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2011.igem.org - User contributions [en]
2024-03-29T07:10:48Z
From 2011.igem.org
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http://2011.igem.org/Team:UNIPV-Pavia/Team
Team:UNIPV-Pavia/Team
2011-11-25T13:16:30Z
<p>Edoardo Baldini: </p>
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nam[5] = "Daniele Sartori";<br />
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des[1] = "My name is Niccolò Franceschi, I was born in Parma but now I live in Pavia to attend University. I am 23 and I am attending the last year in the Master's Degree in Industrial Biotechnologies at the University of Pavia, I also have a Bachelor Degree in Biotechnologies from the University of Parma.<br>Besides the iGEM competition, I am currently working on my thesis on X-ray biodiffractometry.<br><br><b>Contact:</b> niccolo.francheschi01@universitadipavia.it ";<br />
des[2] = "Hi! I'm Davide Bianchini and I'm 24 years old.<br>I earned my Master degree in Biomedical Engineering at the end of March at the Faculty of Engineering of Bologna University, placed in Cesena. I went to Pavia University in order to take part to iGEM 2011 competition, since I believe it is a great opportunity to have insight in Synthetic Biology.<br><br><b>Contact:</b> davide.bianchini@studio.unibo.it";<br />
des[3] = "Bachelor Degree in Biomedical Engineering.<br>Master Degree in Biomedical Engineering.<br>PhD student in Bioengineering and Bioinformatics at the University of Pavia.<br><br>In 2010 I took part in iGEM competition as a student with UNIPV-Pavia Team. The idea to rationally design and implement functions in living systems attracts me a lot, that's why I like synthetic biology.<br>In my free time I like playing football, playing the piano and travelling.<br><br><b>Contact:</b> nicolo.politi@unipv.it ";<br />
des[4] = "I am a Professor of Bioengineering. My teaching and research interests ranging from mathematical model of biological systems to bioinformatics and synthetic biology, from statistics and probability models to medical informatics.<br>I am 42 years old. I am the promoter of the iGEM competition in Pavia. I am an enthusiastic fan of this competition! I think that it represents a big opportunity for a student. I have selected our best students from Biology, Biotechnology and Bioengineering programs and I tried to build a team. Even if I have spent a lot of time in this initiative, I am very happy to partecipate to iGEM 2011 and to have worked with all of them. For 20 years, I was a national referee of Handball. I like skiing and teaching to those students that want to learn.<br><br><b>Contact:</b> paolo.magni@unipv.it ";<br />
des[5] = "Soon to be graduated in Pharmacology at the University of Pavia.<br><br>My decision to be part of the UNIPV-iGEM team arose when I found out of the possible Synthetic Biology applications in pharmaceutical technology or medications pertaining fields. My academic interests are pharmacogenetics, pharmacogenomics, gene therapy, pharmaceutical legislation. I'm very fond of politics, art, reading and music.<br><br><b>Contact:</b> daniele.sartori01@universitadipavia.it ";<br />
des[6] = "Bachelor Degree in Electronic Engineering at the University of Pavia.<br>Currently Master student in Photonics and Optoelectronics, last year.<br><br>My academic interests include Quantum Electronics, Nonlinear Optics, Condensed Matter Physics and Biophotonics. I decided to take part to iGEM competition to explore the points of contact between Synthetic Biology and Photonics/Electronics.<br>Besides university, I spend time playing the piano and leading a musical webmagazine.<br><br><b>Contact:</b> edoardo.baldini01@universitadipavia.it ";<br />
des[7] = "Bachelor Degree in Biomedical Engineering and Master student in Biomedical Engineering at Università degli Studi di Pavia. Member of the Borrhomaic Family and student at IUSS, Sciences & Technologies Class.<br><br> Hi everyone! I come from Bergamo, a city not far from Milan. I' ve been interested in iGEM experience since the first year of university, but this was the right moment to join the competition. I think it's a great opportunity to work with other students, even from different disciplines, and learn a lot of new things that otherwise would not have taken into account.<br><br><b>Contact:</b> tommaso.goggia01@universitadipavia.it ";<br />
des[8] = "Hi! I'm Viola, I'm 23 years old and I come from Pavia.<br>I'm attending the second year of the Master's Degree in Industrial Biotechnologies.<br>I love cooking desserts, reading and travelling! My favorite hobbies are belly dance, playing fighting games and spending some time with my friends. I think iGEM experience is amazing!<br><br><b>Contact:</b> viola.ghio01@universitadipavia.it ";<br />
des[9] = "Bachelor Honor Degree in Biology.<br>Currently Master student in Molecular Biology and Genetics, last year.<br><br>Besides iGEM 2011 activities, I'm working in a DNA Enzymology and Molecular Virology's laboratory.<br>You can't say of me that I'm an athlete but I surely am an art lover; particularly the visual arts (photography, cinema and theater).<br>I love biology and so I hope to meet a lot of good scientist in Amsterdam and Boston! See you at the iGEM competition!<br><br><b>Contact:</b> emanuela.pasi01@universitadipavia.it";<br />
des[10] = "PhD student in Bioengineering and Bioinformatics, third year.<br> <br>Bachelor Degree in Biomedical Engineering and Master Student in Biomedical Engineering at Università degli Studi di Pavia.<br><br>Nice to meet you again, this is my third year at iGEM and I'm very happy to be part of this team! This year our project deals with synthetic biology and mathematical modelling, that are two research area I'm very interested in, and it contributes to make this experience amazing. See you at Jamboree! <br><br><b>Contact:</b> susanna.zucca@unipv.it";<br />
des[11] = "Molecular Biology student.<br><br>This is my second experience in the iGEM competition. Last year, I had the opportunity to appreciate this wonderful world of synthetic biology, so I decided to participate once again.<br>What I like best in synthetic biology is the engineering-based approach, which unfortunately is not thorough in my biology degree. For this reason I'm very happy to be part of this multidisciplinary team. When I'm not in the lab, I spend my time playing the violin and cooking cakes for my friends.<br><br><b>Contact:</b> federica.sampietro02@universitadipavia.it ";<br />
des[12] = "Professor of Anatomy.<br><br><b>Contact:</b> cusella@unipv.it ";<br />
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<h2 class="art-postheader">The Team</h2><br />
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<br />
<br />
<br />
<br />
<div id="teamcont"><br />
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<div id="teamfoto"> <img src="https://static.igem.org/mediawiki/2011/1/1f/UNIPV-team.jpg" width="469" height="312" align="left"></div><br />
<div id="tipo1"> </div><br />
<div id="tipo2"> </div><br />
<div id="tipo3"> </div><br />
<div id="tipo4"> </div><br />
<div id="tipo5"> </div><br />
<div id="tipo6"> </div><br />
<div id="tipo7"> </div><br />
<div id="tipo8"> </div><br />
<div id="tipo9"> </div><br />
<div id="tipo10"> </div><br />
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<div id="students">Students</div><br />
<div id="tipoT1">Niccolò Franceschi</div><br />
<div id="tipoT2">Davide Bianchini</div><br />
<div id="tipoT5">Daniele Sartori</div><br />
<div id="tipoT6">Edoardo Baldini</div><br />
<div id="tipoT7">Tommaso Goggia</div><br />
<div id="tipoT8">Viola Ghio</div><br />
<div id="tipoT9">Emanuela Pasi</div><br />
<div id="tipoT13">Giuseppe Bertoni</div><br />
<div id="advisors">Advisors</div><br />
<div id="tipoT3">Nicolò Politi</div><br />
<div id="tipoT11">Federica Sampietro</div><br />
<div id="tipoT10">Susanna Zucca</div><br />
<div id="instructors">Instructors</div><br />
<div id="tipoT4">Prof. Paolo Magni</div><br />
<div id="tipoT12">Prof. Maria G. Cusella</div><br />
</div><br />
</div><br />
<br />
<br />
<br><br />
<br><br />
<b>ACKNOWLEDGEMENT</b>: <em>a special thanks to Giuseppe Bertoni.</em><br />
<br><br />
<br />
<br />
<br />
</html><br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Team
Team:UNIPV-Pavia/Team
2011-09-22T00:47:39Z
<p>Edoardo Baldini: </p>
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des[1] = "My name is Niccolò Franceschi, I was born in Parma but now I live in Pavia to attend University. I am 23 and I am attending the last year in the Master's Degree in Industrial Biotechnologies at the University of Pavia, I also have a Bachelor Degree in Biotechnologies from the University of Parma.<br>Besides the iGEM competition, I am currently working on my thesis on X-ray biodiffractometry.<br><br><b>Contact:</b> niccolo.francheschi01@universitadipavia.it ";<br />
des[2] = "Hi! I'm Davide Bianchini and I'm 24 years old.<br>I earned my Master degree in Biomedical Engineering at the end of March at the Faculty of Engineering of Bologna University, placed in Cesena. I went to Pavia University in order to take part to iGEM 2011 competition, since I believe it is a great opportunity to have insight in Synthetic Biology.<br><br><b>Contact:</b> davide.bianchini@studio.unibo.it";<br />
des[3] = "Bachelor Degree in Biomedical Engineering.<br>Master Degree in Biomedical Engineering.<br>PhD student in Bioengineering and Bioinformatics at the University of Pavia.<br><br>In 2010 I took part in iGEM competition as a student with UNIPV-Pavia Team. The idea to rationally design and implement functions in living systems attracts me a lot, that's why I like synthetic biology.<br>In my free time I like playing football, playing the piano and travelling.<br><br><b>Contact:</b> nicolo.politi@unipv.it ";<br />
des[4] = "I am a Professor of Bioengineering. My teaching and research interests ranging from mathematical model of biological systems to bioinformatics and synthetic biology, from statistics and probability models to medical informatics.<br>I am 42 years old. I am the promoter of the iGEM competition in Pavia. I am an enthusiastic fan of this competition! I think that it represents a big opportunity for a student. I have selected our best students from Biology, Biotechnology and Bioengineering programs and I tried to build a team. Even if I have spent a lot of time in this initiative, I am very happy to partecipate to iGEM 2011 and to have worked with all of them. For 20 years, I was a national referee of Handball. I like skiing and teaching to those students that want to learn.<br><br><b>Contact:</b> paolo.magni@unipv.it ";<br />
des[5] = "Soon to be graduated in Pharmacology at the University of Pavia.<br><br>My decision to be part of the UNIPV-iGEM team arose when I found out of the possible Synthetic Biology applications in pharmaceutical technology or medications pertaining fields. My academic interests are pharmacogenetics, pharmacogenomics, gene therapy, pharmaceutical legislation. I'm very fond of politics, art, reading and music.<br><br><b>Contact:</b> daniele.sartori01@universitadipavia.it ";<br />
des[6] = "Bachelor Degree in Electronic Engineering at the University of Pavia.<br>Currently Master student in Photonics and Optoelectronics, last year.<br><br>My academic interests include Quantum Electronics, Nonlinear Optics, Condensed Matter Physics and Biophotonics. I decided to take part to iGEM competition to explore the points of contact between Synthetic Biology and Photonics/Electronics.<br>Besides university, I spend time playing the piano and leading a musical webmagazine.<br><br><b>Contact:</b> edoardo.baldini01@universitadipavia.it ";<br />
des[7] = "Bachelor Degree in Biomedical Engineering and Master student in Biomedical Engineering at Università degli Studi di Pavia. Member of the Borrhomaic Family and student at IUSS, Sciences & Technologies Class.<br><br> Hi everyone! I come from Bergamo, a city not far from Milan. I' ve been interested in iGEM experience since the first year of university, but this was the right moment to join the competition. I think it's a great opportunity to work with other students, even from different disciplines, and learn a lot of new things that otherwise would not have taken into account.<br><br><b>Contact:</b> tommaso.goggia01@universitadipavia.it ";<br />
des[8] = "Hi! I'm Viola, I'm 23 years old and I come from Pavia.<br>I'm attending the second year of the Master's Degree in Industrial Biotechnologies.<br>I love cooking desserts, reading and travelling! My favorite hobbies are belly dance, playing fighting games and spending some time with my friends. I think iGEM experience is amazing!<br><br><b>Contact:</b> viola.ghio01@universitadipavia.it ";<br />
des[9] = "Bachelor Honor Degree in Biology.<br>Currently Master student in Molecular Biology and Genetics, last year.<br><br>Besides iGEM 2011 activities, I'm working in a DNA Enzymology and Molecular Virology's laboratory.<br>You can't say of me that I'm an athlete but I surely am an art lover; particularly the visual arts (photography, cinema and theater).<br>I love biology and so I hope to meet a lot of good scientist in Amsterdam and Boston! See you at the iGEM competition!<br><br><b>Contact:</b> emanuela.pasi01@universitadipavia.it";<br />
des[10] = "PhD student in Bioengineering and Bioinformatics, third year.<br> <br>Bachelor Degree in Biomedical Engineering and Master Student in Biomedical Engineering at Università degli Studi di Pavia.<br><br>Nice to meet you again, this is my third year at iGEM and I'm very happy to be part of this team! This year our project deals with synthetic biology and mathematical modelling, that are two research area I'm very interested in, and it contributes to make this experience amazing. See you at Jamboree! <br><br><b>Contact:</b> susanna.zucca@unipv.it";<br />
des[11] = "Molecular Biology student.<br><br>This is my second experience in the iGEM competition. Last year, I had the opportunity to appreciate this wonderful world of synthetic biology, so I decided to participate once again.<br>What I like best in synthetic biology is the engineering-based approach, which unfortunately is not thorough in my biology degree. For this reason I'm very happy to be part of this multidisciplinary team. When I'm not in the lab, I spend my time playing the violin and cooking cakes for my friends.<br><br><b>Contact:</b> federica.sampietro02@universitadipavia.it ";<br />
des[12] = "Professor of Anatomy.<br><br><b>Contact:</b> cusella@unipv.it ";<br />
des[13] = "Bachelor Honor Degree in Biotechnology.<br>Master student in Molecular Biology and Genetics.<br><br><em>Passionate, curious and with an innate creativity. He always gave the best and more even though it was not required. A real talent for research, a good friend and companion in this adventure. He could bring out the best from each of us.</em>";<br />
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<h2 class="art-postheader">The Team</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<br />
<br />
<br />
<div id="teamcont"><br />
<div id="teamcon"><br />
<div id="teamfoto"> <img src="https://static.igem.org/mediawiki/2011/1/1f/UNIPV-team.jpg" width="469" height="312" align="left"></div><br />
<div id="tipo1"> </div><br />
<div id="tipo2"> </div><br />
<div id="tipo3"> </div><br />
<div id="tipo4"> </div><br />
<div id="tipo5"> </div><br />
<div id="tipo6"> </div><br />
<div id="tipo7"> </div><br />
<div id="tipo8"> </div><br />
<div id="tipo9"> </div><br />
<div id="tipo10"> </div><br />
<div id="tipo11"> </div><br />
</div><br />
<div id="teambox"><br />
<div id="teamnam"> </div><br />
<div id="teamful"> </div><br />
</div><br />
<div id="ritratto"> <img src="https://static.igem.org/mediawiki/2011/9/9c/LogoUNIPVanello.jpg" width='195' id="foto"> </div><br />
<div id="box_desc"><br />
<div id="students">Students</div><br />
<div id="tipoT1">Niccolò Franceschi</div><br />
<div id="tipoT2">Davide Bianchini</div><br />
<div id="tipoT5">Daniele Sartori</div><br />
<div id="tipoT6">Edoardo Baldini</div><br />
<div id="tipoT7">Tommaso Goggia</div><br />
<div id="tipoT8">Viola Ghio</div><br />
<div id="tipoT9">Emanuela Pasi</div><br />
<div id="tipoT13">Giuseppe Bertoni</div><br />
<div id="advisors">Advisors</div><br />
<div id="tipoT3">Nicolò Politi</div><br />
<div id="tipoT11">Federica Sampietro</div><br />
<div id="tipoT10">Susanna Zucca</div><br />
<div id="instructors">Instructors</div><br />
<div id="tipoT4">Prof. Paolo Magni</div><br />
<div id="tipoT12">Prof. Maria G. Cusella</div><br />
</div><br />
</div><br />
<br />
<div style='text-align:left'> <br />
<a href="https://2011.igem.org/Team:UNIPV-Pavia/Team/What we did..."><font size="5" font face="Impact">What we did...</font></a><br />
</div><br />
<br><br />
<br><br />
<b>ACKNOWLEDGEMENT</b>: <em>a special thanks to Giuseppe Bertoni.</em><br />
<br><br />
<br />
<br />
<br />
</html><br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Freezer
Team:UNIPV-Pavia/Freezer
2011-09-22T00:43:17Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<html><br />
<h2 class="art-postheader"><br />
Freezer Management<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br><br />
<br />
<div align="center"><br />
<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 />
</p><br><br />
</div><br />
<br />
<center><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>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</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</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</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</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>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Calendar/September/week4
Team:UNIPV-Pavia/Calendar/September/week4
2011-09-22T00:42:26Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{maincal}}<br />
<html><br />
<h2 class="art-postheader"><br />
SEPTEMBER: WEEK 4<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<p style="text-align:left;"><br />
<br />
<p><a name="indice"/><br />
</p><br />
<br />
<div align="justify"><br />
<a name="September.2C_19th"></a><h2> <span class="mw-headline">September, 19th</span></h2><br />
<br />
<p><br />
Wiki update and definition of results.<br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<a name="September.2C_20th"></a><h2> <span class="mw-headline">September, 20th</span></h2><br />
<p><br />
Wiki update and definition of results.<br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<a name="September.2C_21st"></a><h2> <span class="mw-headline">September, 21st</span></h2><br />
<p><br />
Wiki update and definition of results.<br><br />
Definition of poster and presentation.<br />
</p><br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<a name="September.2C_22nd"></a><h2> <span class="mw-headline">September, 22nd</span></h2><br />
<p><br />
Wiki update and definition of results.<br><br />
Definition of poster and presentation.<br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<a name="September.2C_23rd"></a><h2> <span class="mw-headline">September, 23rd</span></h2><br />
<p><br />
Definition of poster and presentation.<br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<a name="September.2C_24th"></a><h2> <span class="mw-headline">September, 24th</span></h2><br />
<p><br />
Definition of poster and presentation.<br />
</p><br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<a name="September.2C_25th"></a><h2> <span class="mw-headline">September, 25th</span></h2><br />
<p><br />
Definition of poster and presentation.<br />
</p><br />
<br />
<br />
<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br><br />
<div><br />
<span style="float:left;"><br />
<a href="/Team:UNIPV-Pavia/Calendar/September/week3" title="Previous week"> <br />
<img src="https://static.igem.org/mediawiki/2011/0/06/Previous_week.png" alt="Previous"></a><br />
</span><br />
<span style="float:right;"><br />
<a href="/Team:UNIPV-Pavia/Calendar/September/week5" title="Next week"><br />
<img src="https://static.igem.org/mediawiki/2011/4/44/Next_week.png" alt="Next week"></a><br />
</span><br />
</div><br />
</div><br />
<br />
</html><br />
<br />
{{endcalendar}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Measurements
Team:UNIPV-Pavia/Measurements
2011-09-22T00:40:59Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<br />
<html><br />
<br />
<br />
<h2 class="art-postheader"><br />
Measurements<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<a name="top_page"></a><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="#Promoters"><span class="tocnumber">1</span> <span class="toctext">Measuring promoters transcriptional strength</span></a></li><br />
<br />
<ul><br />
<li class="toclevel-2"><a href="#pTet_protocol"><span class="tocnumber">1.1</span> <span class="toctext">pTet transcriptional strength</span></a><br />
<li class="toclevel-2"><a href="#pLux_protocol"><span class="tocnumber">1.2</span> <span class="toctext">pLux transcriptional strength</span></a><br />
<li class="toclevel-2"><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">Assaying 3OC<sub><small>6</small></sub>-HSL concentration</span></a></li><br />
</ul><br />
<br />
</td></tr></table><br />
<script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script><br />
<br><br />
</div><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. <br />
</em><br />
<br><br><br><br />
<div class="listcircle"><br />
<div align="justify"><br />
<br />
<br />
<a name="Promoters"></a> <h2 class="art-postheader"><br />
<font size = "5">Measuring promoters transcriptional strength</font><br />
</h2><br />
<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.</p><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.</p><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.</p><br />
<li> Induce cultures in falcon tube with anhydrotetracycline (aTc); final concentrations:</p><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.</p><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:</p><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">^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">^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">^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 />
<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">^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">^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">^top</a></small></div><br />
<br />
<a name="T9002"></a> <h2 class="art-postheader"><br />
Measuring 3OC<sub><small>6</small></sub>-HSL concentration with BBa_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">^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 />
<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> the reference value (i.e. the measure of O.D. at 600 nm of the broth in which cultures grow) was subtracted from each sample of O.D. at 600 nm, (O.D.<sub><small>600</small></sub>)<br />
<li> the reference value (i.e. the red or green fluorescence of a non-fluorescent culture, also known as auto-fluorescence) was also subtracted from each sample of red and green fluorescence <br />
</ul></ol><br />
</p><br />
<div align="right"><small><a href="#top_page">^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: 300px;"><a href="https://static.igem.org/mediawiki/2011/7/7d/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 />
<br />
<div align="right"><small><a href="#top_page">^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: 225px;"><a href="https://static.igem.org/mediawiki/2011/c/c5/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 />
<div align="right"><small><a href="#top_page">^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). 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="https://static.igem.org/mediawiki/2011/7/70/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 />
<br />
<div align="right"><small><a href="#top_page">^top</a></small></div><br />
<br />
<a name="HSL"></a> <h2 class="art-postheader"><br />
Assaying 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 concentrations is useful to estimate the parameters of the activation Hill function of pLux promoter. In this way, for each of our tests, we were able to build a calibration curve for the biosensor:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 350px;"><a class="image"><a href="https://static.igem.org/mediawiki/2011/b/b1/Scell_plux.jpg"><img alt="" src="https://static.igem.org/mediawiki/2011/b/b1/Scell_plux.jpg" class="thumbimage" width="100%" height="70%"></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 evaluate the auto-inducer concentration as follows:<br />
</p><br />
<br />
<div class="center"><div class="thumbinner" style="width: 650px;"><a href="https://static.igem.org/mediawiki/2011/e/e2/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>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Results
Team:UNIPV-Pavia/Project/Results
2011-09-22T00:36:50Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
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<html><br />
<h2 class="art-postheader"> Results </h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"> <br />
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<a name='indice'></a><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<table id="toc" class="toc"><br />
<tr><br />
<td><div id="toctitle"><br />
<h2>Contents</h2><br />
</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><br />
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</table><br />
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<script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script> <br />
<br><em> 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 />
<br><br><br />
For more details on parts characterization, please visit the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized'>parts section</a>. </em> <br><br />
<br><br />
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<a name='assembly'></a><br />
<h1>Parts assembly</h1><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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<a name='characterization'></a><br />
<h1>Characterization of basic modules</h1><br />
<br />
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<!--------pTet and pLux-----------> <br />
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<a name='promoters'></a><br />
<h2>Characterization of promoters pTet and pLux</h2><br />
<p align='justify'><br />
<p>Inducible promoters pLux and pTet were assembled upstream of mRFP 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><br />
<td class="row"><b>BioBrick code</b></td><br />
<td><b> Declared efficiency</b></td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0030 </td><br />
<td class="row"> 0,6</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0031 </td><br />
