 1 ml M9 with the proper antibiotic (Ampicillin when you use <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> or Ampicillin + Chloramphenicol 12.5 mg/ml if you use T9002-ENTERO, see <a href="https://2011.igem.org/Team:UNIPV-Pavia/Freezer">Freezer Management</a>) together with a non-fluorescent culture; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in M9 with the proper antibiotic; let the cultures grow for two hours at 37°C, 220 rpm.<br />
<li> Induce <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with the previously collected supernatants, diluting them 1:20: aliquot 190&mu;l of inducible cultures and 10 &mu;l of supernatants in the wells of the microplate.<br />
<li> Don't forget to build a calibration curve, by inducing <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with known 3OC<sub><small>6</small></sub>-HSL concentrations:<br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.2 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
<li> 1 &mu;M<br />
</ul><br />
<li>Use Tecan Infinite F200 to read O.D. at 600 nm and green fluorescence, setting the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50<br />
<li> O.D. filter: 600 nm<br />
<li> GFP filters: 485 nm (excitation) / 540 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="data_analysis"></a> <h2 class="art-postheader"><br />
<font size = "5">Data analysis</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="preprocessing"></a> <h2 class="art-postheader"><br />
Data pre-processing<br />
</h2><br />
<p><br />
Data from TECAN Infinite F200 need to be pre-processed in order to remove spurious effects affecting measurements. Before evaluating the parameters of interest (for example the synthesis rate per cell, S<sub><small>cell</small></sub>, or Relative Promoter Unit, R.P.U.) blanking is needed:<br />
<ol><ul><br />
<li> each time sample of O.D. at 600 nm, (O.D.<sub><small>600</small></sub>) was subtracted the reference value (i.e. the measure of O.D. at 600 nm of the broth in which cultures grow)<br />
<li> each time sample of red and green fluorescence was subtracted the reference value (i.e. the red or green fluorescence of a non-fluorescent culture)<br />
</ul></ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="doubtime"></a> <h2 class="art-postheader"><br />
Doubling time evaluation<br />
</h2><br />
<p><br />
After blanking O.D.<sub><small>600</small></sub> data you can compute the doubling time of cultures growth, i.e. the time needed to double O.D.<sub><small>600</small></sub>.<br />
<br><br />
Identifying the exponential phase of growth curve can be done by visual inspection, plotting the natural logarithm of O.D.<sub><small>600</small></sub> over time. Linear regression on logarithmic data is performed to estimate the growth rate, named &mu;. Finally doubling time can be evaluated as:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_DoubTime.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/7d/UNIPV_DoubTime.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<br />
<p><br />
When measuring multiple growth for a strain this value was evaluated for each reaplicate and then average was computed.<br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<br />
<a name="scell"></a><h2 class="art-postheader"><br />
Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation<br />
</h2><br />
<p><br />
Another interesting parameter, which describes the activity of a promoter of interest (&phi;), is the S<sub><small>cell</small></sub>; it needs to be measured using reporter genes as RFP or GFP.<br />
<br><br />
Compute the average of the time derivative of the red or green fluorescence, divided by the O.D.<sub><small>600</small></sub>, in the time interval corresponding to the exponential growth phase which boundaries can be identified by visual inspection of the logarithmic growth curve:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_Scell.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c5/UNIPV_Scell.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
The S<sub><small>cell</small></sub> is basic to measure the Relative Promoter Unit, a robust parameter expressing the transcriptional activity of a promoter.<br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="RPU"></a> <h2 class="art-postheader"><br />
R.P.U. evaluation<br />
</h2><br />
<p><br />
As described in Kelly J. et al., 2009, Relative Promoter Units express the activity of a promoter of interest (&phi;), reported to the one of a reference promoter, <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a> (from Anderson promoters collection):<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_RPU.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/70/UNIPV_RPU.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
This procedure has the advantage of being robust to variations in experimental conditions and proportional to PoPS (Polymerase Per Second) if the subsequent specifications are satisfied:<br />
<ol><ul><br />
<li> strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in BBa_J23101 reference standard<br />
<li> the reporter protein must have a half life higher than the experiment duration<br />
</ul></ol><br />
