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| - | <h2 class="art-postheader">
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| - | Results
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| - | <!-----------MENU----------------->
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| - | <table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div>
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| - | <ul>
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| - | <li class="toclevel-1"><a href="#assembly"><span class="tocnumber">1</span> <span class="toctext">Part assembly</span></a>
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| - | <li class="toclevel-1"><a href="#characterization"><span class="tocnumber">1</span> <span class="toctext">Characterization of basic modules</span></a>
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| - | <ul>
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| - | <li class="toclevel-2"><a href="#promoters"><span class="tocnumber">2.1</span> <span class="toctext">Promoter characterization</span></a></li>
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| - | <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>
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| - | <li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.3</span> <span class="toctext">Characterization of RBS efficiency</span></a></li>
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| - | </ul>
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| - | <li class="toclevel-1"><a href="#growth"><span class="tocnumber">3</span> <span class="toctext">Identification of bacterial growth parameters</span></a></li>
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| - | <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>
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| - | <li class="toclevel-1"><a href="#t9002"><span class="tocnumber">5</span> <span class="toctext">Characterization of BBa_T9002 biosensor</span></a></li>
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| - | </ul></td></tr></table>
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| - | <em>
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| - | 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.
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| - | <a name='assembly'></a><h1>Parts assembly</h1>
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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>.
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| - | <div align="right"><small><a href="#indice" title="">^top</a></small></div>
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| - | <a name='characterization'></a><h1>Characterization of basic modules</h1>
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| - | <!--------pTet and pLux----------->
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| - | <a name='promoters'></a><h2>Characterization of promoters pTet and pLux</h2>
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| - | <p align='justify'>
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| - | <p>Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection.</p>
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| - | <p>The assembled RBSs are:</p>
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| - | <div align='center'>
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| - | <table class='data'>
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| - | <tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr>
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| - | <tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr>
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| - | <tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr>
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| - | <tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr>
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| - | <tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr>
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| - | </table></div>
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| - | <br>
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| - | <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>
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| - | <p>The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein.
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| - | 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>
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| - | <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>
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| - | </p>
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| - | <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>
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| - | <p><ol><ul><li><b>
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| - | RPU<sub>max</sub></b> is equal to the α and represents the maximum promoter activity</p>
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| - | </li><p><li><b>
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| - | RPU<sub>min</sub></b> is equal to the α * δ represents the minimum promoter activity</p>
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| - | </li><p><li>
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| - | <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>
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| - | </li>
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| - | <p><li>
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| - | <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>
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| - | </ul></ol>
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| - | </p>
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| - | <p align='justify'>
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| - | 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>.
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| - | </p>
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| - | <table class='data' width='100%'>
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| - | <tr>
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| - | <td class="row"><b>RBS</b></td>
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| - | <td class="row"><b>α<sub>p<sub>Lux</sub></sub> [(AUr/min)/cell]</b></td>
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| - | <td class="row"><b>δ<sub>p<sub>Lux</sub></sub> [-]</b></td>
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| - | <td class="row"><b>η<sub>p<sub>Lux</sub></sub> [-]</b></td>
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| - | <td class="row"><b>k<sub>p<sub>Lux</sub></sub> [ng/ml]</b></td>
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| - | </tr>
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| - | <tr><td class="row">BBa_B0030</td>
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| - | <td class="row">438 [10]</td>
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| - | <td class="row">0.05 [>100]</td>
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| - | <td class="row">2 [47]</td>
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| - | <td class="row">1.88 [27]</td>
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| - | </tr>
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| - | <tr><td class="row">BBa_B0031</td>
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| - | <td class="row">9.8 [7]</td>
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| - | <td class="row">0.11 [57]</td>
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| - | <td class="row">1.2 [29]</td>
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| - | <td class="row">1.5 [26]</td>
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| - | </tr>
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| - | <tr><td class="row">BBa_B0032</td>
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| - | <td class="row">206 [3]</td>
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| - | <td class="row">0 [>>100]</td>
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| - | <td class="row">1.36 [10]</td>
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| - | <td class="row">1.87 [9]</td>
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| - | </tr>
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| - | <tr><td class="row">BBa_B0034</td>
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| - | <td class="row">1105 [6]</td>
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| - | <td class="row">0.02 [>100]</td>
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| - | <td class="row">1.33 [19]</td>
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| - | <td class="row">2.34 [18]</td>
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| - | </tr>
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| - | </table>
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| - | <div align="center">Data are provided as average [CV%].</div><br>
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| - | <p>The operative parameters are summarized in the table below:</p></div>
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| - | <table align='center' class='data' width='100%'>
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| - | <td class='row'><b>RBS</b></td>
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| - | <td class='row'><b>RPU<sub>max</sub></b></td>
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| - | <td class='row'><b>RPU<sub>min</sub></b></td>
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| - | <td class='row'><b>Switch point [nM]</b></td>
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| - | <td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td>
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| - | </tr>
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| - | <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>
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| - | </tr>
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| - | <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>
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| - | </tr>
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| - | <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>
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| - | </tr>
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| - | <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>
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| - | </tr>
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| - | </table>
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| - | <table width='100%'><tr><td width='50%'>
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| - | <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>
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| - | </td>
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| - | <td width='50%'>
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| - | <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>
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| - | </td></tr>
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| - | <tr><td width='50%'>
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| - | <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>
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| - | </td>
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| - | <td width='50%'>
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| - | <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>
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| - | </td></tr>
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| - | </table>
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| - | <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>
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| - | <p>
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| - | 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>
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| - | <center><a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image">
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| - | <img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a>
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| - | </center>
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| - | {{end}}
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