<td class="row"> 0,07</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0032 </td><br />
<td class="row"> 0,3</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0034 </td><br />
<td class="row"> 1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<div class="listcircle"><br />
<div align="justify"><br />
<p>The RBS variation has the purpose to stretch the induction curve, thus modulating the PoPs-OUT range of the inducible device.</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><br />
<p></p><br />
<p><br />
<ol><br />
<ul><br />
<li><b> RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li><b> RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li> <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 />
</p><br />
</li><br />
<p><br />
<li> <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.<br />
<br />
</li><br />
</p><br />
</ul><br />
</ol><br />
</p><br />
<div align="justify"> 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>. </div><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><br />
<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><br />
<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><br />
<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><br />
<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 of pLux are summarized in the table below:</p><br />
</div><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><br />
<td class='row'>4.28</td><br />
<td class='row'>0.20</td><br />
<td class='row'>1.08</td><br />
<td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>4.93</td><br />
<td class='row'>0.55</td><br />
<td class='row'>0.25</td><br />
<td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>9.49</td><br />
<td class='row'>0.02</td><br />
<td class='row'>0.47</td><br />
<td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>21.53</td><br />
<td class='row'>0.51</td><br />
<td class='row'>0.53</td><br />
<td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><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> 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><br />
<br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"> <img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><br />
<p align='justify'> The estimated parameters of the Hill curves described in the figures 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> [(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><br />
<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><br />
<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><br />
<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><br />
<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 />
<div align="center">Data are provided as average [CV%]</div> <br><br />
<br><br />
<p align='justify'> The operative parameters are summarized in the table below: </p><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><br />
<td class='row'>1.53</td><br />
<td class='row'>~0</td><br />
<td class='row'>7.95</td><br />
<td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>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><br />
<td class='row'>B0032</td><br />
<td class='row'>3.16</td><br />
<td class='row'>~0</td><br />
<td class='row'>6.7</td><br />
<td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>2.73</td><br />
<td class='row'>0.23</td><br />
<td class='row'>8.96</td><br />
<td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------AiiA and LuxI-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='enzymes'></a><br />
<h2>Characterization of enzymes AiiA and LuxI</h2><br />
<p align='justify'> 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 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 />
<div align="justify"><br />
<div class="thumbinner" style="width: 850px;"> <a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div><br />
</div><br />
<div align="justify"><br />
<p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p><br />
</div><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><br />
<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><br />
</table><br />
</div><br />
<br><br />
</p><br />
</p><br />
<p align='justify'> 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. </p><br />
<p align='justify'> The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity. </p><br />
<p align='justify'> 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 />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
</table><br />
<br />
<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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------RBS---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='rbs'></a><br />
<h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
RBSs were used for the fine tuning of CTRL+E. Different experimental conditions were assayed. <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 />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>0.40</td><br />
<td class='row'>1.6814</td><br />
<td class='row'>2.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.01</td><br />
<td class='row'>ND</td><br />
<td class='row'>0.04</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.19</td><br />
<td class='row'>0.4193</td><br />
<td class='row'>0.40</td><br />
<td class='row'>0,3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br><br />
Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT, with GeneX=mRFP, AiiA or LuxI: <br><br />
<br><br />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>1.72</td><br />
<td class='row'>0.53</td><br />
<td class='row'>0.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.03</td><br />
<td class='row'>0.83</td><br />
<td class='row'>0.028</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.37</td><br />
<td class='row'>0.50</td><br />
<td class='row'>N.D.</td><br />
<td class='row'>0.3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------growth---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='growth'></a><br />
<h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'> The bacterial growth curve has been modelled as a logistic curve and is represented by the following equation: <br><br />
<div align="center"><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br><br />
N(0)=n<sub>0</sub> <br></div><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> 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><br />
<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 class="disc"><br />
<li>1:1</li><br />
<li>1:10</li><br />
<li>1:100</li><br />
<li>1:1000</li><br />
</ul><br />
in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm. </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 />
</ul><br />
</ol><br />
<p>The results are summarized in the table and in the figure below: </p><br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><b> Culture </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><br />
<td class='row'><b> C.F.U. </b></td><br />
<td class='row'><b> Dilution Factor (10^) </b></td><br />
<td class='row'><b> N </b></td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,367950002 </td><br />
<td class='row'> 0,758004416 </td><br />
<td class='row'> 990 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 990000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,044649998 </td><br />
<td class='row'> 0,091982322 </td><br />
<td class='row'> 141 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 1410000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,004799999 </td><br />
<td class='row'> 0,009888356 </td><br />
<td class='row'> 136 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 136000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,000549998 </td><br />
<td class='row'> 0,001133037 </td><br />
<td class='row'> 20 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 200000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,54840003 </td><br />
<td class='row'> 1,129744917 </td><br />
<td class='row'> 165 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 16500000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,058200002 </td><br />
<td class='row'> 0,119896339 </td><br />
<td class='row'> 23 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 23000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,008700002 </td><br />
<td class='row'> 0,017922652 </td><br />
<td class='row'> 251 </td><br />
<td class='row'> 3 </td><br />
<td class='row'> 2510000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,000100002 </td><br />
<td class='row'> 0,000206011 </td><br />
<td class='row'> 24 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 2400000 </td><br />
</tr><br />
</table><br />
</center><br />
<br><br />
</p><br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png"><img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"><br />
</center><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 />
</p><br />
<center><br />
<table width='50%' class='data'><br />
<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><br />
<td class='row'>1*10<sup>9</sup></td><br />
<td class='row'>0.004925</td><br />
</tr><br />
</table><br />
</center><br />
<br />
<p>The reported value of &mu; corresponds to a doubling time of 142 minutes. </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 />
<a name='HSL'></a><br />
<h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 6</sub></b></td><br />
<td class='row'>32 </td><br />
<td class='row'>0.022 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 7</sub></b></td><br />
<td class='row'>8 </td><br />
<td class='row'>0.087 </td><br />
</tr><br />
</table><br />
</center><br />
<p> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 6</sub></b></td><br />
<td class='row'>&#8734; </td><br />
<td class='row'>0 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 7</sub></b></td><br />
<td class='row'>19 </td><br />
<td class='row'>0.037 </td><br />
</tr><br />
</table><br />
</center><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; </p><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------------t9002--------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='t9002'></a><br />
<h2>Characterization of BBa_T9002 biosensor</h2><br />
<div align="justify">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></div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <a href="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg"><img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage" width="47%" height="50%"></a></div><br />
</div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <a href="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg"><img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage" width="100%"></a></div><br />
</div><br />
<center><br />
<table width='50%' class='data'><br />
<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 />
<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 />
</tr><br />
</table><br />
</center><br />
<br><br />
<br><br />
<div align="justify">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.</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<h1>Conclusions</h1><br />
<br />
<p align='justify'><br />
To sum up the results of our project, we have characterized several basic parts and documented all of our experiments in the experience pages. All the data collected have been reported and synthetic parameters to describe the behavior of promoters, RBSs and genes have been provided. Such parameters can be used for the <em>in silico</em> simulation of mathematical models of complex circuits. <br><br />
Four what concerns CTRL+E, that is a proof of concept of the modular behavior of systems in synthetic biology, all the parameters of pTet, pLux and LuxI have been estimated. AiiA parameters revealed difficult to estimate.<br><br />
Reasonable values for AiiA parameters have been derived from experiments in different contexts. These values were used to simulate the behavior of CTRL+E.<br><br />
<br />
Further experiments are necessary to evaluate the consistence of the derived parameters for AiiA. <br><br />
Once the most promising module combinations will be evaluated, a library of differently tuned controller will be built and characterized in order to assess the correspondence between the model predictions and the real data.<br />
<br />
</p><br />
<br />
<br />
<br />
</html><br />
<br />
<br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling
Team:UNIPV-Pavia/Project/Modelling
2011-09-22T00:32:51Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<br />
<html><br />
<br />
<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 />
</ul> <br />
</td></tr></table><br />
<script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script><br />
<br><br><br />
<div class="listcircle"><br />
<br />
<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 simulations performed, using <em>ODEs</em> with MATLAB. </div></p><br />
<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br />
<br> <br />
<br />
<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 />
<br />
</ul><br />
</div><br />
<div align="right"><small><a href="#indice">^top</a></small></div><br />
<br><br />
<br />
<br />
<br />
<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 />
<br />
<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 />
<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/2011/5/5e/Circuito_finale.jpg" class="thumbimage" width="87%"></a></div></div><br />
</td><br />
</tr><br />
</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 />
<div align="right"><small><a href="#indice">^top</a></small></div><br />
<div style='text-align:center; font-size: 12px; font-style:italic; margin-top=50px; padding-top=50px;'>Schematic description of Ctrl+E system behavior</div><br />
<br><br />
<br />
<br />
<br />
<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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<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 dilution 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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<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" 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">[nM]</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 />
<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; margin-bottom:0px; padding-bottom:0px;'><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 />
<div style='text-align:center; font-size: 12px; font-style:italic; margin-top:-20px; padding-top:-20px;'>Data analysis procedure for the determination of promoters activation curve</div><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 LuxI dependent HSL production, we have:</p><br />
<br />
<table align='center' width='100%' style='margin-bottom:0px; padding-bottom:0px;'><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 />
<div style='text-align:center; font-size: 12px; font-style:italic; margin-top:0px; padding-top:0px;'>Graphical representation of LuxI dependent HSL production, determined through T9002 HSL biosensor</div><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>. In case of pH=6, &gamma;<sub>HSL</sub>=0.<br />
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<a name="Simulations"></a><h1><span class="mw-headline"> <b>Simulations</b> </span></h1><br />
<div><br />
The whole control circuit has been simulated and here the simulation results are presented. <br><br />
All the combinations of pTet-RBSx and pLux-RBSx were simulated using ODEs', in case of aTc=100 ng/ml.<br />
As explained in <a href="https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized#AiiA">AiiA gene - BBa_C0060 section</a>, parameters k<sub>cat</sub> and k<sub>M,AiiA</sub> were difficult to estimate from the collected data.<br />
<br> Here simulations are performed assuming reasonable values for k<sub>cat</sub> and k<sub>M,AiiA</sub> (1*10<sup>-9</sup> [1/(min*cell)] and k<sub>M,AiiA</sub>=5000 [AUr/cell], respectively).<br> <br />
<br />
Obviously, increasing the k<sub>cat</sub> value, the HSL steady-state concentration decreases. <br />
Nevertheless, if you consider 100-fold variation of k<sub>cat</sub> value, the steady state of HSL is in the range about [0.08-0.7] nM. <br />
</div><br />
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<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><a href="https://static.igem.org/mediawiki/2011/0/0d/Ptet_RBS30plux_RBS31simul.jpg"><img alt="" src="https://static.igem.org/mediawiki/2011/0/0d/Ptet_RBS30plux_RBS31simul.jpg" class="thumbimage" width="85%" height="55%"></a></div></div><br />
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<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><a href="https://static.igem.org/mediawiki/2011/f/f7/Ptet_RBS30plux_RBS34simul.jpg"><img alt="" src="https://static.igem.org/mediawiki/2011/f/f7/Ptet_RBS30plux_RBS34simul.jpg" class="thumbimage" width="85%" height="55%"></a></div></div><br />
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<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><a href="https://static.igem.org/mediawiki/2011/4/4a/Ptet_RBS32plux_RBS31simul.jpg"><img alt="" src="https://static.igem.org/mediawiki/2011/4/4a/Ptet_RBS32plux_RBS31simul.jpg" class="thumbimage" width="85%" height="55%"></a></div></div><br />
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<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><a href="https://static.igem.org/mediawiki/2011/d/d8/Ptet_RBS32plux_RBS34simul.jpg"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d8/Ptet_RBS32plux_RBS34simul.jpg" class="thumbimage" width="85%" height="55%"></a></div></div><br />
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<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><a href="https://static.igem.org/mediawiki/2011/0/0c/Ptet_RBS34plux_RBS31simul.jpg"><img alt="" src="https://static.igem.org/mediawiki/2011/0/0c/Ptet_RBS34plux_RBS31simul.jpg" class="thumbimage" width="85%" height="55%"></a></div></div><br />
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<div style='text-align:justify'><div class="thumbinner" style="width: 850px;"><a href="https://static.igem.org/mediawiki/2011/f/fc/Ptet_RBS34plux_RBS34simul.jpg"><img alt="" src="https://static.igem.org/mediawiki/2011/f/fc/Ptet_RBS34plux_RBS34simul.jpg" class="thumbimage" width="85%" height="55%"></a></div></div><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 <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 further simplify the steady state equations. In particular, one concern relates to the number of cells N (in the order of 10^7), which is far lower than N<sub>max</sub> (10^9). The other pertains to &gamma;*HSL parameter, which can be neglected compared to the other two terms of the third equation, considering pH=6. 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 />
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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%;"><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 />
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<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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<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 />
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<p> We can introduce the simplification to remove the &eta;<sub>pLux</sub> 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 relationship 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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<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 />
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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%;"><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 />
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<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"><span style="text-decoration:overline;" > V</span><sub>LuxI</sub></td><br />
<td class="row">(&gamma;<sub>AiiA</sub>+&mu;)*(k<sub>pLux</sub>)<sup>&eta;pLux</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">b</td><br />
<td class="row"><span style="text-decoration:overline;" > V</span><sub>LuxI</sub>*k<sub>M,AiiA</sub></td><br />
<td class="row">-&alpha;<sub>pLux</sub>*&delta;<sub>pLux</sub>*(k<sub>pLux</sub>)<sup>&eta;pLux</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">c</sub></sub></td><br />
<td class="row">k<sub>cat</sub></span></td><br />
<td class="row">-&gamma;<sub>AiiA</sub>-&mu;</td> <br />
</tr><br />
<br />
<tr><br />
<td class="row">d</td><br />
<td class="row">0</td><br />
<td class="row">&alpha;<sub>pLux</sub></td><br />
</tr><br />
<br />
<tr><br />
<td class="row">Horizontal asymptote</td><br />
<td class="row"><span style="text-decoration:overline;" > V</span><sub>LuxI</sub>/k<sub>cat</sub></td><br />
<td class="row">a/c=(k<sub>pLux</sub>)<sup>&eta;pLux</sup></td> <br />
</tr><br />
<br />
<tr><br />
<td class="row">Vertical asymptote</td><br />
<td class="row">0</td><br />
<td class="row">-d/c=&alpha;<sub>pLux</sub>/(&gamma;<sub>AiiA</sub>+&mu;)</td><br />
</tr><br />
</table><br />
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</center><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 />
<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 />
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<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 RH2, 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 />
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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/2011/0/04/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 />
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<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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<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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<a name="References"></a><h1><span class="mw-headline"> <b>References</b> </span></h1><br />
<div style='text-align:justify'><br />
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<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>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling
Team:UNIPV-Pavia/Project/Modelling
2011-09-21T22:38:07Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<br />
<html><br />
<br />
<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 />
</ul> <br />
</td></tr></table><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> <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 />
<br />
</ul><br />
</div><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 />
<br />
<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 />
<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/2011/5/5e/Circuito_finale.jpg" class="thumbimage" width="87%"></a></div></div><br />
</td><br />
</tr><br />
</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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<div style='text-align:center; font-size: 12px; font-style:italic; margin-top=50px; padding-top=50px;'>Schematic description of Ctrl+E system behavior</div><br />
<br><br />
<br />
<br />
<br />
<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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<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 dilution 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 />
<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" 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">[nM]</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 />
<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 />
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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/2011/7/79/Caratterizzazione_pluxN.jpg" class="thumbimage" width="70%"></a></div></div><br />
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</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; margin-bottom:0px; padding-bottom:0px;'><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 />
<div style='text-align:center; font-size: 12px; font-style:italic; margin-top:-20px; padding-top:-20px;'>Data analysis procedure for the determination of promoters activation curve</div><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 />
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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 />
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<div style='text-align:justify'><br />
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<table align='center' width='100%'><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/2011/8/88/Caratterizzazione_aiia.JPG" class="thumbimage" width="32%"></a></div></div><br />
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</table><br />
</div><br />
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<div style='text-align:justify'><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/2011/4/48/Caratterizzazione_luxIN.jpg" class="thumbimage" width="28%"></a></div></div><br />
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</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 LuxI dependent HSL production, we have:</p><br />
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<table align='center' width='100%' style='margin-bottom:0px; padding-bottom:0px;'><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 />
<div style='text-align:center; font-size: 12px; font-style:italic; margin-top:0px; padding-top:0px;'>Graphical representation of LuxI dependent HSL production, determined through T9002 HSL biosensor</div><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 />
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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 />
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<center><br />
<table><br />
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<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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<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 />
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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 <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 further simplify the steady state equations. In particular, one concern relates to the number of cells N (in the order of 10^7), which is far lower than N<sub>max</sub> (10^9). The other pertains to &gamma;*HSL parameter, which can be neglected compared to the other two terms of the third equation, considering pH=6. 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 />
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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%;"><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 />
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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%;"><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;<sub>pLux</sub> 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 relationship 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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<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 />