Moreover the S<sub><small>cell</small></sub> signal of both the promoter of interest and of BBa_J23101 has tp be at the steady state.<br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="HSL"></a> <h2 class="art-postheader"><br />
Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration<br />
</h2><br />
<p><br />
The procedure described below is useful in order to quantify the concentration of 3OC<small><sub>6</sub></small>-HSL in cultures expressing LuxI and AiiA enzyme.<br />
<br><br />
Once collected (according to the <a href = "#T9002">protocol above</a>) and pre-processed data, it is necessary to compute the S<sub><small>cell</small></sub> of every culture.<br />
<br><br />
BBa_T9002 induced with known 3OC<small><sub>6</sub></small>-HSL concentration is useful to estimate the parameters of the activation Hill function of pLux promoter:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_activation_pLux.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f2/UNIPV_activation_pLux.png"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
Provided that the S<sub><small>cell</small></sub> measured for BBa_T9002 induced with supernatants of cultures producing or degrading 3OC<small><sub>6</sub></small>-HSL has a value included in the linear zone of the biosensor, you can easily extrapolate the auto-inducer concentration as follows:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_HSL.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/e/e2/UNIPV_HSL.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
Finally, don't forget to multiply this value for the dilution factor (in our experiments it was 20).<br />
</p><br />
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{{end}}</div>Nickpvhttp://2011.igem.org/File:UNIPV_HSL.PNGFile:UNIPV HSL.PNG2011-09-17T13:30:38Z<p>Nickpv: </p>
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<div></div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/MeasurementsTeam:UNIPV-Pavia/Measurements2011-09-17T12:05:05Z<p>Nickpv: </p>
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div><br />
<ul><br />
<li class="toclevel-1"><a href="#Promoters"><span class="tocnumber">1</span> <span class="toctext">Measuring promoters transcriptional strength</span></a></li><br />
<br />
<ul><br />
<li class="toclevel-1"><a href="#pTet_protocol"><span class="tocnumber">1.1</span> <span class="toctext">pTet transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#pLux_protocol"><span class="tocnumber">1.2</span> <span class="toctext">pLux transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#j101_protocol"><span class="tocnumber">1.3</span> <span class="toctext">Constitutive BBa_J23101x promoters transcriptional strength</span></a><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#Enzyme"><span class="tocnumber">2</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</span></a><br />
<br />
<ul><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.1</span> <span class="toctext">LuxI enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.2</span> <span class="toctext">AiiA enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#Deg"><span class="tocnumber">2.3</span> <span class="toctext">3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH</span></a></li><br />
<li class="toclevel-2"><a href="#T9002"><span class="tocnumber">2.4</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL concentration with BBa_T9002</span></a></li><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#data_analysis"><span class="tocnumber">3</span> <span class="toctext">Data analysis</span></a><br />
<ul><br />
<li class="toclevel-2"><a href="#preprocessing"><span class="tocnumber">3.1</span> <span class="toctext">Data pre-processing</span></a></li><br />
<li class="toclevel-2"><a href="#doubtime"><span class="tocnumber">3.2</span> <span class="toctext">Doubling time evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#scell"><span class="tocnumber">3.3</span> <span class="toctext">Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#RPU"><span class="tocnumber">3.4</span> <span class="toctext">R.P.U. evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#HSL"><span class="tocnumber">3.5</span> <span class="toctext">Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration</span></a></li><br />
</ul><br />
<br />
</td></tr></table><br />
</div><br />
<br />
<a name="Promoters"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring promoters transcriptional strength</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pTet_protocol"></a> <h2 class="art-postheader"><br />
Measuring pTet transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for three hours.<br />
<li> Induce cultures in falcon tube with anhydrotetracycline (aTc); final concentrations:<br />
<ul><br />
<li> 0 ng/ml<br />
<li> 1 ng/ml<br />
<li> 2 ng/ml<br />
<li> 3 ng/ml<br />
<li> 4 ng/ml<br />
<li> 5 ng/ml<br />
<li> 8 ng/ml<br />
<li> 10 ng/ml<br />
<li> 50 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow at 37°C, 220 rpm for three hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pLux_protocol"></a> <h2 class="art-postheader"><br />
Measuring pLux transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours at 37°C, 220 rpm.<br />
<li> Induce cultures in falcon tube with 3OC<sub><small>6</small></sub>-HSL; final concentrations: <br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