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</table><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%;"><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 />
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<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"><span style="text-decoration:overline;" > V</span><sub>LuxI</sub></td><br />
<td class="row">(&gamma;<sub>AiiA</sub>+&mu;)*(k<sub>pLux</sub>)<sup>&eta;pLux</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">b</td><br />
<td class="row"><span style="text-decoration:overline;" > V</span><sub>LuxI</sub>*k<sub>M,AiiA</sub></td><br />
<td class="row">-&alpha;<sub>pLux</sub>*&delta;<sub>pLux</sub>*(k<sub>pLux</sub>)<sup>&eta;pLux</td><br />
</tr><br />
<br />
<tr><br />
<td class="row">c</sub></sub></td><br />
<td class="row">k<sub>cat</sub></span></td><br />
<td class="row">-&gamma;<sub>AiiA</sub>+&mu;</td> <br />
</tr><br />
<br />
<tr><br />
<td class="row">d</td><br />
<td class="row">0</td><br />
<td class="row">&alpha;<sub>pLux</sub></td><br />
</tr><br />
<br />
<tr><br />
<td class="row">Horizontal asymptote</td><br />
<td class="row"><span style="text-decoration:overline;" > V</span><sub>LuxI</sub>/k<sub>cat</sub></td><br />
<td class="row">a/c=(k<sub>pLux</sub>)<sup>&eta;pLux</sup></td> <br />
</tr><br />
<br />
<tr><br />
<td class="row">Vertical asymptote</td><br />
<td class="row">0</td><br />
<td class="row">-d/c=&alpha;<sub>pLux</sub>/(&gamma;<sub>AiiA</sub>+&mu;)</td><br />
</tr><br />
</table><br />
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</center><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 />
<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 RH2, 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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<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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<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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</div><br />
</div><br />
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</html><br />
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{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Team
Team:UNIPV-Pavia/Team
2011-09-21T20:26:38Z
<p>Edoardo Baldini: </p>
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nam[1] = "Niccolò Franceschi";<br />
nam[2] = "Davide Bianchini";<br />
nam[3] = "Nicolò Politi";<br />
nam[4] = "Prof. Paolo Magni";<br />
nam[5] = "Daniele Sartori";<br />
nam[6] = "Edoardo Baldini";<br />
nam[7] = "Tommaso Goggia";<br />
nam[8] = "Viola Ghio";<br />
nam[9] = "Emanuela Pasi";<br />
nam[10] = "Susanna Zucca";<br />
nam[11] = "Federica Sampietro";<br />
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nam[13] = "Giuseppe Bertoni";<br />
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des[1] = "My name is Niccolò Franceschi, I was born in Parma but now I live in Pavia to attend University. I am 23 and I am attending the last year in the Master's Degree in Industrial Biotechnologies at the University of Pavia, I also have a Bachelor Degree in Biotechnologies from the University of Parma.<br>Besides the iGEM competition, I am currently working on my thesis on X-ray biodiffractometry.<br><br><b>Contact:</b> niccolo.francheschi01@universitadipavia.it ";<br />
des[2] = "Hi! I'm Davide Bianchini and I'm 24 years old.<br>I earned my Master degree in Biomedical Engineering at the end of March at the Faculty of Engineering of Bologna University, placed in Cesena. I went to Pavia University in order to take part to iGEM 2011 competition, since I believe it is a great opportunity to have insight in Synthetic Biology.<br><br><b>Contact:</b> davide.bianchini@studio.unibo.it";<br />
des[3] = "Bachelor Degree in Biomedical Engineering.<br>Master Degree in Biomedical Engineering.<br>PhD student in Bioengineering and Bioinformatics at the University of Pavia.<br><br>In 2010 I took part in iGEM competition as a student with UNIPV-Pavia Team. The idea to rationally design and implement functions in living systems attracts me a lot, that's why I like synthetic biology.<br>In my free time I like playing football, playing the piano and travelling.<br><br><b>Contact:</b> nicolo.politi@unipv.it ";<br />
des[4] = "I am a Professor of Bioengineering. My teaching and research interests ranging from mathematical model of biological systems to bioinformatics and synthetic biology, from statistics and probability models to medical informatics.<br>I am 42 years old. I am the promoter of the iGEM competition in Pavia. I am an enthusiastic fan of this competition! I think that it represents a big opportunity for a student. I have selected our best students from Biology, Biotechnology and Bioengineering programs and I tried to build a team. Even if I have spent a lot of time in this initiative, I am very happy to partecipate to iGEM 2011 and to have worked with all of them. For 20 years, I was a national referee of Handball. I like skiing and teaching to those students that want to learn.<br><br><b>Contact:</b> paolo.magni@unipv.it ";<br />
des[5] = "Soon to be graduated in Pharmacology at the University of Pavia.<br><br>My decision to be part of the UNIPV-iGEM team arose when I found out of the possible Synthetic Biology applications in pharmaceutical technology or medications pertaining fields. My academic interests are pharmacogenetics, pharmacogenomics, gene therapy, pharmaceutical legislation. I'm very fond of politics, art, reading and music.<br><br><b>Contact:</b> daniele.sartori01@universitadipavia.it ";<br />
des[6] = "Bachelor Degree in Electronic Engineering at University of Pavia.<br>Currently Master student in Photonics and Optoelectronics, last year.<br><br>My academic interests include Quantum Electronics, Nonlinear Optics, Condensed Matter Physics and Biophotonics. I decided to take part to iGEM competition to explore the points of contact between Synthetic Biology and Photonics/Electronics.<br>Besides university, I spend time playing the piano and leading a musical webmagazine.<br><br><b>Contact:</b> edoardo.baldini01@universitadipavia.it ";<br />
des[7] = "Bachelor Degree in Biomedical Engineering and Master student in Biomedical Engineering at Università degli Studi di Pavia. Member of the Borrhomaic Family and student at IUSS, Sciences & Technologies Class.<br><br> Hi everyone! I come from Bergamo, a city not far from Milan. I' ve been interested in iGEM experience since the first year of university, but this was the right moment to join the competition. I think it's a great opportunity to work with other students, even from different disciplines, and learn a lot of new things that otherwise would not have taken into account.<br><br><b>Contact:</b> tommaso.goggia01@universitadipavia.it ";<br />
des[8] = "Hi! I'm Viola, I'm 23 years old and I come from Pavia.<br>I'm attending the second year of the Master's Degree in Industrial Biotechnologies.<br>I love cooking desserts, reading and travelling! My favorite hobbies are belly dance, playing fighting games and spending some time with my friends. I think iGEM experience is amazing!<br><br><b>Contact:</b> viola.ghio01@universitadipavia.it ";<br />
des[9] = "Bachelor Honor Degree in Biology.<br>Currently Master student in Molecular Biology and Genetics, last year.<br><br>Besides iGEM 2011 activities, I'm working in a DNA Enzymology and Molecular Virology's laboratory.<br>You can't say of me that I'm an athlete but I surely am an art lover; particularly the visual arts (photography, cinema and theater).<br>I love biology and so I hope to meet a lot of good scientist in Amsterdam and Boston! See you at the iGEM competition!<br><br><b>Contact:</b> emanuela.pasi01@universitadipavia.it";<br />
des[10] = "PhD student in Bioengineering and Bioinformatics, third year.<br> <br>Bachelor Degree in Biomedical Engineering and Master Student in Biomedical Engineering at Università degli Studi di Pavia.<br><br>Nice to meet you again, this is my third year at iGEM and I'm very happy to be part of this team! This year our project deals with synthetic biology and mathematical modelling, that are two research area I'm very interested in, and it contributes to make this experience amazing. See you at Jamboree! <br><br><b>Contact:</b> susanna.zucca@unipv.it";<br />
des[11] = "Molecular Biology student.<br><br>This is my second experience in the iGEM competition. Last year, I had the opportunity to appreciate this wonderful world of synthetic biology, so I decided to participate once again.<br>What I like best in synthetic biology is the engineering-based approach, which unfortunately is not thorough in my biology degree. For this reason I'm very happy to be part of this multidisciplinary team. When I'm not in the lab, I spend my time playing the violin and cooking cakes for my friends.<br><br><b>Contact:</b> federica.sampietro02@universitadipavia.it ";<br />
des[12] = "Professor of Anatomy.<br><br><b>Contact:</b> cusella@unipv.it ";<br />
des[13] = "Bachelor Honor Degree in Biotechnology.<br>Master student in Molecular Biology and Genetics.<br><br><em>Passionate, curious and with an innate creativity. He always gave the best and more even though it was not required. A real talent for research, a good friend and companion in this adventure. He could bring out the best from each of us.</em>";<br />
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<h2 class="art-postheader">The Team</h2><br />
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<br />
<br />
<br />
<br />
<div id="teamcont"><br />
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<div id="tipo1"> </div><br />
<div id="tipo2"> </div><br />
<div id="tipo3"> </div><br />
<div id="tipo4"> </div><br />
<div id="tipo5"> </div><br />
<div id="tipo6"> </div><br />
<div id="tipo7"> </div><br />
<div id="tipo8"> </div><br />
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<div id="students">Students</div><br />
<div id="tipoT1">Niccolò Franceschi</div><br />
<div id="tipoT2">Davide Bianchini</div><br />
<div id="tipoT5">Daniele Sartori</div><br />
<div id="tipoT6">Edoardo Baldini</div><br />
<div id="tipoT7">Tommaso Goggia</div><br />
<div id="tipoT8">Viola Ghio</div><br />
<div id="tipoT9">Emanuela Pasi</div><br />
<div id="tipoT13">Giuseppe Bertoni</div><br />
<div id="advisors">Advisors</div><br />
<div id="tipoT3">Nicolò Politi</div><br />
<div id="tipoT11">Federica Sampietro</div><br />
<div id="tipoT10">Susanna Zucca</div><br />
<div id="instructors">Instructors</div><br />
<div id="tipoT4">Prof. Paolo Magni</div><br />
<div id="tipoT12">Prof. Maria G. Cusella</div><br />
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<br />
<div style='text-align:left'> <br />
<a href="https://2011.igem.org/Team:UNIPV-Pavia/Team/What we did..."><font size="5" font face="Impact">What we did...</font></a><br />
</div><br />
<br><br />
<br><br />
<b>ACKNOWLEDGEMENT</b>: <em>a special thanks to Giuseppe Bertoni for his precious contribution to our work.</em><br />
<br><br />
<br />
<br />
<br />
</html><br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Team
Team:UNIPV-Pavia/Team
2011-09-21T20:26:02Z
<p>Edoardo Baldini: </p>
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des[1] = "My name is Niccolò Franceschi, I was born in Parma but now I live in Pavia to attend University. I am 23 and I am attending the last year in the Master's Degree in Industrial Biotechnologies at the University of Pavia, I also have a Bachelor Degree in Biotechnologies from the University of Parma.<br>Besides the iGEM competition, I am currently working on my thesis on X-ray biodiffractometry.<br><br><b>Contact:</b> niccolo.francheschi01@universitadipavia.it ";<br />
des[2] = "Hi! I'm Davide Bianchini and I'm 24 years old.<br>I earned my Master degree in Biomedical Engineering at the end of March at the Faculty of Engineering of Bologna University, placed in Cesena. I went to Pavia University in order to take part to iGEM 2011 competition, since I believe it is a great opportunity to have insight in Synthetic Biology.<br><br><b>Contact:</b> davide.bianchini@studio.unibo.it";<br />
des[3] = "Bachelor Degree in Biomedical Engineering.<br>Master Degree in Biomedical Engineering.<br>PhD student in Bioengineering and Bioinformatics at the University of Pavia.<br><br>In 2010 I took part in iGEM competition as a student with UNIPV-Pavia Team. The idea to rationally design and implement functions in living systems attracts me a lot, that's why I like synthetic biology.<br>In my free time I like playing football, playing the piano and travelling.<br><br><b>Contact:</b> nicolo.politi@unipv.it ";<br />
des[4] = "I am a Professor of Bioengineering. My teaching and research interests ranging from mathematical model of biological systems to bioinformatics and synthetic biology, from statistics and probability models to medical informatics.<br>I am 42 years old. I am the promoter of the iGEM competition in Pavia. I am an enthusiastic fan of this competition! I think that it represents a big opportunity for a student. I have selected our best students from Biology, Biotechnology and Bioengineering programs and I tried to build a team. Even if I have spent a lot of time in this initiative, I am very happy to partecipate to iGEM 2011 and to have worked with all of them. For 20 years, I was a national referee of Handball. I like skiing and teaching to those students that want to learn.<br><br><b>Contact:</b> paolo.magni@unipv.it ";<br />
des[5] = "Soon to be graduated in Pharmacology at the University of Pavia.<br><br>My decision to be part of the UNIPV-iGEM team arose when I found out of the possible Synthetic Biology applications in pharmaceutical technology or medications pertaining fields. My academic interests are pharmacogenetics, pharmacogenomics, gene therapy, pharmaceutical legislation.<br>I'm very fond of politics, art, reading and music.<br><br><b>Contact:</b> daniele.sartori01@universitadipavia.it ";<br />
des[6] = "Bachelor Degree in Electronic Engineering at University of Pavia.<br>Currently Master student in Photonics and Optoelectronics, last year.<br><br>My academic interests include Quantum Electronics, Nonlinear Optics, Condensed Matter Physics and Biophotonics. I decided to take part to iGEM competition to explore the points of contact between Synthetic Biology and Photonics/Electronics.<br>Besides university, I spend time playing the piano and leading a musical webmagazine.<br><br><b>Contact:</b> edoardo.baldini01@universitadipavia.it ";<br />
des[7] = "Bachelor Degree in Biomedical Engineering and Master student in Biomedical Engineering at Università degli Studi di Pavia. Member of the Borrhomaic Family and student at IUSS, Sciences & Technologies Class.<br><br> Hi everyone! I come from Bergamo, a city not far from Milan. I' ve been interested in iGEM experience since the first year of university, but this was the right moment to join the competition. I think it's a great opportunity to work with other students, even from different disciplines, and learn a lot of new things that otherwise would not have taken into account.<br><br><b>Contact:</b> tommaso.goggia01@universitadipavia.it ";<br />
des[8] = "Hi! I'm Viola, I'm 23 years old and I come from Pavia.<br>I'm attending the second year of the Master's Degree in Industrial Biotechnologies.<br>I love cooking desserts, reading and travelling! My favorite hobbies are belly dance, playing fighting games and spending some time with my friends. I think iGEM experience is amazing!<br><br><b>Contact:</b> viola.ghio01@universitadipavia.it ";<br />
des[9] = "Bachelor Honor Degree in Biology.<br>Currently Master student in Molecular Biology and Genetics, last year.<br><br>Besides iGEM 2011 activities, I'm working in a DNA Enzymology and Molecular Virology's laboratory.<br>You can't say of me that I'm an athlete but I surely am an art lover; particularly the visual arts (photography, cinema and theater).<br>I love biology and so I hope to meet a lot of good scientist in Amsterdam and Boston! See you at the iGEM competition!<br><br><b>Contact:</b> emanuela.pasi01@universitadipavia.it";<br />
des[10] = "PhD student in Bioengineering and Bioinformatics, third year.<br> <br>Bachelor Degree in Biomedical Engineering and Master Student in Biomedical Engineering at Università degli Studi di Pavia.<br><br>Nice to meet you again, this is my third year at iGEM and I'm very happy to be part of this team! This year our project deals with synthetic biology and mathematical modelling, that are two research area I'm very interested in, and it contributes to make this experience amazing. See you at Jamboree! <br><br><b>Contact:</b> susanna.zucca@unipv.it";<br />
des[11] = "Molecular Biology student.<br><br>This is my second experience in the iGEM competition. Last year, I had the opportunity to appreciate this wonderful world of synthetic biology, so I decided to participate once again.<br>What I like best in synthetic biology is the engineering-based approach, which unfortunately is not thorough in my biology degree. For this reason I'm very happy to be part of this multidisciplinary team. When I'm not in the lab, I spend my time playing the violin and cooking cakes for my friends.<br><br><b>Contact:</b> federica.sampietro02@universitadipavia.it ";<br />
des[12] = "Professor of Anatomy.<br><br><b>Contact:</b> cusella@unipv.it ";<br />
des[13] = "Bachelor Honor Degree in Biotechnology.<br>Master student in Molecular Biology and Genetics.<br><br><em>Passionate, curious and with an innate creativity. He always gave the best and more even though it was not required. A real talent for research, a good friend and companion in this adventure. He could bring out the best from each of us.</em>";<br />
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<h2 class="art-postheader">The Team</h2><br />
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<br />
<br />
<br />
<br />
<div id="teamcont"><br />
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<div id="teamfoto"> <img src="https://static.igem.org/mediawiki/2011/1/1f/UNIPV-team.jpg" width="469" height="312" align="left"></div><br />
<div id="tipo1"> </div><br />
<div id="tipo2"> </div><br />
<div id="tipo3"> </div><br />
<div id="tipo4"> </div><br />
<div id="tipo5"> </div><br />
<div id="tipo6"> </div><br />
<div id="tipo7"> </div><br />
<div id="tipo8"> </div><br />
<div id="tipo9"> </div><br />
<div id="tipo10"> </div><br />
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<div id="students">Students</div><br />
<div id="tipoT1">Niccolò Franceschi</div><br />
<div id="tipoT2">Davide Bianchini</div><br />
<div id="tipoT5">Daniele Sartori</div><br />
<div id="tipoT6">Edoardo Baldini</div><br />
<div id="tipoT7">Tommaso Goggia</div><br />
<div id="tipoT8">Viola Ghio</div><br />
<div id="tipoT9">Emanuela Pasi</div><br />
<div id="tipoT13">Giuseppe Bertoni</div><br />
<div id="advisors">Advisors</div><br />
<div id="tipoT3">Nicolò Politi</div><br />
<div id="tipoT11">Federica Sampietro</div><br />
<div id="tipoT10">Susanna Zucca</div><br />
<div id="instructors">Instructors</div><br />
<div id="tipoT4">Prof. Paolo Magni</div><br />
<div id="tipoT12">Prof. Maria G. Cusella</div><br />
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<br />
<div style='text-align:left'> <br />
<a href="https://2011.igem.org/Team:UNIPV-Pavia/Team/What we did..."><font size="5" font face="Impact">What we did...</font></a><br />
</div><br />
<br><br />
<br><br />
<b>ACKNOWLEDGEMENT</b>: <em>a special thanks to Giuseppe Bertoni for his precious contribution to our work.</em><br />
<br><br />
<br />
<br />
<br />
</html><br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Team
Team:UNIPV-Pavia/Team
2011-09-21T20:25:22Z
<p>Edoardo Baldini: </p>
<hr />
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nam[5] = "Daniele Sartori";<br />
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nam[8] = "Viola Ghio";<br />
nam[9] = "Emanuela Pasi";<br />
nam[10] = "Susanna Zucca";<br />
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des[1] = "My name is Niccolò Franceschi, I was born in Parma but now I live in Pavia to attend University. I am 23 and I am attending the last year in the Master's Degree in Industrial Biotechnologies at the University of Pavia, I also have a Bachelor Degree in Biotechnologies from the University of Parma.<br>Besides the iGEM competition, I am currently working on my thesis on X-ray biodiffractometry.<br><br><b>Contact:</b> niccolo.francheschi01@universitadipavia.it ";<br />
des[2] = "Hi! I'm Davide Bianchini and I'm 24 years old.<br>I earned my Master degree in Biomedical Engineering at the end of March at the Faculty of Engineering of Bologna University, placed in Cesena. I went to Pavia University in order to take part to iGEM 2011 competition, since I believe it is a great opportunity to have insight in Synthetic Biology.<br><br><b>Contact:</b> davide.bianchini@studio.unibo.it";<br />
des[3] = "Bachelor Degree in Biomedical Engineering.<br>Master Degree in Biomedical Engineering.<br>PhD student in Bioengineering and Bioinformatics at the University of Pavia.<br><br>In 2010 I took part in iGEM competition as a student with UNIPV-Pavia Team. The idea to rationally design and implement functions in living systems attracts me a lot, that's why I like synthetic biology.<br>In my free time I like playing football, playing the piano and travelling.<br><br><b>Contact:</b> nicolo.politi@unipv.it ";<br />
des[4] = "I am a Professor of Bioengineering. My teaching and research interests ranging from mathematical model of biological systems to bioinformatics and synthetic biology, from statistics and probability models to medical informatics.<br>I am 42 years old. I am the promoter of the iGEM competition in Pavia. I am an enthusiastic fan of this competition! I think that it represents a big opportunity for a student. I have selected our best students from Biology, Biotechnology and Bioengineering programs and I tried to build a team. Even if I have spent a lot of time in this initiative, I am very happy to partecipate to iGEM 2011 and to have worked with all of them. For 20 years, I was a national referee of Handball. I like skiing and teaching to those students that want to learn.<br><br><b>Contact:</b> paolo.magni@unipv.it ";<br />
des[5] = "Soon to be graduated in Pharmacology at the University of Pavia.<br><br>My decision to be part of the UNIPV-iGEM team arose when I found out of the possible Synthetic Biology applications in pharmaceutical technology or medications pertaining fields.<br>My academic interests are pharmacogenetics, pharmacogenomics, gene therapy, pharmaceutical legislation.<br>I'm very fond of politics, art, reading and music.<br><br><b>Contact:</b> daniele.sartori01@universitadipavia.it ";<br />
des[6] = "Bachelor Degree in Electronic Engineering at University of Pavia.<br>Currently Master student in Photonics and Optoelectronics, last year.<br><br>My academic interests include Quantum Electronics, Nonlinear Optics, Condensed Matter Physics and Biophotonics. I decided to take part to iGEM competition to explore the points of contact between Synthetic Biology and Photonics/Electronics.<br>Besides university, I spend time playing the piano and leading a musical webmagazine.<br><br><b>Contact:</b> edoardo.baldini01@universitadipavia.it ";<br />
des[7] = "Bachelor Degree in Biomedical Engineering and Master student in Biomedical Engineering at Università degli Studi di Pavia. Member of the Borrhomaic Family and student at IUSS, Sciences & Technologies Class.<br><br> Hi everyone! I come from Bergamo, a city not far from Milan. I' ve been interested in iGEM experience since the first year of university, but this was the right moment to join the competition. I think it's a great opportunity to work with other students, even from different disciplines, and learn a lot of new things that otherwise would not have taken into account.<br><br><b>Contact:</b> tommaso.goggia01@universitadipavia.it ";<br />
des[8] = "Hi! I'm Viola, I'm 23 years old and I come from Pavia.<br>I'm attending the second year of the Master's Degree in Industrial Biotechnologies.<br>I love cooking desserts, reading and travelling! My favorite hobbies are belly dance, playing fighting games and spending some time with my friends. I think iGEM experience is amazing!<br><br><b>Contact:</b> viola.ghio01@universitadipavia.it ";<br />
des[9] = "Bachelor Honor Degree in Biology.<br>Currently Master student in Molecular Biology and Genetics, last year.<br><br>Besides iGEM 2011 activities, I'm working in a DNA Enzymology and Molecular Virology's laboratory.<br>You can't say of me that I'm an athlete but I surely am an art lover; particularly the visual arts (photography, cinema and theater).<br>I love biology and so I hope to meet a lot of good scientist in Amsterdam and Boston! See you at the iGEM competition!<br><br><b>Contact:</b> emanuela.pasi01@universitadipavia.it";<br />
des[10] = "PhD student in Bioengineering and Bioinformatics, third year.<br> <br>Bachelor Degree in Biomedical Engineering and Master Student in Biomedical Engineering at Università degli Studi di Pavia.<br><br>Nice to meet you again, this is my third year at iGEM and I'm very happy to be part of this team! This year our project deals with synthetic biology and mathematical modelling, that are two research area I'm very interested in, and it contributes to make this experience amazing. See you at Jamboree! <br><br><b>Contact:</b> susanna.zucca@unipv.it";<br />
des[11] = "Molecular Biology student.<br><br>This is my second experience in the iGEM competition. Last year, I had the opportunity to appreciate this wonderful world of synthetic biology, so I decided to participate once again.<br>What I like best in synthetic biology is the engineering-based approach, which unfortunately is not thorough in my biology degree. For this reason I'm very happy to be part of this multidisciplinary team. When I'm not in the lab, I spend my time playing the violin and cooking cakes for my friends.<br><br><b>Contact:</b> federica.sampietro02@universitadipavia.it ";<br />
des[12] = "Professor of Anatomy.<br><br><b>Contact:</b> cusella@unipv.it ";<br />
des[13] = "Bachelor Honor Degree in Biotechnology.<br>Master student in Molecular Biology and Genetics.<br><br><em>Passionate, curious and with an innate creativity. He always gave the best and more even though it was not required. A real talent for research, a good friend and companion in this adventure. He could bring out the best from each of us.</em>";<br />
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<h2 class="art-postheader">The Team</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<br />
<br />
<br />
<div id="teamcont"><br />
<div id="teamcon"><br />