</ul><br />
<li> Let the cultures grow for three hours at 37°C, 220 rpm.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="j101_protocol"></a> <h2 class="art-postheader"><br />
Constitutive BBa_J23101x promoters transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for six hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Enzyme"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="LuxI"></a> <h2 class="art-postheader"><br />
LuxI enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and grow them for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of induction, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="AiiA"></a> <h2 class="art-postheader"><br />
AiiA enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow for one more hour at 37°C, 220 rpm.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Deg"></a> <h2 class="art-postheader"><br />
3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks not expressing lactonases in 1 ml of M9 with the proper antibiotic. Use M9 at different pHs, for example pH = 6.0 and pH = 7.0. Let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 with the proper antibiotic in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Prepare falcon tubes with M9 at pH = 6.0 and pH = 7.0.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL to each falcon tube.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="T9002"></a> <h2 class="art-postheader"><br />
Measuring 3OC<sub><small>6</small></sub>-HSL concentration withBBa_T9002<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> in 1 ml M9 with the proper antibiotic (Ampicillin when you use <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> or Ampicillin + Chloramphenicol 12.5 mg/ml if you use T9002-ENTERO, see <a href="https://2011.igem.org/Team:UNIPV-Pavia/Freezer">Freezer Management</a>) together with a non-fluorescent culture; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in M9 with the proper antibiotic; let the cultures grow for two hours at 37°C, 220 rpm.<br />
<li> Induce <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with the previously collected supernatants, diluting them 1:20: aliquot 190&mu;l of inducible cultures and 10 &mu;l of supernatants in the wells of the microplate.<br />
<li> Don't forget to build a calibration curve, by inducing <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with known 3OC<sub><small>6</small></sub>-HSL concentrations:<br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.2 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
<li> 1 &mu;M<br />
</ul><br />
<li>Use Tecan Infinite F200 to read O.D. at 600 nm and green fluorescence, setting the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50<br />
<li> O.D. filter: 600 nm<br />
<li> GFP filters: 485 nm (excitation) / 540 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="data_analysis"></a> <h2 class="art-postheader"><br />
<font size = "5">Data analysis</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="preprocessing"></a> <h2 class="art-postheader"><br />
Data pre-processing<br />
</h2><br />
<p><br />
Data from TECAN Infinite F200 need to be pre-processed in order to remove spurious effects affecting measurements. Before evaluating the parameters of interest (for example the synthesis rate per cell, S<sub><small>cell</small></sub>, or Relative Promoter Unit, R.P.U.) blanking is needed:<br />
<ol><ul><br />
<li> each time sample of O.D. at 600 nm, (O.D.<sub><small>600</small></sub>) was subtracted the reference value (i.e. the measure of O.D. at 600 nm of the broth in which cultures grow)<br />
<li> each time sample of red and green fluorescence was subtracted the reference value (i.e. the red or green fluorescence of a non-fluorescent culture)<br />
</ul></ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="doubtime"></a> <h2 class="art-postheader"><br />
Doubling time evaluation<br />
</h2><br />
<p><br />
After blanking O.D.<sub><small>600</small></sub> data you can compute the doubling time of cultures growth, i.e. the time needed to double O.D.<sub><small>600</small></sub>.<br />
<br><br />
Identifying the exponential phase of growth curve can be done by visual inspection, plotting the natural logarithm of O.D.<sub><small>600</small></sub> over time. Linear regression on logarithmic data is performed to estimate the growth rate, named &mu;. Finally doubling time can be evaluated as:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_DoubTime.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/7d/UNIPV_DoubTime.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<br />
<p><br />
When measuring multiple growth for a strain this value was evaluated for each reaplicate and then average was computed.<br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<br />
<a name="scell"></a><h2 class="art-postheader"><br />
Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation<br />
</h2><br />
<p><br />
Another interesting parameter, which describes the activity of a promoter of interest (&phi;), is the S<sub><small>cell</small></sub>; it needs to be measured using reporter genes as RFP or GFP.<br />
<br><br />