<div id="teamfoto"> <img src="https://static.igem.org/mediawiki/2011/1/1f/UNIPV-team.jpg" width="469" height="312" align="left"></div><br />
<div id="tipo1"> </div><br />
<div id="tipo2"> </div><br />
<div id="tipo3"> </div><br />
<div id="tipo4"> </div><br />
<div id="tipo5"> </div><br />
<div id="tipo6"> </div><br />
<div id="tipo7"> </div><br />
<div id="tipo8"> </div><br />
<div id="tipo9"> </div><br />
<div id="tipo10"> </div><br />
<div id="tipo11"> </div><br />
</div><br />
<div id="teambox"><br />
<div id="teamnam"> </div><br />
<div id="teamful"> </div><br />
</div><br />
<div id="ritratto"> <img src="https://static.igem.org/mediawiki/2011/9/9c/LogoUNIPVanello.jpg" width='195' id="foto"> </div><br />
<div id="box_desc"><br />
<div id="students">Students</div><br />
<div id="tipoT1">Niccolò Franceschi</div><br />
<div id="tipoT2">Davide Bianchini</div><br />
<div id="tipoT5">Daniele Sartori</div><br />
<div id="tipoT6">Edoardo Baldini</div><br />
<div id="tipoT7">Tommaso Goggia</div><br />
<div id="tipoT8">Viola Ghio</div><br />
<div id="tipoT9">Emanuela Pasi</div><br />
<div id="tipoT13">Giuseppe Bertoni</div><br />
<div id="advisors">Advisors</div><br />
<div id="tipoT3">Nicolò Politi</div><br />
<div id="tipoT11">Federica Sampietro</div><br />
<div id="tipoT10">Susanna Zucca</div><br />
<div id="instructors">Instructors</div><br />
<div id="tipoT4">Prof. Paolo Magni</div><br />
<div id="tipoT12">Prof. Maria G. Cusella</div><br />
</div><br />
</div><br />
<br />
<div style='text-align:left'> <br />
<a href="https://2011.igem.org/Team:UNIPV-Pavia/Team/What we did..."><font size="5" font face="Impact">What we did...</font></a><br />
</div><br />
<br><br />
<br><br />
<b>ACKNOWLEDGEMENT</b>: <em>a special thanks to Giuseppe Bertoni for his precious contribution to our work.</em><br />
<br><br />
<br />
<br />
<br />
</html><br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Instruments
Team:UNIPV-Pavia/Instruments
2011-09-21T20:19:33Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
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<h2 class="art-postheader">Instruments</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<p><a name="indice"> </a></p><br />
<a name="indice"><br />
<table id="toc" class="toc" summary="Contents"><br />
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<td><div id="toctitle"><br />
<h2>Contents</h2><br />
<span class="toctoggle">[<a href="javascript:toggleToc()" class="internal" id="togglelink">hide</a>]</span></div><br />
<ul><br />
<li class="toclevel-1"><a href="#TECAN_Infinite_2000"><span class="tocnumber">1</span> <span class="toctext">TECAN Infinite 2000</span></a></li><br />
<li class="toclevel-1"><a href="#Nanodrop_ND_1000_spectrophotometer"><span class="tocnumber">2</span> <span class="toctext">Nanodrop ND 1000 spectrophotometer</span></a></li><br />
<li class="toclevel-1"><a href="#PCR_thermocyclers"><span class="tocnumber">3</span> <span class="toctext">PCR thermocyclers</span></a></li><br />
<li class="toclevel-1"><a href="#Instruments_For_Electrophoresis"><span class="tocnumber">4</span> <span class="toctext">Instruments for electrophoresis</span></a></li><br />
<li class="toclevel-1"><a href="#Gel_Image_Acquisition_System"><span class="tocnumber">5</span> <span class="toctext">Gel image acquisition system</span></a></li><br />
<li class="toclevel-1"><a href="#Incubator_For_Plates"><span class="tocnumber">6</span> <span class="toctext">Incubator for plates</span></a></li><br />
<li class="toclevel-1"><a href="#Incubator_And_Shaker_For_Liquid_Cultures"><span class="tocnumber">7</span> <span class="toctext">Incubator and shaker for liquid cultures</span></a></li><br />
<li class="toclevel-1"><a href="#Hood"><span class="tocnumber">8</span> <span class="toctext">Laminar flow cabinet</span></a></li><br />
<li class="toclevel-1"><a href="#Autoclave"><span class="tocnumber">9</span> <span class="toctext">Autoclave</span></a></li><br />
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</tbody><br />
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<a name="TECAN_Infinite_2000"></a><br />
<h2> <span class="mw-headline">TECAN Infinite 2000</span></h2><br />
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<p align="justify"> Tecan Infinite F200 is a multifunctional microplate filter-based reader with injector option, that provides high performance for the vast majority of microplate applications and research. It has been designed as a general purpose laboratory instrument for professional use, supporting common 6 to 384-well microplates. <br />
This instrument allows long incubation of the microplate, the chance to set the shaking movements, the duration of all operations, including cycles, and the dispensation of liquid material in the wells. These actions are managed by a user interface operated by i-Control software, ranging from scheduling experiments to the cleaning of injectors. <br />
We used this instrument for the following measurement techniques: </p><br />
<div class="listcircle"><br />
<div style="text-align:justify"><br />
<div class="art-postcontent" style="margin-left: 15pt;"><br />
<li> fluorescence intensity; </li><br />
<li> absorbance. </li><br />
</div></div></td><br />
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<tbody><br />
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<td><div class="floatright"><span><img alt="TECAN Infinite 2000" src="https://static.igem.org/mediawiki/2010/5/5f/UNIPV_Pavia_tecan.jpg" border="0" height="198" width="250"></span></div></td><br />
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<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>TECAN infinite 2000</span></div></td><br />
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<p><br><br />
</p><br />
<br />
<a name="Nanodrop_ND_1000_spectrophotometer"></a><br />
<h2> <span class="mw-headline">NanoDrop ND 1000 spectrophotometer</span></h2><br />
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<p align="justify"> The Thermo Scientific NanoDrop™ 1000 Spectrophotometer (v 3.7) is a full-spectrum (220 - 750nm) spectrophotometer. This instrument is a really helpfull instrument that elimante the needs of cuvettes and is capable to measure highly concentrated samples without dilution. We use NanoDrop to measure:<br />
<div class="listcircle"><br />
<div style="text-align:justify"><br />
<div class="art-postcontent" style="margin-left: 15pt;"><br />
<div class="listcircle"><br />
<li> concentration of DNA in a micro-volume of sample (1 microliter);</li><br />
<li> and OD in cell cultures (2 microliter).</li></p><br />
<p></p></div></div></td><br />
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<td><div class="floatright"><span><img alt="Nanodrop" src="https://static.igem.org/mediawiki/2011/3/36/Nanodrop_ND1000.jpg" border="0" width="250"></span></div></td><br />
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<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>NanoDrop ND 1000</span> </div></td><br />
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<p><br><br />
</p><br />
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<a name="PCR_thermocyclers"></a><br />
<h2> <span class="mw-headline">PCR thermocyclers</span></h2><br />
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<p align="justify"> The laboratory is equipped with:<br />
<div class="listcircle"><br />
<div style="text-align:justify"><br />
<div class="art-postcontent" style="margin-left: 15pt;"><br />
<li>Gene Amp PCR System 9700 (Applied Biosystem) thermal cycler: used to amplify segments of DNA via the polymerase chain reaction (PCR) process; </li><br />
<li>MiniOpticon Real-Time PCR Instrument (BIO-RAD): this machine combines the functions of a thermal cycler and a fluorimeter. It is able to amplify and detect DNA.</li></p><br />
<p></p></td><br />
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<td><div class="floatright"><span><img alt="Real time" src="https://static.igem.org/mediawiki/2008/d/dd/Pv_pcr_parallel2.jpg" border="0" height="188" width="250"></span></div></td><br />
</tr><br />
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<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>MiniOpticon Real-Time PCR</span> </div></td><br />
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<table border="0"><br />
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<td><div class="floatright"><span><img alt="thermocycler" src="https://static.igem.org/mediawiki/2008/0/08/Pv_pcr_parallel1.jpg" border="0" height="188" width="250"></span></div></td><br />
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<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>Gene Amp PCR System 9700</span> </div></td><br />
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<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<p><br><br />
</p><br />
<br />
<a name="Instruments_For_Electrophoresis"></a><br />
<h2> <span class="mw-headline">Instruments for electrophoresis</span></h2><br />
<table width="100%"><br />
<tbody><br />
<tr><br />
<td align="left" valign="top"><p></p><br />
<p align="justify"> Gel electrophoresis apparatus - Agarose gel is placed in this TBE buffer filled box and electrical field is applied via the power supply to the rear. The negative terminal is the black one while the positive is the red one. In this way DNA, that is naturally negatively charged, migrates to the positive pole. </p><br />
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<td><div class="floatright"><span><img alt="Instruments for electrophoresis" src="https://static.igem.org/mediawiki/2010/3/31/UNIPV_Pavia_electrophoresis.jpg" border="0" height="188" width="250"></span></div></td><br />
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<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>Large, medium and small electrophoresis apparatuses</span></div></td><br />
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<p><br><br />
</p><br />
<br />
<a name="Gel_Image_Acquisition_System"></a><br />
<h2> <span class="mw-headline">Gel image acquisition system</span></h2><br />
<table width="100%"><br />
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<td align="left" valign="top"><p></p><br />
<p align="justify"> It is a UV system with camera used to acquire gel run snapshots. </p><br />
<p></p></td><br />
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<tbody><br />
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<td><div class="floatright"><span><img alt="Gel image acquisition system" src="https://static.igem.org/mediawiki/2011/b/bb/Gelacquisition.jpg" border="0" height="188" width="250"></span></div></td><br />
</tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>UV system and camera</span> </div></td><br />
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<p><br><br />
</p><br />
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<br />
<a name="Incubator_For_Plates"></a><br />
<h2> <span class="mw-headline">Incubator for plates</span></h2><br />
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<td align="left" valign="top"><p></p><br />
<p align="justify"> In order to grow bacteria a culture media is needed, together with plates (petri-dishes), some laboratory supplies and incubators.<br />
The incubator is an oven able to keep bacteria at the desired temperature; the typical temperature needed to allow a rapid growth of bacteria is 37 °C.<br />
</p><br />
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<td><div class="floatright"><span><img alt="Incubator for plates" src="https://static.igem.org/mediawiki/2010/0/0a/UNIPV_Pavia_incubatorforplates.jpg" border="0" height="188" width="250"></span></div></td><br />
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<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>Incubator for plates</span> </div></td><br />
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<p><br><br />
</p><br />
<br />
<br />
<a name="Incubator_And_Shaker_For_Liquid_Cultures"></a><br />
<h2> <span class="mw-headline">Incubator and shaker for liquid cultures</span></h2><br />
<table width="100%"><br />
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<td align="left" valign="top"><p></p><br />
<p align="justify"> Shaking incubators, also known as environmental shakers, are often used for cell culturing, cell aeration and solubility studies. In addition to stable temperature conditions, they use an orbital agitation at variable speeds to affect the growth of cell cultures, displaced in a liquid media. All major functions (temperature, RPM and time) have alarms that alert the user to deviations from set parameters. Overtemperature protection is provided via a safety thermostat. Digital keypad operation offers the ability to calibrate the temperature controller to a reference thermometer.</p><br />
<p></p></td><br />
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<tbody><br />
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<td><div class="floatright"><span><img alt="Incubator and shaker for liquid cultures" src="https://static.igem.org/mediawiki/2010/5/5f/UNIPV_Pavia_incubatorshaker.jpg" border="0" height="188" width="250"></span></div></td><br />
</tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>Incubator and shaker for liquid cultures</span> </div></td><br />
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<p><br><br />
</p><br />
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<br />
<a name="Hood"></a><br />
<h2> <span class="mw-headline">Laminar flow cabinet</span></h2><br />
<table width="100%"><br />
<tbody><br />
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<td align="left" valign="top"><p></p><br />
<p align="justify"> The lab is equipped with laminar flow cabinet. It is a carefully enclosed bench designed to prevent contamination of biological samples. Air is drawn through a HEPA filter and blown in a very smooth, laminar flow towards the user. The hood is provided of UV-C germicidal lamp for the sterilization. </p><br />
<p></p></td><br />
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<td><div class="floatright"><span><img alt="Hood" src="https://static.igem.org/mediawiki/2010/d/d4/UNIPV_Pavia_hood.jpg" border="0" height="238" width="250"></span></div></td><br />
</tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>Laminar flow cabinet</span> </div></td><br />
</tr><br />
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<p><br><br />
</p><br />
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<a name="Autoclave"></a><br />
<h2> <span class="mw-headline">Autoclave</span></h2><br />
<table width="100%"><br />
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<p align="justify"> An autoclave is a self-locking apparatus used to sterilize equipment and supplies by subjecting them to high pressure saturated steam.<br />
It allows steam to flow around each article placed in the chamber. The vapor penetrates cloth or paper used to package the articles being sterilized. Autoclaving brings to the destruction of all types of microorganisms, including spores. Therefore, autoclave is widely used in microbiology, medicine, veterinary science, mycology or chiropody; its size varies on the media it is sterilizing.<br />
The amount of time and degree of temperature necessary for sterilization depend on the articles to be sterilized and whether they are wrapped or left directly exposed to the steam. Typically the process is run at 121°C or more for 15 to 20 minutes and it is essential to ensure that all the trapped air is removed, because hot air is less efficient than steam to achieve sterility.<br />
</p><br />
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<td><div class="floatright"><span><img alt="Autoclave" src="https://static.igem.org/mediawiki/2010/7/7d/UNIPV_Pavia_autoclave.jpg" border="0" height="188" width="250"></span></div></td><br />
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<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>The autoclave</span> </div></td><br />
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{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Instruments
Team:UNIPV-Pavia/Instruments
2011-09-21T20:15:08Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<br />
<html><br />
<br />
<br />
<br />
<h2 class="art-postheader">Instruments</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<p><a name="indice"> </a></p><br />
<a name="indice"><br />
<table id="toc" class="toc" summary="Contents"><br />
<tbody><br />
<tr><br />
<td><div id="toctitle"><br />
<h2>Contents</h2><br />
<span class="toctoggle">[<a href="javascript:toggleToc()" class="internal" id="togglelink">hide</a>]</span></div><br />
<ul><br />
<li class="toclevel-1"><a href="#TECAN_Infinite_2000"><span class="tocnumber">1</span> <span class="toctext">TECAN Infinite 2000</span></a></li><br />
<li class="toclevel-1"><a href="#Nanodrop_ND_1000_spectrophotometer"><span class="tocnumber">2</span> <span class="toctext">Nanodrop ND 1000 spectrophotometer</span></a></li><br />
<li class="toclevel-1"><a href="#PCR_thermocyclers"><span class="tocnumber">3</span> <span class="toctext">PCR thermocyclers</span></a></li><br />
<li class="toclevel-1"><a href="#Instruments_For_Electrophoresis"><span class="tocnumber">4</span> <span class="toctext">Instruments for electrophoresis</span></a></li><br />
<li class="toclevel-1"><a href="#Gel_Image_Acquisition_System"><span class="tocnumber">5</span> <span class="toctext">Gel image acquisition system</span></a></li><br />
<li class="toclevel-1"><a href="#Incubator_For_Plates"><span class="tocnumber">6</span> <span class="toctext">Incubator for plates</span></a></li><br />
<li class="toclevel-1"><a href="#Incubator_And_Shaker_For_Liquid_Cultures"><span class="tocnumber">7</span> <span class="toctext">Incubator and shaker for liquid cultures</span></a></li><br />
<li class="toclevel-1"><a href="#Hood"><span class="tocnumber">8</span> <span class="toctext">Laminar flow cabinet</span></a></li><br />
<li class="toclevel-1"><a href="#Autoclave"><span class="tocnumber">9</span> <span class="toctext">Autoclave</span></a></li><br />
</ul></td><br />
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</tbody><br />
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<script type="text/javascript"> if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script> <br><br />
<a name="TECAN_Infinite_2000"></a><br />
<h2> <span class="mw-headline">TECAN Infinite 2000</span></h2><br />
<table width="100%"><br />
<tbody><br />
<tr><br />
<td align="left" valign="top"><p></p><br />
<p align="justify"> Tecan Infinite F200 is a multifunctional microplate filter-based reader with injector option, that provides high performance for the vast majority of microplate applications and research. It has been designed as a general purpose laboratory instrument for professional use, supporting common 6 to 384-well microplates. <br />
This instrument allows long incubation of the microplate, the chance to set the shaking movements, the duration of all operations, including cycles, and the dispensation of liquid material in the wells. These actions are managed by a user interface operated by i-Control software, ranging from scheduling experiments to the cleaning of injectors. <br />
We used this instrument for the following measurement techniques: </p><br />
<div class="listcircle"><br />
<div style="text-align:justify"><br />
<div class="art-postcontent" style="margin-left: 15pt;"><br />
<li> fluorescence intensity; </li><br />
<li> absorbance. </li><br />
</div></div></td><br />
<td align="right" valign="top" width="20%"><table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="TECAN Infinite 2000" src="https://static.igem.org/mediawiki/2010/thumb/5/5f/UNIPV_Pavia_tecan.jpg/250px-UNIPV_Pavia_tecan.jpg" border="0" height="198" width="250"></span></div></td><br />
</tr><br />
<tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>TECAN infinite 2000</span></div></td><br />
</tr><br />
</tbody><br />
</table></td><br />
</tr><br />
</tbody><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<p><br><br />
</p><br />
<br />
<a name="Nanodrop_ND_1000_spectrophotometer"></a><br />
<h2> <span class="mw-headline">NanoDrop ND 1000 spectrophotometer</span></h2><br />
<table width="100%"><br />
<tbody><br />
<tr><br />
<td align="left" valign="top"><p></p><br />
<p align="justify"> The Thermo Scientific NanoDrop™ 1000 Spectrophotometer (v 3.7) is a full-spectrum (220 - 750nm) spectrophotometer. This instrument is a really helpfull instrument that elimante the needs of cuvettes and is capable to measure highly concentrated samples without dilution. We use NanoDrop to measure:<br />
<div class="listcircle"><br />
<div style="text-align:justify"><br />
<div class="art-postcontent" style="margin-left: 15pt;"><br />
<div class="listcircle"><br />
<li> concentration of DNA in a micro-volume of sample (1 microliter);</li><br />
<li> and OD in cell cultures (2 microliter).</li></p><br />
<p></p></div></div></td><br />
<td align="right" valign="top" width="20%"><table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="Nanodrop" src="https://static.igem.org/mediawiki/2011/3/36/Nanodrop_ND1000.jpg" border="0" width="250"></span></div></td><br />
</tr><br />
<tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>NanoDrop ND 1000</span> </div></td><br />
</tr><br />
</tbody><br />
</table></td><br />
</tr><br />
</tbody><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<p><br><br />
</p><br />
<br />
<a name="PCR_thermocyclers"></a><br />
<h2> <span class="mw-headline">PCR thermocyclers</span></h2><br />
<table width="100%"><br />
<tbody><br />
<tr><br />
<td align="left" valign="top"><p></p><br />
<p align="justify"> The laboratory is equipped with:<br />
<div class="listcircle"><br />
<div style="text-align:justify"><br />
<div class="art-postcontent" style="margin-left: 15pt;"><br />
<li>Gene Amp PCR System 9700 (Applied Biosystem) thermal cycler: used to amplify segments of DNA via the polymerase chain reaction (PCR) process; </li><br />
<li>MiniOpticon Real-Time PCR Instrument (BIO-RAD): this machine combines the functions of a thermal cycler and a fluorimeter. It is able to amplify and detect DNA.</li></p><br />
<p></p></td><br />
<td align="right" valign="top" width="20%"><table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="Real time" src="https://static.igem.org/mediawiki/2008/d/dd/Pv_pcr_parallel2.jpg" border="0" height="188" width="250"></span></div></td><br />
</tr><br />
<tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>MiniOpticon Real-Time PCR</span> </div></td><br />
</tr><br />
</tbody><br />
</table><br />
<table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="thermocycler" src="https://static.igem.org/mediawiki/2008/0/08/Pv_pcr_parallel1.jpg" border="0" height="188" width="250"></span></div></td><br />
</tr><br />
</tr><br />
<tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>Gene Amp PCR System 9700</span> </div></td><br />
</tr><br />
</tbody><br />
</table></td><br />
</tr><br />
</tbody><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<p><br><br />
</p><br />
<br />
<a name="Instruments_For_Electrophoresis"></a><br />
<h2> <span class="mw-headline">Instruments for electrophoresis</span></h2><br />
<table width="100%"><br />
<tbody><br />
<tr><br />
<td align="left" valign="top"><p></p><br />
<p align="justify"> Gel electrophoresis apparatus - Agarose gel is placed in this TBE buffer filled box and electrical field is applied via the power supply to the rear. The negative terminal is the black one while the positive is the red one. In this way DNA, that is naturally negatively charged, migrates to the positive pole. </p><br />
<p></p></td><br />
<td align="right" valign="top" width="20%"><table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="Instruments for electrophoresis" src="https://static.igem.org/mediawiki/2010/thumb/3/31/UNIPV_Pavia_electrophoresis.jpg/250px-UNIPV_Pavia_electrophoresis.jpg" border="0" height="188" width="250"></span></div></td><br />
</tr><br />
<tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>Large, medium and small electrophoresis apparatuses</span></div></td><br />
</tr><br />
</tbody><br />
</table></td><br />
</tr><br />
</tbody><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<p><br><br />
</p><br />
<br />
<a name="Gel_Image_Acquisition_System"></a><br />
<h2> <span class="mw-headline">Gel image acquisition system</span></h2><br />
<table width="100%"><br />
<tbody><br />
<tr><br />
<td align="left" valign="top"><p></p><br />
<p align="justify"> It is a UV system with camera used to acquire gel run snapshots. </p><br />
<p></p></td><br />
<td align="right" valign="top" width="20%"><table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="Gel image acquisition system" src="https://static.igem.org/mediawiki/2011/b/bb/Gelacquisition.jpg" border="0" height="188" width="250"></span></div></td><br />
</tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>UV system and camera</span> </div></td><br />
</tr><br />
<br />
</tbody><br />
</table></td><br />
</tr><br />
</tbody><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<p><br><br />
</p><br />
<br />
<br />
<a name="Incubator_For_Plates"></a><br />
<h2> <span class="mw-headline">Incubator for plates</span></h2><br />
<table width="100%"><br />
<tbody><br />
<tr><br />
<td align="left" valign="top"><p></p><br />
<p align="justify"> In order to grow bacteria a culture media is needed, together with plates (petri-dishes), some laboratory supplies and incubators.<br />
The incubator is an oven able to keep bacteria at the desired temperature; the typical temperature needed to allow a rapid growth of bacteria is 37 °C.<br />
</p><br />
<p></p></td><br />
<td align="right" valign="top" width="20%"><table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="Incubator for plates" src="https://static.igem.org/mediawiki/2010/thumb/0/0a/UNIPV_Pavia_incubatorforplates.jpg/250px-UNIPV_Pavia_incubatorforplates.jpg" border="0" height="188" width="250"></span></div></td><br />
</tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>Incubator for plates</span> </div></td><br />
</tr><br />
<br />
</tbody><br />
</table></td><br />
</tr><br />
</tbody><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<p><br><br />
</p><br />
<br />
<br />
<a name="Incubator_And_Shaker_For_Liquid_Cultures"></a><br />
<h2> <span class="mw-headline">Incubator and shaker for liquid cultures</span></h2><br />
<table width="100%"><br />
<tbody><br />
<tr><br />
<td align="left" valign="top"><p></p><br />
<p align="justify"> Shaking incubators, also known as environmental shakers, are often used for cell culturing, cell aeration and solubility studies. In addition to stable temperature conditions, they use an orbital agitation at variable speeds to affect the growth of cell cultures, displaced in a liquid media. All major functions (temperature, RPM and time) have alarms that alert the user to deviations from set parameters. Overtemperature protection is provided via a safety thermostat. Digital keypad operation offers the ability to calibrate the temperature controller to a reference thermometer.</p><br />
<p></p></td><br />
<td align="right" valign="top" width="20%"><table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="Incubator and shaker for liquid cultures" src="https://static.igem.org/mediawiki/2010/5/5f/UNIPV_Pavia_incubatorshaker.jpg" border="0" height="188" width="250"></span></div></td><br />
</tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>Incubator and shaker for liquid cultures</span> </div></td><br />
</tr><br />
</tbody><br />
</table></td><br />
</tr><br />
</tbody><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<p><br><br />
</p><br />
<br />
<br />
<a name="Hood"></a><br />
<h2> <span class="mw-headline">Laminar flow cabinet</span></h2><br />
<table width="100%"><br />
<tbody><br />
<tr><br />
<td align="left" valign="top"><p></p><br />
<p align="justify"> The lab is equipped with laminar flow cabinet. It is a carefully enclosed bench designed to prevent contamination of biological samples. Air is drawn through a HEPA filter and blown in a very smooth, laminar flow towards the user. The hood is provided of UV-C germicidal lamp for the sterilization. </p><br />
<p></p></td><br />
<td align="right" valign="top" width="20%"><table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="Hood" src="https://static.igem.org/mediawiki/2010/thumb/d/d4/UNIPV_Pavia_hood.jpg/250px-UNIPV_Pavia_hood.jpg" border="0" height="238" width="250"></span></div></td><br />
</tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>Laminar flow cabinet</span> </div></td><br />
</tr><br />
</tbody><br />
</table></td><br />
</tr><br />
</tbody><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<p><br><br />
</p><br />
<br />
<br />
<a name="Autoclave"></a><br />
<h2> <span class="mw-headline">Autoclave</span></h2><br />
<table width="100%"><br />
<tbody><br />
<tr><br />
<td align="left" valign="top"><p></p><br />
<p align="justify"> An autoclave is a self-locking apparatus used to sterilize equipment and supplies by subjecting them to high pressure saturated steam.<br />
It allows steam to flow around each article placed in the chamber. The vapor penetrates cloth or paper used to package the articles being sterilized. Autoclaving brings to the destruction of all types of microorganisms, including spores. Therefore, autoclave is widely used in microbiology, medicine, veterinary science, mycology or chiropody; its size varies on the media it is sterilizing.<br />
The amount of time and degree of temperature necessary for sterilization depend on the articles to be sterilized and whether they are wrapped or left directly exposed to the steam. Typically the process is run at 121°C or more for 15 to 20 minutes and it is essential to ensure that all the trapped air is removed, because hot air is less efficient than steam to achieve sterility.<br />
</p><br />
<p></p></td><br />
<td align="right" valign="top" width="20%"><table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="Autoclave" src="https://static.igem.org/mediawiki/2010/thumb/7/7d/UNIPV_Pavia_autoclave.jpg/250px-UNIPV_Pavia_autoclave.jpg" border="0" height="188" width="250"></span></div></td><br />
</tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>The autoclave</span> </div></td><br />
</tr><br />
</tbody><br />
</table></td><br />
</tr><br />
</tbody><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<p><br><br />
</p><br />
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</html><br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Instruments
Team:UNIPV-Pavia/Instruments
2011-09-21T20:13:25Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<br />
<html><br />
<br />
<br />
<br />
<h2 class="art-postheader">Instruments</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<p><a name="indice"> </a></p><br />
<a name="indice"><br />
<table id="toc" class="toc" summary="Contents"><br />
<tbody><br />
<tr><br />
<td><div id="toctitle"><br />
<h2>Contents</h2><br />
<span class="toctoggle">[<a href="javascript:toggleToc()" class="internal" id="togglelink">hide</a>]</span></div><br />
<ul><br />
<li class="toclevel-1"><a href="#TECAN_Infinite_2000"><span class="tocnumber">1</span> <span class="toctext">TECAN Infinite 2000</span></a></li><br />
<li class="toclevel-1"><a href="#Nanodrop_ND_1000_spectrophotometer"><span class="tocnumber">2</span> <span class="toctext">Nanodrop ND 1000 spectrophotometer</span></a></li><br />
<li class="toclevel-1"><a href="#PCR_thermocyclers"><span class="tocnumber">3</span> <span class="toctext">PCR thermocyclers</span></a></li><br />
<li class="toclevel-1"><a href="#Instruments_For_Electrophoresis"><span class="tocnumber">4</span> <span class="toctext">Instruments for electrophoresis</span></a></li><br />
<li class="toclevel-1"><a href="#Gel_Image_Acquisition_System"><span class="tocnumber">5</span> <span class="toctext">Gel image acquisition system</span></a></li><br />
<li class="toclevel-1"><a href="#Incubator_For_Plates"><span class="tocnumber">6</span> <span class="toctext">Incubator for plates</span></a></li><br />
<li class="toclevel-1"><a href="#Incubator_And_Shaker_For_Liquid_Cultures"><span class="tocnumber">7</span> <span class="toctext">Incubator and shaker for liquid cultures</span></a></li><br />
<li class="toclevel-1"><a href="#Hood"><span class="tocnumber">8</span> <span class="toctext">Laminar flow cabinet</span></a></li><br />
<li class="toclevel-1"><a href="#Autoclave"><span class="tocnumber">9</span> <span class="toctext">Autoclave</span></a></li><br />
</ul></td><br />
<br />
</tr><br />
</tbody><br />
</table><br />
<script type="text/javascript"> if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script> <br><br />
<a name="TECAN_Infinite_2000"></a><br />
<h2> <span class="mw-headline">TECAN Infinite 2000</span></h2><br />
<table width="100%"><br />
<tbody><br />
<tr><br />
<td align="left" valign="top"><p></p><br />
<p align="justify"> Tecan Infinite F200 is a multifunctional microplate filter-based reader with injector option, that provides high performance for the vast majority of microplate applications and research. It has been designed as a general purpose laboratory instrument for professional use, supporting common 6 to 384-well microplates. <br />
This instrument allows long incubation of the microplate, the chance to set the shaking movements, the duration of all operations, including cycles, and the dispensation of liquid material in the wells. These actions are managed by a user interface operated by i-Control software, ranging from scheduling experiments to the cleaning of injectors. <br />
We used this instrument for the following measurement techniques: </p><br />
<div class="listcircle"><br />
<div style="text-align:justify"><br />
<div class="art-postcontent" style="margin-left: 15pt;"><br />
<li> fluorescence intensity; </li><br />
<li> absorbance. </li><br />
</div></div></td><br />
<td align="right" valign="top" width="20%"><table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="TECAN Infinite 2000" src="https://static.igem.org/mediawiki/2010/thumb/5/5f/UNIPV_Pavia_tecan.jpg/250px-UNIPV_Pavia_tecan.jpg" border="0" height="198" width="250"></span></div></td><br />
</tr><br />
<tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>TECAN infinite 2000</span></div></td><br />
</tr><br />
</tbody><br />
</table></td><br />
</tr><br />
</tbody><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<p><br><br />
</p><br />
<br />
<a name="Nanodrop_ND_1000_spectrophotometer"></a><br />
<h2> <span class="mw-headline">NanoDrop ND 1000 spectrophotometer</span></h2><br />
<table width="100%"><br />
<tbody><br />
<tr><br />
<td align="left" valign="top"><p></p><br />
<p align="justify"> The Thermo Scientific NanoDrop™ 1000 Spectrophotometer (v 3.7) is a full-spectrum (220 - 750nm) spectrophotometer. This instrument is a really helpfull instrument that elimante the needs of cuvettes and is capable to measure highly concentrated samples without dilution. We use NanoDrop to measure:<br />
<div class="listcircle"><br />
<div style="text-align:justify"><br />
<div class="art-postcontent" style="margin-left: 15pt;"><br />
<div class="listcircle"><br />
<li> concentration of DNA in a micro-volume of sample (1 microliter);</li><br />
<li> and OD in cell cultures (2 microliter).</li></p><br />
<p></p></div></div></td><br />
<td align="right" valign="top" width="20%"><table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="Nanodrop" src="https://static.igem.org/mediawiki/2011/3/36/Nanodrop_ND1000.jpg" border="0" width="250"></span></div></td><br />
</tr><br />
<tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>NanoDrop ND 1000</span> </div></td><br />
</tr><br />
</tbody><br />
</table></td><br />
</tr><br />
</tbody><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<p><br><br />
</p><br />
<br />
<a name="PCR_thermocyclers"></a><br />
<h2> <span class="mw-headline">PCR thermocyclers</span></h2><br />
<table width="100%"><br />
<tbody><br />
<tr><br />
<td align="left" valign="top"><p></p><br />
<p align="justify"> The laboratory is equipped with:<br />
<div class="listcircle"><br />
<div style="text-align:justify"><br />
<div class="art-postcontent" style="margin-left: 15pt;"><br />
<li>Gene Amp PCR System 9700 (Applied Biosystem) thermal cycler: used to amplify segments of DNA via the polymerase chain reaction (PCR) process; </li><br />
<li>MiniOpticon Real-Time PCR Instrument (BIO-RAD): this machine combines the functions of a thermal cycler and a fluorimeter. It is able to amplify and detect DNA.</li></p><br />
<p></p></td><br />
<td align="right" valign="top" width="20%"><table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="Real time" src="https://static.igem.org/mediawiki/2008/d/dd/Pv_pcr_parallel2.jpg" border="0" height="188" width="250"></span></div></td><br />
</tr><br />
<tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>MiniOpticon Real-Time PCR</span> </div></td><br />
</tr><br />
</tbody><br />
</table><br />
<table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="thermocycler" src="https://static.igem.org/mediawiki/2008/0/08/Pv_pcr_parallel1.jpg" border="0" height="188" width="250"></span></div></td><br />
</tr><br />
</tr><br />
<tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>Gene Amp PCR System 9700</span> </div></td><br />
</tr><br />
</tbody><br />
</table></td><br />
</tr><br />
</tbody><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<p><br><br />
</p><br />
<br />
<a name="Instruments_For_Electrophoresis"></a><br />
<h2> <span class="mw-headline">Instruments for electrophoresis</span></h2><br />
<table width="100%"><br />
<tbody><br />
<tr><br />
<td align="left" valign="top"><p></p><br />
<p align="justify"> Gel electrophoresis apparatus - Agarose gel is placed in this TBE buffer filled box and electrical field is applied via the power supply to the rear. The negative terminal is the black one while the positive is the red one. In this way DNA, that is naturally negatively charged, migrates to the positive pole. </p><br />
<p></p></td><br />
<td align="right" valign="top" width="20%"><table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="Instruments for electrophoresis" src="https://static.igem.org/mediawiki/2010/thumb/3/31/UNIPV_Pavia_electrophoresis.jpg/250px-UNIPV_Pavia_electrophoresis.jpg" border="0" height="188" width="250"></span></div></td><br />
</tr><br />
<tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>Large, medium and small electrophoresis apparatuses</span></div></td><br />
</tr><br />
</tbody><br />
</table></td><br />
</tr><br />
</tbody><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<p><br><br />
</p><br />
<br />
<a name="Gel_Image_Acquisition_System"></a><br />
<h2> <span class="mw-headline">Gel image acquisition system</span></h2><br />
<table width="100%"><br />
<tbody><br />
<tr><br />
<td align="left" valign="top"><p></p><br />
<p align="justify"> It is a UV system with camera used to acquire gel run snapshots. </p><br />
<p></p></td><br />
<td align="right" valign="top" width="20%"><table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="Gel image acquisition system" src="https://static.igem.org/mediawiki/2011/b/bb/Gelacquisition.jpg" border="0" height="188" width="250"></span></div></td><br />
</tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>UV system and camera</span> </div></td><br />
</tr><br />
<br />
</tbody><br />
</table></td><br />
</tr><br />
</tbody><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<p><br><br />
</p><br />
<br />
<br />
<a name="Incubator_For_Plates"></a><br />
<h2> <span class="mw-headline">Incubator for plates</span></h2><br />
<table width="100%"><br />
<tbody><br />
<tr><br />
<td align="left" valign="top"><p></p><br />
<p align="justify"> In order to grow bacteria a culture media is needed, together with plates (petri-dishes), some laboratory supplies and incubators.<br />
The incubator is an oven able to keep bacteria at the desired temperature; the typical temperature needed to allow a rapid growth of bacteria is 37 °C.<br />
</p><br />
<p></p></td><br />
<td align="right" valign="top" width="20%"><table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="Incubator for plates" src="https://static.igem.org/mediawiki/2010/thumb/0/0a/UNIPV_Pavia_incubatorforplates.jpg/250px-UNIPV_Pavia_incubatorforplates.jpg" border="0" height="188" width="250"></span></div></td><br />
</tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>Incubator for plates</span> </div></td><br />
</tr><br />
<br />
</tbody><br />
</table></td><br />
</tr><br />
</tbody><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<p><br><br />
</p><br />
<br />
<br />
<a name="Incubator_And_Shaker_For_Liquid_Cultures"></a><br />
<h2> <span class="mw-headline">Incubator and shaker for liquid cultures</span></h2><br />
<table width="100%"><br />
<tbody><br />
<tr><br />
<td align="left" valign="top"><p></p><br />
<p align="justify"> Shaking incubators, also known as environmental shakers, are often used for cell culturing, cell aeration and solubility studies. In addition to stable temperature conditions, they use an orbital agitation at variable speeds to affect the growth of cell cultures, displaced in a liquid media. All major functions (temperature, RPM and time) have alarms that alert the user to deviations from set parameters. Overtemperature protection is provided via a safety thermostat. Digital keypad operation offers the ability to calibrate the temperature controller to a reference thermometer.</p><br />
<p></p></td><br />
<td align="right" valign="top" width="20%"><table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="Incubator and shaker for liquid cultures" src="https://static.igem.org/mediawiki/2010/thumb/5/5f/UNIPV_Pavia_incubatorshaker.jpg/250px-UNIPV_Pavia_incubatorshaker.jpg" border="0" height="188" width="250"></span></div></td><br />
</tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>Incubator and shaker for liquid cultures</span> </div></td><br />
</tr><br />
</tbody><br />
</table></td><br />
</tr><br />
</tbody><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<p><br><br />
</p><br />
<br />
<br />
<a name="Hood"></a><br />
<h2> <span class="mw-headline">Laminar flow cabinet</span></h2><br />
<table width="100%"><br />
<tbody><br />
<tr><br />
<td align="left" valign="top"><p></p><br />
<p align="justify"> The lab is equipped with laminar flow cabinet. It is a carefully enclosed bench designed to prevent contamination of biological samples. Air is drawn through a HEPA filter and blown in a very smooth, laminar flow towards the user. The hood is provided of UV-C germicidal lamp for the sterilization. </p><br />
<p></p></td><br />
<td align="right" valign="top" width="20%"><table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="Hood" src="https://static.igem.org/mediawiki/2010/thumb/d/d4/UNIPV_Pavia_hood.jpg/250px-UNIPV_Pavia_hood.jpg" border="0" height="238" width="250"></span></div></td><br />
</tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>Laminar flow cabinet</span> </div></td><br />
</tr><br />
</tbody><br />
</table></td><br />
</tr><br />
</tbody><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<p><br><br />
</p><br />
<br />
<br />
<a name="Autoclave"></a><br />
<h2> <span class="mw-headline">Autoclave</span></h2><br />
<table width="100%"><br />
<tbody><br />
<tr><br />
<td align="left" valign="top"><p></p><br />
<p align="justify"> An autoclave is a self-locking apparatus used to sterilize equipment and supplies by subjecting them to high pressure saturated steam.<br />
It allows steam to flow around each article placed in the chamber. The vapor penetrates cloth or paper used to package the articles being sterilized. Autoclaving brings to the destruction of all types of microorganisms, including spores. Therefore, autoclave is widely used in microbiology, medicine, veterinary science, mycology or chiropody; its size varies on the media it is sterilizing.<br />
The amount of time and degree of temperature necessary for sterilization depend on the articles to be sterilized and whether they are wrapped or left directly exposed to the steam. Typically the process is run at 121°C or more for 15 to 20 minutes and it is essential to ensure that all the trapped air is removed, because hot air is less efficient than steam to achieve sterility.<br />
</p><br />
<p></p></td><br />
<td align="right" valign="top" width="20%"><table border="0"><br />
<tbody><br />
<tr><br />
<td><div class="floatright"><span><img alt="Autoclave" src="https://static.igem.org/mediawiki/2010/thumb/7/7d/UNIPV_Pavia_autoclave.jpg/250px-UNIPV_Pavia_autoclave.jpg" border="0" height="188" width="250"></span></div></td><br />
</tr><br />
<td><div style='text-align:center; font-size: 12px; font-style:italic;'><span>The autoclave</span> </div></td><br />
</tr><br />
</tbody><br />
</table></td><br />
</tr><br />
</tbody><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<p><br><br />
</p><br />
<br />
<br />
<br />
<br />
<br />
</html><br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/File:Gelacquisition.jpg
File:Gelacquisition.jpg
2011-09-21T20:12:23Z
<p>Edoardo Baldini: </p>
<hr />
<div></div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Parts/DataPage
Team:UNIPV-Pavia/Parts/DataPage
2011-09-21T20:09:26Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<html><br />
<h2 class="art-postheader"><br />
Data Page<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<br />
<p><a name="indice"/> </p><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/f/fc/QS-based_control_system.png"><img src="https://static.igem.org/mediawiki/2011/f/fc/QS-based_control_system.png" <br />
class="thumbimage" width="100%"></a></div></div> </div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<nowiki><div class="art-postcontent"><h1><span class="mw-headline"> <b>Data For Our Favorite New Parts</b> </span></h1></div></nowiki> <br />
<div style="text-align:justify"><br />
<br><br><br />
</div><br />
<br />
<br />
<br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>BBa_K516210</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
<br><br />
</div><br />
<br />
<div class="listcircle"><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/f/f5/LuxI_animazione.gif"><br />
<img src="https://static.igem.org/mediawiki/2011/f/f5/LuxI_animazione.gif" <br />
class="thumbimage" width="60%"></a></div></div> </div></div><br />
</td><br />
</tr><br />
</table><br />
<br><br><br />
<br />
<p align='justify'><br />
<A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_K516210">BBa_K516210</a> (p<sub>Tet</sub>-RBS30-LuxI) - HSL synthesis device, aTc inducible. <br> This is one of four systems with different RBSs (output modulation) tested to quantify the produced HSL. The results obtained by testing this part allowed the identification of important model parameters to predict the behavior of the whole CTRL+E circuit. <br />
</p><br />
<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
<br />
<br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>BBa_K516334</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
<br><br />
</div><br />
<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/d/d5/Plux_animazione.gif"><br />
<img src="https://static.igem.org/mediawiki/2011/d/d5/Plux_animazione.gif" <br />
class="thumbimage" width="60%"></a></div></div> </div></div><br />
</td><br />
</tr><br />
</table><br />
<p align='justify'><br />
<A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_K516334">BBa_K516334</a> (p<sub>Lambda</sub>-RBS30-LuxR-T-p<sub>Lux</sub>-RBS34-mRFP-TT) - 3OC<sub>6</sub>-HSL biosensor, mRFP output <br><br />
This is one of the four biosensors (different RBSs) built and characterized. This measurement system senses the HSL concentration in the medium and responds with an RFP output. Several RBSs have been used to modulate the output range. This is a simple measurement system, used to identify important model parameters used to simulate the behavior of the whole circuit. <br />
</p><br />
<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>BBa_K516230</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
<br><br />
</div><br />
<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/2011/1/1f/PTet_animazione.gif"><br />
<img src="https://static.igem.org/mediawiki/2011/1/1f/PTet_animazione.gif" <br />
class="thumbimage" width="60%"></a></div></div> </div></div><br />
</td><br />
</tr><br />
</table><br />
<br><br><br />
<p align='justify'><br />
<A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_K516230">BBa_K516230</a> (p<sub>Tet</sub>-RBS34-mRFP-TT) - aTc inducible, mRFP output <br><br />
This is one of the four p<sub>Tet</sub> measurement systems (different RBSs) built and characterized. This measurement system senses the aTc concentration in the medium and responds with an RFP output. Several RBSs have been used to modulate the output range. This is a simple measurement system, used to identify important model parameters used to simulate the behavior of the whole circuit. <br />
</p><br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
<nowiki><div class="art-postcontent"><h1><span class="mw-headline"> <b>Data For existing Parts</b> </span></h1></div></nowiki> <br />
<div style="text-align:justify"><br />
</div><br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>RBSs from the community collection</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
</div><br />
<br />
<p align='justify'><br />
<ol><br />
<ul><br />
<li><a href='http://partsregistry.org/Part:BBa_B0030:Experience'>Experience page of BBa_B0030</a> <br />
</li><br />
<li><a href='http://partsregistry.org/Part:BBa_B0031:Experience'>Experience page of BBa_B0031</a><br />
</li><br />
<li><a href='http://partsregistry.org/Part:BBa_B0032:Experience'>Experience page of BBa_B0032</a><br />
</li><br />
<li><a href='http://partsregistry.org/Part:BBa_B0034:Experience'>Experience page of BBa_B0034</a><br />
</li><br />
</ul><br />
</ol><br />
<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 />
</p><br />
<br><br><br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>BBa_K081022</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
</div><br />
<ol><ul><br><br />
<li><a href='http://partsregistry.org/Part:BBa_K081022:Experience'>Experience page of BBa_K081022</a><br />
</li></ul></ol><br><br />
<p align='justify'><br />
This device has been characterized with several RBSs in order to modulate the output range. In this way, this device can be used to tightly control the expression of downstream encoded protein from a very weak production (using weak RBS) to a very intensive production (with strong RBSs). <br><br>The Hill's estimated parameters are reported in the table below:<br />
<br><br><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><br />
The operative parameters are reported in the table below:<br />
<br><br><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 />
<br />
</p><br />
<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
<br />
<br />
<br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>BBa_R0040</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
<br><br />
</div><br />
<br />
<p align='justify'><br />
<ol><ul><br />
<li><a href='http://partsregistry.org/Part:BBa_R0040:Experience'>Experience page of BBa_R0040</a><br />
</li></ul></ol><br />
<br><br />
pTet promoter was characterized with various RBSs and mRFP. <br />
<br><br>The Hill's estimated parameters are reported in the table below:<br />
<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> [(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 />
<div align="center">Data are provided as average [CV%]</div><br />
<br><br><br />
The operative parameters are reported in the table below:<br />
<br><br><br />
<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 />
</p><br />
<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
<br />
<br />
<nowiki><div class="art-postcontent"><h1><span class="mw-headline"> <b>Improved existing parts</b> </span></h1></div></nowiki> <br />
<div style="text-align:justify"><br />
</div><br />
<br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>BBa_K516999</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
</div><br />
<br />
<ol><ul><br />
<li><a href='http://partsregistry.org/Part:BBa_K516999'>BBa_K516999</a><br />
</li></ul></ol><br />
<br><br><br />
<p align='justify'><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 />
This vector was designed and realized in order to facilitate the cloning of coding sequences downstream of the strong promoter p<sub>Tet</sub>.<br />
</p><br />
<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
<br />
<br />
<br />
<nowiki><div class="art-postcontent"><h1><span class="mw-headline"> <b>We've Also Characterized the Following Parts</b> </span></h1></div></nowiki> <br />
<div style="text-align:justify"><br />
<br><br />
</div><br />
For a detailed description of all the parts characterized, please visit the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized'>Parts Characterized</a> wiki page.<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
</html><br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Parts/DataPage
Team:UNIPV-Pavia/Parts/DataPage
2011-09-21T20:07:56Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<html><br />
<h2 class="art-postheader"><br />
Data Page<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<br />
<p><a name="indice"/> </p><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/f/fc/QS-based_control_system.png"><img src="https://static.igem.org/mediawiki/2011/f/fc/QS-based_control_system.png" <br />