Compute the average of the time derivative of the red or green fluorescence, divided by the O.D.<sub><small>600</small></sub>, in the time interval corresponding to the exponential growth phase which boundaries can be identified by visual inspection of the logarithmic growth curve:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_Scell.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c5/UNIPV_Scell.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
The S<sub><small>cell</small></sub> is basic to measure the Relative Promoter Unit, a robust parameter expressing the transcriptional activity of a promoter.<br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="RPU"></a> <h2 class="art-postheader"><br />
R.P.U. evaluation<br />
</h2><br />
<p><br />
As described in Kelly J. et al., 2009, Relative Promoter Units express the activity of a promoter of interest (&phi;), reported to the one of a reference promoter, <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a> (from Anderson promoters collection):<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_RPU.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/70/UNIPV_RPU.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
This procedure has the advantage of being robust to variations in experimental conditions and proportional to PoPS (Polymerase Per Second) if the subsequent specifications are satisfied:<br />
<ol><ul><br />
<li> strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in BBa_J23101 reference standard<br />
<li> the reporter protein must have a half life higher than the experiment duration<br />
</ul></ol><br />
Moreover the S<sub><small>cell</small></sub> signal of both the promoter of interest and of BBa_J23101 has tp be at the steady state.<br />
</p><br />
<br />
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<a name="HSL"></a> <h2 class="art-postheader"><br />
Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration<br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/MeasurementsTeam:UNIPV-Pavia/Measurements2011-09-17T11:55:32Z<p>Nickpv: </p>
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div><br />
<ul><br />
<li class="toclevel-1"><a href="#Promoters"><span class="tocnumber">1</span> <span class="toctext">Measuring promoters transcriptional strength</span></a></li><br />
<br />
<ul><br />
<li class="toclevel-1"><a href="#pTet_protocol"><span class="tocnumber">1.1</span> <span class="toctext">pTet transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#pLux_protocol"><span class="tocnumber">1.2</span> <span class="toctext">pLux transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#j101_protocol"><span class="tocnumber">1.3</span> <span class="toctext">Constitutive BBa_J23101x promoters transcriptional strength</span></a><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#Enzyme"><span class="tocnumber">2</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</span></a><br />
<br />
<ul><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.1</span> <span class="toctext">LuxI enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.2</span> <span class="toctext">AiiA enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#Deg"><span class="tocnumber">2.3</span> <span class="toctext">3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH</span></a></li><br />
<li class="toclevel-2"><a href="#T9002"><span class="tocnumber">2.4</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL concentration with BBa_T9002</span></a></li><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#data_analysis"><span class="tocnumber">3</span> <span class="toctext">Data analysis</span></a><br />
<ul><br />
<li class="toclevel-2"><a href="#preprocessing"><span class="tocnumber">3.1</span> <span class="toctext">Data pre-processing</span></a></li><br />
<li class="toclevel-2"><a href="#doubtime"><span class="tocnumber">3.2</span> <span class="toctext">Doubling time evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#scell"><span class="tocnumber">3.3</span> <span class="toctext">Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#RPU"><span class="tocnumber">3.4</span> <span class="toctext">R.P.U. evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#HSL"><span class="tocnumber">3.5</span> <span class="toctext">Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration</span></a></li><br />
</ul><br />
<br />
</td></tr></table><br />
</div><br />
<br />
<a name="Promoters"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring promoters transcriptional strength</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pTet_protocol"></a> <h2 class="art-postheader"><br />
Measuring pTet transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for three hours.<br />
<li> Induce cultures in falcon tube with anhydrotetracycline (aTc); final concentrations:<br />
<ul><br />
<li> 0 ng/ml<br />
<li> 1 ng/ml<br />
<li> 2 ng/ml<br />
<li> 3 ng/ml<br />
<li> 4 ng/ml<br />
<li> 5 ng/ml<br />
<li> 8 ng/ml<br />
<li> 10 ng/ml<br />
<li> 50 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow at 37°C, 220 rpm for three hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pLux_protocol"></a> <h2 class="art-postheader"><br />
Measuring pLux transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours at 37°C, 220 rpm.<br />
<li> Induce cultures in falcon tube with 3OC<sub><small>6</small></sub>-HSL; final concentrations: <br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