class="thumbimage" width="100%"></a></div></div> </div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<nowiki><div class="art-postcontent"><h1><span class="mw-headline"> <b>Data For Our Favorite New Parts</b> </span></h1></div></nowiki> <br />
<div style="text-align:justify"><br />
<br><br><br />
</div><br />
<br />
<br />
<br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>BBa_K516210</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
<br><br />
</div><br />
<br />
<div class="listcircle"><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/f/f5/LuxI_animazione.gif"><br />
<img src="https://static.igem.org/mediawiki/2011/f/f5/LuxI_animazione.gif" <br />
class="thumbimage" width="60%"></a></div></div> </div></div><br />
</td><br />
</tr><br />
</table><br />
<br><br><br />
<br />
<p align='justify'><br />
<A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_K516210">BBa_K516210</a> (p<sub>Tet</sub>-RBS30-LuxI) - HSL synthesis device, aTc inducible. <br> This is one of four systems with different RBSs (output modulation) tested to quantify the produced HSL. The results obtained by testing this part allowed the identification of important model parameters to predict the behavior of the whole CTRL+E circuit. <br />
</p><br />
<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
<br />
<br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>BBa_K516334</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
<br><br />
</div><br />
<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/d/d5/Plux_animazione.gif"><br />
<img src="https://static.igem.org/mediawiki/2011/d/d5/Plux_animazione.gif" <br />
class="thumbimage" width="60%"></a></div></div> </div></div><br />
</td><br />
</tr><br />
</table><br />
<p align='justify'><br />
<A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_K516334">BBa_K516334</a> (p<sub>Lambda</sub>-RBS30-LuxR-T-p<sub>Lux</sub>-RBS34-mRFP-TT) - 3OC<sub>6</sub>-HSL biosensor, mRFP output <br><br />
This is one of the four biosensors (different RBSs) built and characterized. This measurement system senses the HSL concentration in the medium and responds with an RFP output. Several RBSs have been used to modulate the output range. This is a simple measurement system, used to identify important model parameters used to simulate the behavior of the whole circuit. <br />
</p><br />
<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>BBa_K516230</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
<br><br />
</div><br />
<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/2011/1/1f/PTet_animazione.gif"><br />
<img src="https://static.igem.org/mediawiki/2011/1/1f/PTet_animazione.gif" <br />
class="thumbimage" width="60%"></a></div></div> </div></div><br />
</td><br />
</tr><br />
</table><br />
<br><br><br />
<p align='justify'><br />
<A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_K516230">BBa_K516230</a> (p<sub>Tet</sub>-RBS34-mRFP-TT) - aTc inducible, mRFP output <br><br />
This is one of the four p<sub>Tet</sub> measurement systems (different RBSs) built and characterized. This measurement system senses the aTc concentration in the medium and responds with an RFP output. Several RBSs have been used to modulate the output range. This is a simple measurement system, used to identify important model parameters used to simulate the behavior of the whole circuit. <br />
</p><br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
<nowiki><div class="art-postcontent"><h1><span class="mw-headline"> <b>Data For existing Parts</b> </span></h1></div></nowiki> <br />
<div style="text-align:justify"><br />
</div><br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>RBSs from the community collection</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
</div><br />
<br />
<p align='justify'><br />
<ol><br />
<ul><br />
<li><a href='http://partsregistry.org/Part:BBa_B0030:Experience'>Experience page of BBa_B0030</a> <br />
</li><br />
<li><a href='http://partsregistry.org/Part:BBa_B0031:Experience'>Experience page of BBa_B0031</a><br />
</li><br />
<li><a href='http://partsregistry.org/Part:BBa_B0032:Experience'>Experience page of BBa_B0032</a><br />
</li><br />
<li><a href='http://partsregistry.org/Part:BBa_B0034:Experience'>Experience page of BBa_B0034</a><br />
</li><br />
</ul><br />
</ol><br />
<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 />
</p><br />
<br><br><br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>BBa_K081022</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
</div><br />
<ol><ul><br><br />
<li><a href='http://partsregistry.org/Part:BBa_K081022:Experience'>Experience page of BBa_K081022</a><br />
</li></ul></ol><br><br />
<p align='justify'><br />
This device has been characterized with several RBSs in order to modulate the output range. In this way, this device can be used to tightly control the expression of downstream encoded protein from a very weak production (using weak RBS) to a very intensive production (with strong RBSs). <br><br>The Hill's estimated parameters are reported in the table below:<br />
<br><br><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><br />
The operative parameters are reported in the table below:<br />
<br><br><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 />
<br />
</p><br />
<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
<br />
<br />
<br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>BBa_R0040</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
<br><br />
</div><br />
<br />
<p align='justify'><br />
<ol><ul><br />
<li><a href='http://partsregistry.org/Part:BBa_R0040:Experience'>Experience page of BBa_R0040</a><br />
</li></ul></ol><br />
<br><br />
pTet promoter was characterized with various RBSs and mRFP. <br />
<br><br>The Hill's estimated parameters are reported in the table below:<br />
<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> [(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 />
The operative parameters are reported in the table below:<br />
<br><br><br />
<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 />
</p><br />
<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
<br />
<br />
<nowiki><div class="art-postcontent"><h1><span class="mw-headline"> <b>Improved existing parts</b> </span></h1></div></nowiki> <br />
<div style="text-align:justify"><br />
</div><br />
<br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>BBa_K516999</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
</div><br />
<br />
<ol><ul><br />
<li><a href='http://partsregistry.org/Part:BBa_K516999'>BBa_K516999</a><br />
</li></ul></ol><br />
<br><br><br />
<p align='justify'><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 />
This vector was designed and realized in order to facilitate the cloning of coding sequences downstream of the strong promoter p<sub>Tet</sub>.<br />
</p><br />
<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
<br />
<br />
<br />
<nowiki><div class="art-postcontent"><h1><span class="mw-headline"> <b>We've Also Characterized the Following Parts</b> </span></h1></div></nowiki> <br />
<div style="text-align:justify"><br />
<br><br><br />
</div><br />
For a detailed description of all the parts characterized, please visit the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized'>Parts Characterized</a> wiki page.<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
</html><br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Parts/DataPage
Team:UNIPV-Pavia/Parts/DataPage
2011-09-21T20:06:33Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<html><br />
<h2 class="art-postheader"><br />
Data Page<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<br />
<p><a name="indice"/> </p><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/f/fc/QS-based_control_system.png"><img src="https://static.igem.org/mediawiki/2011/f/fc/QS-based_control_system.png" <br />
class="thumbimage" width="100%"></a></div></div> </div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<nowiki><div class="art-postcontent"><h1><span class="mw-headline"> <b>Data For Our Favorite New Parts</b> </span></h1></div></nowiki> <br />
<div style="text-align:justify"><br />
<br><br><br />
</div><br />
<br />
<br />
<br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>BBa_K516210</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
<br><br />
</div><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/f/f5/LuxI_animazione.gif"><br />
<img src="https://static.igem.org/mediawiki/2011/f/f5/LuxI_animazione.gif" <br />
class="thumbimage" width="60%"></a></div></div> </div></div><br />
</td><br />
</tr><br />
</table><br />
<br><br><br />
<br />
<p align='justify'><br />
<A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_K516210">BBa_K516210</a> (p<sub>Tet</sub>-RBS30-LuxI) - HSL synthesis device, aTc inducible. <br> This is one of four systems with different RBSs (output modulation) tested to quantify the produced HSL. The results obtained by testing this part allowed the identification of important model parameters to predict the behavior of the whole CTRL+E circuit. <br />
</p><br />
<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
<br />
<br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>BBa_K516334</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
<br><br />
</div><br />
<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/d/d5/Plux_animazione.gif"><br />
<img src="https://static.igem.org/mediawiki/2011/d/d5/Plux_animazione.gif" <br />
class="thumbimage" width="60%"></a></div></div> </div></div><br />
</td><br />
</tr><br />
</table><br />
<p align='justify'><br />
<A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_K516334">BBa_K516334</a> (p<sub>Lambda</sub>-RBS30-LuxR-T-p<sub>Lux</sub>-RBS34-mRFP-TT) - 3OC<sub>6</sub>-HSL biosensor, mRFP output <br><br />
This is one of the four biosensors (different RBSs) built and characterized. This measurement system senses the HSL concentration in the medium and responds with an RFP output. Several RBSs have been used to modulate the output range. This is a simple measurement system, used to identify important model parameters used to simulate the behavior of the whole circuit. <br />
</p><br />
<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>BBa_K516230</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
<br><br />
</div><br />
<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/2011/1/1f/PTet_animazione.gif"><br />
<img src="https://static.igem.org/mediawiki/2011/1/1f/PTet_animazione.gif" <br />
class="thumbimage" width="60%"></a></div></div> </div></div><br />
</td><br />
</tr><br />
</table><br />
<br><br><br />
<p align='justify'><br />
<A HREF="http://partsregistry.org/wiki/index.php/Part:BBa_K516230">BBa_K516230</a> (p<sub>Tet</sub>-RBS34-mRFP-TT) - aTc inducible, mRFP output <br><br />
This is one of the four p<sub>Tet</sub> measurement systems (different RBSs) built and characterized. This measurement system senses the aTc concentration in the medium and responds with an RFP output. Several RBSs have been used to modulate the output range. This is a simple measurement system, used to identify important model parameters used to simulate the behavior of the whole circuit. <br />
</p><br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
<nowiki><div class="art-postcontent"><h1><span class="mw-headline"> <b>Data For existing Parts</b> </span></h1></div></nowiki> <br />
<div style="text-align:justify"><br />
</div><br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>RBSs from the community collection</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
</div><br />
<br />
<p align='justify'><br />
<ol><br />
<ul><br />
<li><a href='http://partsregistry.org/Part:BBa_B0030:Experience'>Experience page of BBa_B0030</a> <br />
</li><br />
<li><a href='http://partsregistry.org/Part:BBa_B0031:Experience'>Experience page of BBa_B0031</a><br />
</li><br />
<li><a href='http://partsregistry.org/Part:BBa_B0032:Experience'>Experience page of BBa_B0032</a><br />
</li><br />
<li><a href='http://partsregistry.org/Part:BBa_B0034:Experience'>Experience page of BBa_B0034</a><br />
</li><br />
</ul><br />
</ol><br />
<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 />
</p><br />
<br><br><br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>BBa_K081022</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
</div><br />
<ol><ul><br><br><br />
<li><a href='http://partsregistry.org/Part:BBa_K081022:Experience'>Experience page of BBa_K081022</a><br />
</li></ul></ol><br><br><br />
<p align='justify'><br />
This device has been characterized with several RBSs in order to modulate the output range. In this way, this device can be used to tightly control the expression of downstream encoded protein from a very weak production (using weak RBS) to a very intensive production (with strong RBSs). <br><br>The Hill's estimated parameters are reported in the table below:<br />
<br><br><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><br />
The operative parameters are reported in the table below:<br />
<br><br><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 />
<br />
</p><br />
<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
<br />
<br />
<br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>BBa_R0040</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
<br><br><br />
</div><br />
<br />
<p align='justify'><br />
<ol><ul><br />
<li><a href='http://partsregistry.org/Part:BBa_R0040:Experience'>Experience page of BBa_R0040</a><br />
</li></ul></ol><br />
<br><br><br />
pTet promoter was characterized with various RBSs and mRFP. <br />
<br><br>The Hill's estimated parameters are reported in the table below:<br />
<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> [(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 />
The operative parameters are reported in the table below:<br />
<br><br><br />
<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 />
</p><br />
<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
<br />
<br />
<nowiki><div class="art-postcontent"><h1><span class="mw-headline"> <b>Improved existing parts</b> </span></h1></div></nowiki> <br />
<div style="text-align:justify"><br />
</div><br />
<br />
<nowiki><div class="art-postcontent"><h2><span class="mw-headline"> <b>BBa_K516999</b> </span></h2></div></nowiki> <br />
<div style="text-align:justify"><br />
</div><br />
<br />
<ol><ul><br />
<li><a href='http://partsregistry.org/Part:BBa_K516999'>BBa_K516999</a><br />
</li></ul></ol><br />
<br><br><br />
<p align='justify'><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 />
This vector was designed and realized in order to facilitate the cloning of coding sequences downstream of the strong promoter p<sub>Tet</sub>.<br />
</p><br />
<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
<br />
<br />
<br />
<nowiki><div class="art-postcontent"><h1><span class="mw-headline"> <b>We've Also Characterized the Following Parts</b> </span></h1></div></nowiki> <br />
<div style="text-align:justify"><br />
<br><br><br />
</div><br />
For a detailed description of all the parts characterized, please visit the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Parts/Characterized'>Parts Characterized</a> wiki page.<br />
<div align="right"><small><a href="#indice">^top</a></small></div><br><br><br />
<br />
</html><br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Results
Team:UNIPV-Pavia/Project/Results
2011-09-21T20:03:32Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<br />
<html><br />
<h2 class="art-postheader"> Results </h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"> <br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------MENU-----------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br><br />
<a name='indice'></a><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<table id="toc" class="toc"><br />
<tr><br />
<td><div id="toctitle"><br />
<h2>Contents</h2><br />
</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><br />
</tr><br />
</table><br />
</div><br />
<script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script> <br />
<br><em> 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. </em> <br><br />
<br><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------ASSEMBLY-------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='assembly'></a><br />
<h1>Parts assembly</h1><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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------CHARACTERIZATION---------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='characterization'></a><br />
<h1>Characterization of basic modules</h1><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------pTet and pLux-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='promoters'></a><br />
<h2>Characterization of promoters pTet and pLux</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><br />
<td class="row"><b>BioBrick code</b></td><br />
<td><b> Declared efficiency</b></td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0030 </td><br />
<td class="row"> 0,6</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0031 </td><br />
<td class="row"> 0,07</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0032 </td><br />
<td class="row"> 0,3</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0034 </td><br />
<td class="row"> 1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<div class="listcircle"><br />
<div align="justify"><br />
<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><br />
<p></p><br />
<p><br />
<ol><br />
<ul><br />
<li><b> RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li><b> RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li> <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 />
</p><br />
</li><br />
<p><br />
<li> <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.<br />
<br />
</li><br />
</p><br />
</ul><br />
</ol><br />
</p><br />
<div align="justify"> 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>. </div><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><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><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><br />
<td class='row'>4.28</td><br />
<td class='row'>0.20</td><br />
<td class='row'>1.08</td><br />
<td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>4.93</td><br />
<td class='row'>0.55</td><br />
<td class='row'>0.25</td><br />
<td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>9.49</td><br />
<td class='row'>0.02</td><br />
<td class='row'>0.47</td><br />
<td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>21.53</td><br />
<td class='row'>0.51</td><br />
<td class='row'>0.53</td><br />
<td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><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> 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><br />
<br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"> <img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><br />
<p align='justify'> The estimated parameters of the Hill curves described in the figures 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> [(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><br />
<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><br />
<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><br />
<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><br />
<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 />
<div align="center">Data are provided as average [CV%]</div> <br><br />
<br><br />
<p align='justify'> The operative parameters are summarized in the table below: </p><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><br />
<td class='row'>1.53</td><br />
<td class='row'>~0</td><br />
<td class='row'>7.95</td><br />
<td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>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><br />
<td class='row'>B0032</td><br />
<td class='row'>3.16</td><br />
<td class='row'>~0</td><br />
<td class='row'>6.7</td><br />
<td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>2.73</td><br />
<td class='row'>0.23</td><br />
<td class='row'>8.96</td><br />
<td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------AiiA and LuxI-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='enzymes'></a><br />
<h2>Characterization of enzymes AiiA and LuxI</h2><br />
<p align='justify'> 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 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 />
<div align="justify"><br />
<div class="thumbinner" style="width: 850px;"> <a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div><br />
</div><br />
<div align="justify"><br />
<p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p><br />
</div><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><br />
<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><br />
</table><br />
</div><br />
<br><br />
</p><br />
</p><br />
<p align='justify'> 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. </p><br />
<p align='justify'> The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity. </p><br />
<p align='justify'> 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 />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
</table><br />
<br />
<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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------RBS---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='rbs'></a><br />
<h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
RBSs were used for the fine tuning of CTRL+E. Different experimental conditions were assayed. <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 />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>0.40</td><br />
<td class='row'>1.6814</td><br />
<td class='row'>2.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.01</td><br />
<td class='row'>ND</td><br />
<td class='row'>0.04</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.19</td><br />
<td class='row'>0.4193</td><br />
<td class='row'>0.40</td><br />
<td class='row'>0,3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br><br />
Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT, with GeneX=mRFP, AiiA or LuxI: <br><br />
<br><br />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>1.72</td><br />
<td class='row'>0.53</td><br />
<td class='row'>0.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.03</td><br />
<td class='row'>0.83</td><br />
<td class='row'>0.028</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.37</td><br />
<td class='row'>0.50</td><br />
<td class='row'>N.D.</td><br />
<td class='row'>0.3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------growth---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='growth'></a><br />
<h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'> The bacterial growth curve has been modelled as a logistic curve and is represented by the following equation: <br><br />
<div align="center"><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br><br />
N(0)=n<sub>0</sub> <br></div><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> 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><br />
<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 class="disc"><br />
<li>1:1</li><br />
<li>1:10</li><br />
<li>1:100</li><br />
<li>1:1000</li><br />
</ul><br />
in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm. </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 />
</ul><br />
</ol><br />
<p>The results are summarized in the table and in the figure below: </p><br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><b> Culture </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><br />
<td class='row'><b> C.F.U. </b></td><br />
<td class='row'><b> Dilution Factor (10^) </b></td><br />
<td class='row'><b> N </b></td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,367950002 </td><br />
<td class='row'> 0,758004416 </td><br />
<td class='row'> 990 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 990000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,044649998 </td><br />
<td class='row'> 0,091982322 </td><br />
<td class='row'> 141 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 1410000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,004799999 </td><br />
<td class='row'> 0,009888356 </td><br />
<td class='row'> 136 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 136000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,000549998 </td><br />
<td class='row'> 0,001133037 </td><br />
<td class='row'> 20 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 200000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,54840003 </td><br />
<td class='row'> 1,129744917 </td><br />
<td class='row'> 165 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 16500000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,058200002 </td><br />
<td class='row'> 0,119896339 </td><br />
<td class='row'> 23 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 23000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,008700002 </td><br />
<td class='row'> 0,017922652 </td><br />
<td class='row'> 251 </td><br />
<td class='row'> 3 </td><br />
<td class='row'> 2510000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,000100002 </td><br />
<td class='row'> 0,000206011 </td><br />
<td class='row'> 24 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 2400000 </td><br />
</tr><br />
</table><br />
</center><br />
<br><br />
</p><br />
<center><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"><br />
</center><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 />
</p><br />
<center><br />
<table width='50%' class='data'><br />
<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><br />
<td class='row'>1*10<sup>9</sup></td><br />
<td class='row'>0.004925</td><br />
</tr><br />
</table><br />
</center><br />
<br />
<p>The reported value of &mu; corresponds to a doubling time of 142 minutes. </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 />
<a name='HSL'></a><br />
<h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 6</sub></b></td><br />
<td class='row'>32 </td><br />
<td class='row'>0.022 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 7</sub></b></td><br />
<td class='row'>8 </td><br />
<td class='row'>0.087 </td><br />
</tr><br />
</table><br />
</center><br />
<p> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 6</sub></b></td><br />
<td class='row'>&#8734; </td><br />
<td class='row'>0 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 7</sub></b></td><br />
<td class='row'>19 </td><br />
<td class='row'>0.037 </td><br />
</tr><br />
</table><br />
</center><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; </p><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------------t9002--------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='t9002'></a><br />
<h2>Characterization of BBa_T9002 biosensor</h2><br />
<div align="justify">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></div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage" width="47%" height="50%"></a></div><br />
</div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage" width="100%"></a></div><br />
</div><br />
<center><br />
<table width='50%' class='data'><br />
<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 />
<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 />
</tr><br />
</table><br />
</center><br />
<br><br />
<br><br />
<div align="justify">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.</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
</html><br />
<br />
<br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Results
Team:UNIPV-Pavia/Project/Results
2011-09-21T20:02:14Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<br />
<html><br />
<h2 class="art-postheader"> Results </h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"> <br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------MENU-----------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br><br />
<a name='indice'></a><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<table id="toc" class="toc"><br />
<tr><br />
<td><div id="toctitle"><br />
<h2>Contents</h2><br />
</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><br />
</tr><br />
</table><br />
</div><br />
<script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script> <br />
<br><em> 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. </em> <br><br />
<br><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------ASSEMBLY-------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='assembly'></a><br />
<h1>Parts assembly</h1><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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------CHARACTERIZATION---------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='characterization'></a><br />
<h1>Characterization of basic modules</h1><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------pTet and pLux-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='promoters'></a><br />
<h2>Characterization of promoters pTet and pLux</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><br />
<td class="row"><b>BioBrick code</b></td><br />
<td><b> Declared efficiency</b></td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0030 </td><br />
<td class="row"> 0,6</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0031 </td><br />
<td class="row"> 0,07</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0032 </td><br />
<td class="row"> 0,3</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0034 </td><br />
<td class="row"> 1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<div class="listcircle"><br />
<div align="justify"><br />
<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><br />
<p></p><br />
<p><br />
<ol><br />
<ul><br />
<li><b> RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li><b> RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li> <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 />
</p><br />
</li><br />
<p><br />
<li> <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.<br />
<br />
</li><br />
</p><br />
</ul><br />
</ol><br />
</p><br />
<p align='justify'> 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>. </p><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><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><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><br />
<td class='row'>4.28</td><br />
<td class='row'>0.20</td><br />
<td class='row'>1.08</td><br />
<td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>4.93</td><br />
<td class='row'>0.55</td><br />
<td class='row'>0.25</td><br />
<td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>9.49</td><br />
<td class='row'>0.02</td><br />
<td class='row'>0.47</td><br />
<td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>21.53</td><br />
<td class='row'>0.51</td><br />
<td class='row'>0.53</td><br />
<td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><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> 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><br />
<br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"> <img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><br />
<p align='justify'> The estimated parameters of the Hill curves described in the figures 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> [(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><br />
<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><br />
<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><br />
<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><br />
<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 />
<div align="center">Data are provided as average [CV%]</div> <br><br />
<br><br />
<p align='justify'> The operative parameters are summarized in the table below: </p><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><br />
<td class='row'>1.53</td><br />
<td class='row'>~0</td><br />
<td class='row'>7.95</td><br />
<td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>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><br />
<td class='row'>B0032</td><br />
<td class='row'>3.16</td><br />
<td class='row'>~0</td><br />
<td class='row'>6.7</td><br />
<td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>2.73</td><br />
<td class='row'>0.23</td><br />
<td class='row'>8.96</td><br />
<td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------AiiA and LuxI-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='enzymes'></a><br />
<h2>Characterization of enzymes AiiA and LuxI</h2><br />
<p align='justify'> 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 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 />
<div align="justify"><br />
<div class="thumbinner" style="width: 850px;"> <a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div><br />
</div><br />
<div align="justify"><br />
<p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p><br />
</div><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><br />
<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><br />
</table><br />
</div><br />
<br><br />
</p><br />
</p><br />
<p align='justify'> 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. </p><br />
<p align='justify'> The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity. </p><br />
<p align='justify'> 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 />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
</table><br />
<br />
<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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------RBS---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='rbs'></a><br />
<h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
RBSs were used for the fine tuning of CTRL+E. Different experimental conditions were assayed. <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 />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>0.40</td><br />
<td class='row'>1.6814</td><br />
<td class='row'>2.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.01</td><br />
<td class='row'>ND</td><br />
<td class='row'>0.04</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.19</td><br />
<td class='row'>0.4193</td><br />
<td class='row'>0.40</td><br />
<td class='row'>0,3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br><br />
Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT, with GeneX=mRFP, AiiA or LuxI: <br><br />
<br><br />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>1.72</td><br />
<td class='row'>0.53</td><br />
<td class='row'>0.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.03</td><br />
<td class='row'>0.83</td><br />
<td class='row'>0.028</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.37</td><br />
<td class='row'>0.50</td><br />
<td class='row'>N.D.</td><br />
<td class='row'>0.3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------growth---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='growth'></a><br />
<h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'> The bacterial growth curve has been modelled as a logistic curve and is represented by the following equation: <br><br />
<div align="center"><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br><br />
N(0)=n<sub>0</sub> <br></div><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> 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><br />
<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 class="disc"><br />
<li>1:1</li><br />
<li>1:10</li><br />
<li>1:100</li><br />
<li>1:1000</li><br />
</ul><br />
in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm. </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 />
</ul><br />
</ol><br />
<p>The results are summarized in the table and in the figure below: </p><br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><b> Culture </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><br />
<td class='row'><b> C.F.U. </b></td><br />
<td class='row'><b> Dilution Factor (10^) </b></td><br />
<td class='row'><b> N </b></td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,367950002 </td><br />
<td class='row'> 0,758004416 </td><br />
<td class='row'> 990 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 990000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,044649998 </td><br />
<td class='row'> 0,091982322 </td><br />
<td class='row'> 141 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 1410000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,004799999 </td><br />
<td class='row'> 0,009888356 </td><br />
<td class='row'> 136 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 136000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,000549998 </td><br />
<td class='row'> 0,001133037 </td><br />
<td class='row'> 20 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 200000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,54840003 </td><br />
<td class='row'> 1,129744917 </td><br />
<td class='row'> 165 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 16500000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,058200002 </td><br />
<td class='row'> 0,119896339 </td><br />
<td class='row'> 23 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 23000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,008700002 </td><br />
<td class='row'> 0,017922652 </td><br />
<td class='row'> 251 </td><br />
<td class='row'> 3 </td><br />
<td class='row'> 2510000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,000100002 </td><br />
<td class='row'> 0,000206011 </td><br />
<td class='row'> 24 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 2400000 </td><br />
</tr><br />
</table><br />
</center><br />
<br><br />
</p><br />
<center><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"><br />
</center><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 />
</p><br />
<center><br />
<table width='50%' class='data'><br />
<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><br />
<td class='row'>1*10<sup>9</sup></td><br />
<td class='row'>0.004925</td><br />
</tr><br />
</table><br />
</center><br />
<br />
<p>The reported value of &mu; corresponds to a doubling time of 142 minutes. </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 />
<a name='HSL'></a><br />
<h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 6</sub></b></td><br />
<td class='row'>32 </td><br />
<td class='row'>0.022 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 7</sub></b></td><br />
<td class='row'>8 </td><br />
<td class='row'>0.087 </td><br />
</tr><br />
</table><br />
</center><br />
<p> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 6</sub></b></td><br />
<td class='row'>&#8734; </td><br />
<td class='row'>0 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 7</sub></b></td><br />
<td class='row'>19 </td><br />
<td class='row'>0.037 </td><br />
</tr><br />
</table><br />
</center><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; </p><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------------t9002--------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='t9002'></a><br />
<h2>Characterization of BBa_T9002 biosensor</h2><br />
<div align="justify">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></div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage" width="47%" height="50%"></a></div><br />
</div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage" width="100%"></a></div><br />
</div><br />
<center><br />
<table width='50%' class='data'><br />
<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 />
<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 />
</tr><br />
</table><br />
</center><br />
<br><br />
<br><br />
<div align="justify">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.</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
</html><br />
<br />
<br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Results
Team:UNIPV-Pavia/Project/Results
2011-09-21T20:01:06Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<br />
<html><br />
<h2 class="art-postheader"> Results </h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"> <br />
<br />
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<br><br />
<a name='indice'></a><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<table id="toc" class="toc"><br />
<tr><br />
<td><div id="toctitle"><br />
<h2>Contents</h2><br />
</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><br />
</tr><br />
</table><br />
</div><br />
<script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script> <br />
<br><em> 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. </em> <br><br />
<br><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------ASSEMBLY-------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='assembly'></a><br />
<h1>Parts assembly</h1><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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------CHARACTERIZATION---------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='characterization'></a><br />
<h1>Characterization of basic modules</h1><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------pTet and pLux-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='promoters'></a><br />
<h2>Characterization of promoters pTet and pLux</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><br />
<td class="row"><b>BioBrick code</b></td><br />
<td><b> Declared efficiency</b></td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0030 </td><br />
<td class="row"> 0,6</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0031 </td><br />
<td class="row"> 0,07</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0032 </td><br />
<td class="row"> 0,3</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0034 </td><br />
<td class="row"> 1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<div class="listcircle"><br />
<div align="justify"><br />
<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><br />
<p></p><br />
<p><br />
<ol><br />
<ul><br />
<li><b> RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li><b> RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li> <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 />
</p><br />
</li><br />
<p><br />
<li> <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.<br />
<br />
</li><br />
</p><br />
</ul><br />
</ol><br />
</p><br />
<p align='justify'> 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>. </p><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><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><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><br />
<td class='row'>4.28</td><br />
<td class='row'>0.20</td><br />
<td class='row'>1.08</td><br />
<td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>4.93</td><br />
<td class='row'>0.55</td><br />
<td class='row'>0.25</td><br />
<td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>9.49</td><br />
<td class='row'>0.02</td><br />
<td class='row'>0.47</td><br />
<td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>21.53</td><br />
<td class='row'>0.51</td><br />
<td class='row'>0.53</td><br />
<td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><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> 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><br />
<br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"> <img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><br />
<p align='justify'> The estimated parameters of the Hill curves described in the figures 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> [(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><br />
<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><br />
<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><br />
<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><br />
<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 />
<p align='justify'> The operative parameters are summarized in the table below: </p><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><br />
<td class='row'>1.53</td><br />
<td class='row'>~0</td><br />
<td class='row'>7.95</td><br />
<td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>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><br />
<td class='row'>B0032</td><br />
<td class='row'>3.16</td><br />
<td class='row'>~0</td><br />
<td class='row'>6.7</td><br />
<td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>2.73</td><br />
<td class='row'>0.23</td><br />
<td class='row'>8.96</td><br />
<td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------AiiA and LuxI-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='enzymes'></a><br />
<h2>Characterization of enzymes AiiA and LuxI</h2><br />
<p align='justify'> 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 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 />
<div align="justify"><br />
<div class="thumbinner" style="width: 850px;"> <a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div><br />
</div><br />
<div align="justify"><br />
<p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p><br />
</div><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><br />
<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><br />
</table><br />
</div><br />
<br><br />
</p><br />
</p><br />
<p align='justify'> 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. </p><br />
<p align='justify'> The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity. </p><br />
<p align='justify'> 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 />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
</table><br />
<br />
<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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------RBS---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='rbs'></a><br />
<h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
RBSs were used for the fine tuning of CTRL+E. Different experimental conditions were assayed. <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 />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>0.40</td><br />
<td class='row'>1.6814</td><br />
<td class='row'>2.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.01</td><br />
<td class='row'>ND</td><br />
<td class='row'>0.04</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.19</td><br />
<td class='row'>0.4193</td><br />
<td class='row'>0.40</td><br />
<td class='row'>0,3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br><br />
Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT, with GeneX=mRFP, AiiA or LuxI: <br><br />
<br><br />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>1.72</td><br />
<td class='row'>0.53</td><br />
<td class='row'>0.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.03</td><br />
<td class='row'>0.83</td><br />
<td class='row'>0.028</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.37</td><br />
<td class='row'>0.50</td><br />
<td class='row'>N.D.</td><br />
<td class='row'>0.3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------growth---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='growth'></a><br />
<h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'> The bacterial growth curve has been modelled as a logistic curve and is represented by the following equation: <br><br />
<div align="center"><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br><br />
N(0)=n<sub>0</sub> <br></div><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> 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><br />
<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 class="disc"><br />
<li>1:1</li><br />
<li>1:10</li><br />
<li>1:100</li><br />
<li>1:1000</li><br />
</ul><br />
in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm. </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 />
</ul><br />
</ol><br />
<p>The results are summarized in the table and in the figure below: </p><br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><b> Culture </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><br />
<td class='row'><b> C.F.U. </b></td><br />
<td class='row'><b> Dilution Factor (10^) </b></td><br />
<td class='row'><b> N </b></td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,367950002 </td><br />
<td class='row'> 0,758004416 </td><br />
<td class='row'> 990 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 990000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,044649998 </td><br />
<td class='row'> 0,091982322 </td><br />
<td class='row'> 141 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 1410000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,004799999 </td><br />
<td class='row'> 0,009888356 </td><br />
<td class='row'> 136 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 136000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,000549998 </td><br />
<td class='row'> 0,001133037 </td><br />
<td class='row'> 20 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 200000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,54840003 </td><br />
<td class='row'> 1,129744917 </td><br />
<td class='row'> 165 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 16500000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,058200002 </td><br />
<td class='row'> 0,119896339 </td><br />
<td class='row'> 23 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 23000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,008700002 </td><br />
<td class='row'> 0,017922652 </td><br />
<td class='row'> 251 </td><br />
<td class='row'> 3 </td><br />
<td class='row'> 2510000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,000100002 </td><br />
<td class='row'> 0,000206011 </td><br />
<td class='row'> 24 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 2400000 </td><br />
</tr><br />
</table><br />
</center><br />
<br><br />
</p><br />
<center><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"><br />
</center><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 />
</p><p>&nbsp;</p><br />
<center><br />
<table width='50%' class='data'><br />
<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><br />
<td class='row'>1*10<sup>9</sup></td><br />
<td class='row'>0.004925</td><br />
</tr><br />
</table><br />
</center><br />
<br />
<p>The reported value of &mu; corresponds to a doubling time of 142 minutes. </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 />
<a name='HSL'></a><br />
<h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 6</sub></b></td><br />
<td class='row'>32 </td><br />
<td class='row'>0.022 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 7</sub></b></td><br />
<td class='row'>8 </td><br />
<td class='row'>0.087 </td><br />
</tr><br />
</table><br />
</center><br />
<p> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 6</sub></b></td><br />
<td class='row'>&#8734; </td><br />
<td class='row'>0 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 7</sub></b></td><br />
<td class='row'>19 </td><br />
<td class='row'>0.037 </td><br />
</tr><br />
</table><br />
</center><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; </p><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------------t9002--------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='t9002'></a><br />
<h2>Characterization of BBa_T9002 biosensor</h2><br />
<div align="justify">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></div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage" width="47%" height="50%"></a></div><br />
</div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage" width="100%"></a></div><br />
</div><br />
<center><br />
<table width='50%' class='data'><br />
<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 />
<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 />
</tr><br />
</table><br />
</center><br />
<br><br />
<br><br />
<div align="justify">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.</div><br />
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<br />
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</html><br />
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Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Results
Team:UNIPV-Pavia/Project/Results
2011-09-21T19:59:59Z
<p>Edoardo Baldini: </p>
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<h2 class="art-postheader"> Results </h2><br />
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<h2>Contents</h2><br />
</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 />
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<br><em> 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. </em> <br><br />
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<a name='assembly'></a><br />
<h1>Parts assembly</h1><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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<a name='characterization'></a><br />
<h1>Characterization of basic modules</h1><br />
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<!--------pTet and pLux-----------> <br />
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<a name='promoters'></a><br />
<h2>Characterization of promoters pTet and pLux</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><br />
<td class="row"><b>BioBrick code</b></td><br />
<td><b> Declared efficiency</b></td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0030 </td><br />
<td class="row"> 0,6</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0031 </td><br />
<td class="row"> 0,07</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0032 </td><br />
<td class="row"> 0,3</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0034 </td><br />
<td class="row"> 1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<div class="listcircle"><br />
<div align="justify"><br />
<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><br />
<p></p><br />
<p><br />
<ol><br />
<ul><br />
<li><b> RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li><b> RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li> <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 />
</p><br />
</li><br />
<p><br />
<li> <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.<br />