</ul><br />
<li> Let the cultures grow for three hours at 37°C, 220 rpm.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="j101_protocol"></a> <h2 class="art-postheader"><br />
Constitutive BBa_J23101x promoters transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for six hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Enzyme"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="LuxI"></a> <h2 class="art-postheader"><br />
LuxI enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and grow them for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of induction, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
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<br />
<a name="AiiA"></a> <h2 class="art-postheader"><br />
AiiA enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow for one more hour at 37°C, 220 rpm.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Deg"></a> <h2 class="art-postheader"><br />
3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks not expressing lactonases in 1 ml of M9 with the proper antibiotic. Use M9 at different pHs, for example pH = 6.0 and pH = 7.0. Let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 with the proper antibiotic in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Prepare falcon tubes with M9 at pH = 6.0 and pH = 7.0.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL to each falcon tube.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
<br />
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<br />
<a name="T9002"></a> <h2 class="art-postheader"><br />
Measuring 3OC<sub><small>6</small></sub>-HSL concentration withBBa_T9002<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> in 1 ml M9 with the proper antibiotic (Ampicillin when you use <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> or Ampicillin + Chloramphenicol 12.5 mg/ml if you use T9002-ENTERO, see <a href="https://2011.igem.org/Team:UNIPV-Pavia/Freezer">Freezer Management</a>) together with a non-fluorescent culture; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in M9 with the proper antibiotic; let the cultures grow for two hours at 37°C, 220 rpm.<br />
<li> Induce <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with the previously collected supernatants, diluting them 1:20: aliquot 190&mu;l of inducible cultures and 10 &mu;l of supernatants in the wells of the microplate.<br />
<li> Don't forget to build a calibration curve, by inducing <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with known 3OC<sub><small>6</small></sub>-HSL concentrations:<br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.2 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
<li> 1 &mu;M<br />
</ul><br />
<li>Use Tecan Infinite F200 to read O.D. at 600 nm and green fluorescence, setting the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50<br />
<li> O.D. filter: 600 nm<br />
<li> GFP filters: 485 nm (excitation) / 540 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
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<br />
<a name="data_analysis"></a> <h2 class="art-postheader"><br />
<font size = "5">Data analysis</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="preprocessing"></a> <h2 class="art-postheader"><br />
Data pre-processing<br />
</h2><br />
<p><br />
Data from TECAN Infinite F200 need to be pre-processed in order to remove spurious effects affecting measurements. Before evaluating the parameters of interest (for example the synthesis rate per cell, S<sub><small>cell</small></sub>, or Relative Promoter Unit, R.P.U.) blanking is needed:<br />
<ol><ul><br />
<li> each time sample of O.D. at 600 nm, (O.D.<sub><small>600</small></sub>) was subtracted the reference value (i.e. the measure of O.D. at 600 nm of the broth in which cultures grow)<br />
<li> each time sample of red and green fluorescence was subtracted the reference value (i.e. the red or green fluorescence of a non-fluorescent culture)<br />
</ul></ol><br />
</p><br />
<br />
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<br />
<a name="doubtime"></a> <h2 class="art-postheader"><br />
Doubling time evaluation<br />
</h2><br />
<p><br />
After blanking O.D.<sub><small>600</small></sub> data you can compute the doubling time of cultures growth, i.e. the time needed to double O.D.<sub><small>600</small></sub>.<br />
<br><br />
Identifying the exponential phase of growth curve can be done by visual inspection, plotting the natural logarithm of O.D.<sub><small>600</small></sub> over time. Linear regression on logarithmic data is performed to estimate the growth rate, named &mu;. Finally doubling time can be evaluated as:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_DoubTime.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/7d/UNIPV_DoubTime.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<br />
<p><br />
When measuring multiple growth for a strain this value was evaluated for each reaplicate and then average was computed.<br />
</p><br />
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<br />
<br />
<a name="scell"></a><h2 class="art-postheader"><br />
Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation<br />
</h2><br />
<p><br />
Another interesting parameter, which describes the activity of a promoter of interest (&phi;), is the S<sub><small>cell</small></sub>; it needs to be measured using reporter genes as RFP or GFP.<br />
<br><br />
Compute the average of the time derivative of the red or green fluorescence, divided by the O.D.<sub><small>600</small></sub>, in the time interval corresponding to the exponential growth phase which boundaries can be identified by visual inspection of the logarithmic growth curve:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_Scell.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c5/UNIPV_Scell.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
The S<sub><small>cell</small></sub> is basic to measure the Relative Promoter Unit, a robust parameter expressing the transcriptional activity of a promoter.<br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="RPU"></a> <h2 class="art-postheader"><br />
R.P.U. evaluation<br />
</h2><br />
<p><br />
As described in Kelly J. et al., 2009, Relative Promoter Units express the activity of a promoter of interest (&phi;), reported to the one of a reference promoter, <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a> (from Anderson promoters collection):<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_RPU.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/70/UNIPV_RPU.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
This procedure has the advantage of being robust to variations in experimental conditions and proportional to PoPS (Polymerase Per Second) if the subsequent specifications are satisfied:<br />
<ol><ul><br />
<li> strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in BBa_J23101 reference standard<br />
<li> the reporter protein must have a half life higher than the experiment duration<br />
</ul></ol><br />
Moreover the S<sub><small>cell</small></sub> of both the promoter of interest and of BBa_J23101 has to be constant.<br />
</p><br />
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<a name="HSL"></a> <h2 class="art-postheader"><br />
Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration<br />
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{{end}}</div>Nickpvhttp://2011.igem.org/Team:UNIPV-Pavia/MeasurementsTeam:UNIPV-Pavia/Measurements2011-09-17T11:53:44Z<p>Nickpv: </p>
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<table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div><br />
<ul><br />
<li class="toclevel-1"><a href="#Promoters"><span class="tocnumber">1</span> <span class="toctext">Measuring promoters transcriptional strength</span></a></li><br />
<br />
<ul><br />
<li class="toclevel-1"><a href="#pTet_protocol"><span class="tocnumber">1.1</span> <span class="toctext">pTet transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#pLux_protocol"><span class="tocnumber">1.2</span> <span class="toctext">pLux transcriptional strength</span></a><br />
<li class="toclevel-1"><a href="#j101_protocol"><span class="tocnumber">1.3</span> <span class="toctext">Constitutive BBa_J23101x promoters transcriptional strength</span></a><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#Enzyme"><span class="tocnumber">2</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</span></a><br />
<br />
<ul><br />
<li class="toclevel-2"><a href="#LuxI"><span class="tocnumber">2.1</span> <span class="toctext">LuxI enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#AiiA"><span class="tocnumber">2.2</span> <span class="toctext">AiiA enzyme activity</span></a></li><br />
<li class="toclevel-2"><a href="#Deg"><span class="tocnumber">2.3</span> <span class="toctext">3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH</span></a></li><br />
<li class="toclevel-2"><a href="#T9002"><span class="tocnumber">2.4</span> <span class="toctext">Measuring 3OC<sub><small>6</small></sub>-HSL concentration with BBa_T9002</span></a></li><br />
</ul><br />
<br />
<li class="toclevel-1"><a href="#data_analysis"><span class="tocnumber">3</span> <span class="toctext">Data analysis</span></a><br />
<ul><br />
<li class="toclevel-2"><a href="#preprocessing"><span class="tocnumber">3.1</span> <span class="toctext">Data pre-processing</span></a></li><br />
<li class="toclevel-2"><a href="#doubtime"><span class="tocnumber">3.2</span> <span class="toctext">Doubling time evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#scell"><span class="tocnumber">3.3</span> <span class="toctext">Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#RPU"><span class="tocnumber">3.4</span> <span class="toctext">R.P.U. evaluation</span></a></li><br />
<li class="toclevel-2"><a href="#HSL"><span class="tocnumber">3.5</span> <span class="toctext">Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration</span></a></li><br />
</ul><br />
<br />
</td></tr></table><br />
</div><br />
<br />
<a name="Promoters"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring promoters transcriptional strength</font><br />
</h2><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pTet_protocol"></a> <h2 class="art-postheader"><br />
Measuring pTet transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for three hours.<br />
<li> Induce cultures in falcon tube with anhydrotetracycline (aTc); final concentrations:<br />
<ul><br />
<li> 0 ng/ml<br />
<li> 1 ng/ml<br />
<li> 2 ng/ml<br />
<li> 3 ng/ml<br />
<li> 4 ng/ml<br />
<li> 5 ng/ml<br />
<li> 8 ng/ml<br />
<li> 10 ng/ml<br />
<li> 50 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow at 37°C, 220 rpm for three hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="pLux_protocol"></a> <h2 class="art-postheader"><br />
Measuring pLux transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours at 37°C, 220 rpm.<br />
<li> Induce cultures in falcon tube with 3OC<sub><small>6</small></sub>-HSL; final concentrations: <br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
</ul><br />
<li> Let the cultures grow for three hours at 37°C, 220 rpm.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="j101_protocol"></a> <h2 class="art-postheader"><br />
Constitutive BBa_J23101x promoters transcriptional strength<br />
</h2><br />
<p><br />
<ol><br />
<li> Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.<br />
<li> Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.<br />
<li> Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for six hours.<br />
<li> Aliquot 200 &mu;l of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50 - 80<br />
<li> O.D. filter: 600 nm<br />
<li> RFP filters: 535 nm (excitation) / 620 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
<br />
<div align="right"><small><a href="#top_page" title="">^top</a></small></div><br />
<br />
<a name="Enzyme"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring 3OC<sub><small>6</small></sub>-HSL synthesis and degradation</font><br />
</h2><br />
<br />
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<a name="LuxI"></a> <h2 class="art-postheader"><br />
LuxI enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and grow them for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of induction, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
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<a name="AiiA"></a> <h2 class="art-postheader"><br />
AiiA enzyme activity<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Induce cultures with aTc; final concentrations:<br />
<ul><br />
<li> 6 ng/ml<br />
<li> 8 ng/ml<br />
<li> 100 ng/ml<br />
</ul><br />
<li> Let the cultures grow for one more hour at 37°C, 220 rpm.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according to the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
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<a name="Deg"></a> <h2 class="art-postheader"><br />
3OC<sub><small>6</small></sub>-HSL degradation in M9 medium and cultures not expressing lactonases varying pH<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l of long term glycerol stocks not expressing lactonases in 1 ml of M9 with the proper antibiotic. Use M9 at different pHs, for example pH = 6.0 and pH = 7.0. Let the cultures grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in 4 ml M9 with the proper antibiotic in falcon tubes and let them grow for two hours at 37°C, 220 rpm.<br />
<li> Prepare falcon tubes with M9 at pH = 6.0 and pH = 7.0.<br />
<li> Add 100 nM 3OC<sub><small>6</small></sub>-HSL to each falcon tube.<br />
<li> Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC<sub><small>6</small></sub>-HSL addition, after 1 hour, 2 hours and 4 hours by:<br />
<ul><br />
<li> take 250 &mu;l of cultures<br />
<li> centrifuge them 13.300 rpm, 4 minutes<br />
<li> collect 200&mu;l of supernatants (without resupsending the pelleted bacteria)<br />
<li> let the cultures grow at 37°C, 220 rpm until the next sampling<br />
</ul><br />
<li> Store supernatants at -20°C and measure 3OC<sub><small>6</small></sub>-HSL concentration according the <a href="#T9002">protocol</a> based on <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> biosensor.<br />
</ol><br />
</p><br />
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<a name="T9002"></a> <h2 class="art-postheader"><br />
Measuring 3OC<sub><small>6</small></sub>-HSL concentration withBBa_T9002<br />
</h2><br />
<p><br />
<ol><br />
<li> Inoculate 5 &mu;l <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> in 1 ml M9 with the proper antibiotic (Ampicillin when you use <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> or Ampicillin + Chloramphenicol 12.5 mg/ml if you use T9002-ENTERO, see <a href="https://2011.igem.org/Team:UNIPV-Pavia/Freezer">Freezer Management</a>) together with a non-fluorescent culture; let them grow over night at 37°C, 220 rpm.<br />
<li> Dilute cultures 1:100 in M9 with the proper antibiotic; let the cultures grow for two hours at 37°C, 220 rpm.<br />
<li> Measure 3OC<sub><small>6</small></sub>-HSL concentration of the previously collected supernatants (diluting them 1:20), inducing <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures: aliquot 190&mu;l of inducible cultures and 10 &mu;l of supernatants in the wells of the microplate.<br />
<li> Don't forget to build a calibration curve, by inducing <a href="http://partsregistry.org/Part:BBa_T9002">BBa_T9002</a> cultures with known 3OC<sub><small>6</small></sub>-HSL concentrations:<br />
<ul><br />
<li> 0 M<br />
<li> 0.1 nM<br />
<li> 0.2 nM<br />
<li> 0.5 nM<br />
<li> 1 nM<br />
<li> 2 nM<br />
<li> 5 nM<br />
<li> 10 nM<br />
<li> 100 nM<br />
<li> 1 &mu;M<br />
</ul><br />
<li>Use Tecan Infinite F200 to read O.D. at 600 nm and green fluorescence, setting the automatic procedure:<br />
<ul><br />
<li> temperature: 37°C<br />
<li> sampling time: 5 minutes<br />
<li> 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements<br />
<li> fluorescence gain: 50<br />
<li> O.D. filter: 600 nm<br />
<li> GFP filters: 485 nm (excitation) / 540 nm (emission)<br />
<li> duration time: 10 - 15 hours<br />
</ul><br />
</ol><br />
</p><br />
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<a name="data_analysis"></a> <h2 class="art-postheader"><br />
<font size = "5">Data analysis</font><br />
</h2><br />
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<a name="preprocessing"></a> <h2 class="art-postheader"><br />
Data pre-processing<br />
</h2><br />
<p><br />
Data from TECAN Infinite F200 need to be pre-processed in order to remove spurious effects affecting measurements. Before evaluating the parameters of interest (for example the synthesis rate per cell, S<sub><small>cell</small></sub>, or Relative Promoter Unit, R.P.U.) blanking is needed:<br />
<ol><ul><br />
<li> each time sample of O.D. at 600 nm, (O.D.<sub><small>600</small></sub>) was subtracted the reference value (i.e. the measure of O.D. at 600 nm of the broth in which cultures grow)<br />
<li> each time sample of red and green fluorescence was subtracted the reference value (i.e. the red or green fluorescence of a non-fluorescent culture)<br />
</ul></ol><br />
</p><br />
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<a name="doubtime"></a> <h2 class="art-postheader"><br />
Doubling time evaluation<br />
</h2><br />
<p><br />
After blanking O.D.<sub><small>600</small></sub> data you can compute the doubling time of cultures growth, i.e. the time needed to double O.D.<sub><small>600</small></sub>.<br />
<br><br />
Identifying the exponential phase of growth curve can be done by visual inspection, plotting the natural logarithm of O.D.<sub><small>600</small></sub> over time. Linear regression on logarithmic data is performed to estimate the growth rate, named &mu;. Finally doubling time can be evaluated as:<br />
</p><br />
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<div class="center"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_DoubTime.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/7d/UNIPV_DoubTime.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
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<p><br />
When measuring multiple growth for a strain this value was evaluated for each reaplicate and then average was computed.<br />
</p><br />
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<a name="scell"></a><h2 class="art-postheader"><br />
Synthesis rate per cell (S<sub><small>cell</small></sub>) evaluation<br />
</h2><br />
<p><br />
Another interesting parameter, which describes the activity of a promoter of interest (&phi;), is the S<sub><small>cell</small></sub>; it needs to be measured using reporter genes as RFP or GFP.<br />
<br><br />
Compute the average of the time derivative of the red or green fluorescence, divided by the O.D.<sub><small>600</small></sub>, in the time interval corresponding to the exponential growth phase which boundaries can be identified by visual inspection of the logarithmic growth curve:<br />
</p><br />
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<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_Scell.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/c/c5/UNIPV_Scell.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
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<p><br />
The S<sub><small>cell</small></sub> is basic to measure the Relative Promoter Unit, a robust parameter expressing the transcriptional activity of a promoter.<br />
</p><br />
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<a name="RPU"></a> <h2 class="art-postheader"><br />
R.P.U. evaluation<br />
</h2><br />
<p><br />
As described in Kelly J. et al., 2009, Relative Promoter Units express the activity of a promoter of interest (&phi;), reported to the one of a reference promoter, <a href="http://partsregistry.org/Part:BBa_J23101">BBa_J23101</a> (from Anderson promoters collection):<br />
</p><br />
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<div class="center"><div class="thumbinner" style="width: 300px;"><a href="/File:UNIPV_RPU.PNG" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/7/70/UNIPV_RPU.PNG"class="thumbimage" height="80%" width="80%"></a></div></div><br />
<br />
<p><br />
This procedure has the advantage of being robust to variations in experimental conditions and proportional to PoPS (Polymerase Per Second) if the subsequent specifications are satisfied:<br />
<ol><ul><br />
<li> strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in BBa_J23101 reference standard<br />
<li> the reporter protein must have a half life higher than the experiment duration<br />
</ul></ol><br />
Moreover the S<sub><small>cell</small></sub> of both the promoter of interest and of BBa_J23101 has to be constant.<br />
</p><br />
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<a name="HSL"></a> <h2 class="art-postheader"><br />
Extrapolating 3OC<sub><small>6</small></sub>-HSL concentration<br />
</h2><br />
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