<br />
</li><br />
</p><br />
</ul><br />
</ol><br />
</p><br />
<p align='justify'> 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>. </p><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><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><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><br />
<td class='row'>4.28</td><br />
<td class='row'>0.20</td><br />
<td class='row'>1.08</td><br />
<td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>4.93</td><br />
<td class='row'>0.55</td><br />
<td class='row'>0.25</td><br />
<td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>9.49</td><br />
<td class='row'>0.02</td><br />
<td class='row'>0.47</td><br />
<td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>21.53</td><br />
<td class='row'>0.51</td><br />
<td class='row'>0.53</td><br />
<td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><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> 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><br />
<br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"> <img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><br />
<p align='justify'> The estimated parameters of the Hill curves described in the figures 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> [(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><br />
<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><br />
<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><br />
<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><br />
<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 />
<p align='justify'> The operative parameters are summarized in the table below: </p><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><br />
<td class='row'>1.53</td><br />
<td class='row'>~0</td><br />
<td class='row'>7.95</td><br />
<td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>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><br />
<td class='row'>B0032</td><br />
<td class='row'>3.16</td><br />
<td class='row'>~0</td><br />
<td class='row'>6.7</td><br />
<td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>2.73</td><br />
<td class='row'>0.23</td><br />
<td class='row'>8.96</td><br />
<td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<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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<br />
<a name='enzymes'></a><br />
<h2>Characterization of enzymes AiiA and LuxI</h2><br />
<p align='justify'> 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 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 />
<div align="justify"><br />
<div class="thumbinner" style="width: 850px;"> <a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div><br />
</div><br />
<div align="justify"><br />
<p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p><br />
</div><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><br />
<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><br />
</table><br />
</div><br />
<br><br />
</p><br />
</p><br />
<p align='justify'> 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. </p><br />
<p align='justify'> The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity. </p><br />
<p align='justify'> 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 />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
</table><br />
<br />
<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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------RBS---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='rbs'></a><br />
<h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
RBSs were used for the fine tuning of CTRL+E. Different experimental conditions were assayed. <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 />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>0.40</td><br />
<td class='row'>1.6814</td><br />
<td class='row'>2.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.01</td><br />
<td class='row'>ND</td><br />
<td class='row'>0.04</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.19</td><br />
<td class='row'>0.4193</td><br />
<td class='row'>0.40</td><br />
<td class='row'>0,3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br><br />
Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT, with GeneX=mRFP, AiiA or LuxI: <br><br />
<br><br />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>1.72</td><br />
<td class='row'>0.53</td><br />
<td class='row'>0.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.03</td><br />
<td class='row'>0.83</td><br />
<td class='row'>0.028</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.37</td><br />
<td class='row'>0.50</td><br />
<td class='row'>N.D.</td><br />
<td class='row'>0.3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------growth---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='growth'></a><br />
<h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'> The bacterial growth curve has been modelled as a logistic curve and is represented by the following equation: <br><br />
<div align="center"><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br><br />
N(0)=n<sub>0</sub> <br></div><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> 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><br />
<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 class="disc"><br />
<li>1:1</li><br />
<li>1:10</li><br />
<li>1:100</li><br />
<li>1:1000</li><br />
</ul><br />
in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm. </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 />
</ul><br />
</ol><br />
<p>The results are summarized in the table and in the figure below: </p><br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><b> Culture </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><br />
<td class='row'><b> C.F.U. </b></td><br />
<td class='row'><b> Dilution Factor (10^) </b></td><br />
<td class='row'><b> N </b></td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,367950002 </td><br />
<td class='row'> 0,758004416 </td><br />
<td class='row'> 990 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 990000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,044649998 </td><br />
<td class='row'> 0,091982322 </td><br />
<td class='row'> 141 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 1410000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,004799999 </td><br />
<td class='row'> 0,009888356 </td><br />
<td class='row'> 136 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 136000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,000549998 </td><br />
<td class='row'> 0,001133037 </td><br />
<td class='row'> 20 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 200000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,54840003 </td><br />
<td class='row'> 1,129744917 </td><br />
<td class='row'> 165 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 16500000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,058200002 </td><br />
<td class='row'> 0,119896339 </td><br />
<td class='row'> 23 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 23000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,008700002 </td><br />
<td class='row'> 0,017922652 </td><br />
<td class='row'> 251 </td><br />
<td class='row'> 3 </td><br />
<td class='row'> 2510000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,000100002 </td><br />
<td class='row'> 0,000206011 </td><br />
<td class='row'> 24 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 2400000 </td><br />
</tr><br />
</table><br />
</center><br />
<br><br />
</p><br />
<center><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"><br />
</center><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 />
</p><br />
<center><br />
<table width='50%' class='data'><br />
<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><br />
<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. </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 />
<a name='HSL'></a><br />
<h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 6</sub></b></td><br />
<td class='row'>32 </td><br />
<td class='row'>0.022 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 7</sub></b></td><br />
<td class='row'>8 </td><br />
<td class='row'>0.087 </td><br />
</tr><br />
</table><br />
</center><br />
<p> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 6</sub></b></td><br />
<td class='row'>&#8734; </td><br />
<td class='row'>0 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 7</sub></b></td><br />
<td class='row'>19 </td><br />
<td class='row'>0.037 </td><br />
</tr><br />
</table><br />
</center><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; </p><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------------t9002--------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='t9002'></a><br />
<h2>Characterization of BBa_T9002 biosensor</h2><br />
<div align="justify">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></div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage" width="47%" height="50%"></a></div><br />
</div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage" width="100%"></a></div><br />
</div><br />
<center><br />
<table width='50%' class='data'><br />
<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 />
<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 />
</tr><br />
</table><br />
</center><br />
<br><br />
<br><br />
<div align="justify">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.</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
</html><br />
<br />
<br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Results
Team:UNIPV-Pavia/Project/Results
2011-09-21T19:57:48Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<br />
<html><br />
<h2 class="art-postheader"> Results </h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"> <br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------MENU-----------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br><br />
<a name='indice'></a><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<table id="toc" class="toc"><br />
<tr><br />
<td><div id="toctitle"><br />
<h2>Contents</h2><br />
</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><br />
</tr><br />
</table><br />
</div><br />
<script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script> <br />
<br><em> 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. </em> <br><br />
<br><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------ASSEMBLY-------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='assembly'></a><br />
<h1>Parts assembly</h1><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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------CHARACTERIZATION---------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='characterization'></a><br />
<h1>Characterization of basic modules</h1><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------pTet and pLux-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='promoters'></a><br />
<h2>Characterization of promoters pTet and pLux</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><br />
<td class="row"><b>BioBrick code</b></td><br />
<td><b> Declared efficiency</b></td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0030 </td><br />
<td class="row"> 0,6</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0031 </td><br />
<td class="row"> 0,07</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0032 </td><br />
<td class="row"> 0,3</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0034 </td><br />
<td class="row"> 1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<div class="listcircle"><br />
<div align="justify"><br />
<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><br />
<p></p><br />
<p><br />
<ol><br />
<ul><br />
<li><b> RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li><b> RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li> <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 />
</p><br />
</li><br />
<p><br />
<li> <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.<br />
<br />
</li><br />
</p><br />
</ul><br />
</ol><br />
</p><br />
<p align='justify'> 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>. </p><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><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><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><br />
<td class='row'>4.28</td><br />
<td class='row'>0.20</td><br />
<td class='row'>1.08</td><br />
<td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>4.93</td><br />
<td class='row'>0.55</td><br />
<td class='row'>0.25</td><br />
<td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>9.49</td><br />
<td class='row'>0.02</td><br />
<td class='row'>0.47</td><br />
<td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>21.53</td><br />
<td class='row'>0.51</td><br />
<td class='row'>0.53</td><br />
<td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><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> 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><br />
<br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"> <img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><br />
<p align='justify'> The estimated parameters of the Hill curves described in the figures 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> [(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><br />
<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><br />
<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><br />
<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><br />
<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 />
<p align='justify'> The operative parameters are summarized in the table below: </p><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><br />
<td class='row'>1.53</td><br />
<td class='row'>~0</td><br />
<td class='row'>7.95</td><br />
<td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>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><br />
<td class='row'>B0032</td><br />
<td class='row'>3.16</td><br />
<td class='row'>~0</td><br />
<td class='row'>6.7</td><br />
<td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>2.73</td><br />
<td class='row'>0.23</td><br />
<td class='row'>8.96</td><br />
<td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------AiiA and LuxI-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='enzymes'></a><br />
<h2>Characterization of enzymes AiiA and LuxI</h2><br />
<p align='justify'> 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 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 />
<div align="justify"><br />
<div class="thumbinner" style="width: 850px;"> <a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div><br />
</div><br />
<div align="justify"><br />
<p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p><br />
</div><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><br />
<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><br />
</table><br />
</div><br />
<br><br />
</p><br />
</p><br />
<p align='justify'> 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. </p><br />
<p align='justify'> The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity. </p><br />
<p align='justify'> 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 />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
</table><br />
<br />
<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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------RBS---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='rbs'></a><br />
<h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
RBSs were used for the fine tuning of CTRL+E. Different experimental conditions were assayed. <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 />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>0.40</td><br />
<td class='row'>1.6814</td><br />
<td class='row'>2.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.01</td><br />
<td class='row'>ND</td><br />
<td class='row'>0.04</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.19</td><br />
<td class='row'>0.4193</td><br />
<td class='row'>0.40</td><br />
<td class='row'>0,3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br><br />
Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT, with GeneX=mRFP, AiiA or LuxI: <br><br />
<br><br />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>1.72</td><br />
<td class='row'>0.53</td><br />
<td class='row'>0.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.03</td><br />
<td class='row'>0.83</td><br />
<td class='row'>0.028</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.37</td><br />
<td class='row'>0.50</td><br />
<td class='row'>N.D.</td><br />
<td class='row'>0.3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------growth---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='growth'></a><br />
<h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'> The bacterial growth curve has been modelled as a logistic curve and is represented by the following equation: <br><br />
<div align="center"><br><br />
dN/dt=N*&mu;*(N<sub>max</sub>-N)/N<sub>max</sub><br><br />
N(0)=n<sub>0</sub> <br></div><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> 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><br />
<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><br />
<li>1:1</li><br />
<li>1:10</li><br />
<li>1:100</li><br />
<li>1:1000</li><br />
</ul><br />
in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm. </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 />
</ul><br />
</ol><br />
<p>The results are summarized in the table and in the figure below: </p><br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><b> Culture </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><br />
<td class='row'><b> C.F.U. </b></td><br />
<td class='row'><b> Dilution Factor (10^) </b></td><br />
<td class='row'><b> N </b></td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,367950002 </td><br />
<td class='row'> 0,758004416 </td><br />
<td class='row'> 990 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 990000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,044649998 </td><br />
<td class='row'> 0,091982322 </td><br />
<td class='row'> 141 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 1410000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,004799999 </td><br />
<td class='row'> 0,009888356 </td><br />
<td class='row'> 136 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 136000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,000549998 </td><br />
<td class='row'> 0,001133037 </td><br />
<td class='row'> 20 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 200000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,54840003 </td><br />
<td class='row'> 1,129744917 </td><br />
<td class='row'> 165 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 16500000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,058200002 </td><br />
<td class='row'> 0,119896339 </td><br />
<td class='row'> 23 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 23000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,008700002 </td><br />
<td class='row'> 0,017922652 </td><br />
<td class='row'> 251 </td><br />
<td class='row'> 3 </td><br />
<td class='row'> 2510000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,000100002 </td><br />
<td class='row'> 0,000206011 </td><br />
<td class='row'> 24 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 2400000 </td><br />
</tr><br />
</table><br />
</center><br />
<br><br />
</p><br />
<center><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"><br />
</center><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 />
</p><br />
<center><br />
<table width='50%' class='data'><br />
<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><br />
<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. </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 />
<a name='HSL'></a><br />
<h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 6</sub></b></td><br />
<td class='row'>32 </td><br />
<td class='row'>0.022 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 7</sub></b></td><br />
<td class='row'>8 </td><br />
<td class='row'>0.087 </td><br />
</tr><br />
</table><br />
</center><br />
<p> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 6</sub></b></td><br />
<td class='row'>&#8734; </td><br />
<td class='row'>0 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 7</sub></b></td><br />
<td class='row'>19 </td><br />
<td class='row'>0.037 </td><br />
</tr><br />
</table><br />
</center><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; </p><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------------t9002--------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='t9002'></a><br />
<h2>Characterization of BBa_T9002 biosensor</h2><br />
<div align="justify">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></div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage" width="47%" height="50%"></a></div><br />
</div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage" width="100%"></a></div><br />
</div><br />
<center><br />
<table width='50%' class='data'><br />
<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 />
<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 />
</tr><br />
</table><br />
</center><br />
<br><br />
<br><br />
<div align="justify">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.</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
</html><br />
<br />
<br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Results
Team:UNIPV-Pavia/Project/Results
2011-09-21T19:56:41Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<br />
<html><br />
<h2 class="art-postheader"> Results </h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"> <br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------MENU-----------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br><br />
<a name='indice'></a><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<table id="toc" class="toc"><br />
<tr><br />
<td><div id="toctitle"><br />
<h2>Contents</h2><br />
</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><br />
</tr><br />
</table><br />
</div><br />
<script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script> <br />
<br><em> 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. </em> <br><br />
<br><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------ASSEMBLY-------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='assembly'></a><br />
<h1>Parts assembly</h1><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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------CHARACTERIZATION---------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='characterization'></a><br />
<h1>Characterization of basic modules</h1><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------pTet and pLux-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='promoters'></a><br />
<h2>Characterization of promoters pTet and pLux</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><br />
<td class="row"><b>BioBrick code</b></td><br />
<td><b> Declared efficiency</b></td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0030 </td><br />
<td class="row"> 0,6</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0031 </td><br />
<td class="row"> 0,07</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0032 </td><br />
<td class="row"> 0,3</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0034 </td><br />
<td class="row"> 1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<div class="listcircle"><br />
<div align="justify"><br />
<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><br />
<p></p><br />
<p><br />
<ol><br />
<ul><br />
<li><b> RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li><b> RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li> <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 />
</p><br />
</li><br />
<p><br />
<li> <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.<br />
<br />
</li><br />
</p><br />
</ul><br />
</ol><br />
</p><br />
<p align='justify'> 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>. </p><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><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><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><br />
<td class='row'>4.28</td><br />
<td class='row'>0.20</td><br />
<td class='row'>1.08</td><br />
<td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>4.93</td><br />
<td class='row'>0.55</td><br />
<td class='row'>0.25</td><br />
<td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>9.49</td><br />
<td class='row'>0.02</td><br />
<td class='row'>0.47</td><br />
<td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>21.53</td><br />
<td class='row'>0.51</td><br />
<td class='row'>0.53</td><br />
<td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><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> 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><br />
<br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"> <img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><br />
<p align='justify'> The estimated parameters of the Hill curves described in the figures 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> [(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><br />
<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><br />
<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><br />
<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><br />
<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 />
<p align='justify'> The operative parameters are summarized in the table below: </p><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><br />
<td class='row'>1.53</td><br />
<td class='row'>~0</td><br />
<td class='row'>7.95</td><br />
<td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>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><br />
<td class='row'>B0032</td><br />
<td class='row'>3.16</td><br />
<td class='row'>~0</td><br />
<td class='row'>6.7</td><br />
<td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>2.73</td><br />
<td class='row'>0.23</td><br />
<td class='row'>8.96</td><br />
<td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------AiiA and LuxI-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='enzymes'></a><br />
<h2>Characterization of enzymes AiiA and LuxI</h2><br />
<p align='justify'> 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 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 />
<div align="justify"><br />
<div class="thumbinner" style="width: 850px;"> <a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div><br />
</div><br />
<div align="justify"><br />
<p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p><br />
</div><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><br />
<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><br />
</table><br />
</div><br />
<br><br />
</p><br />
</p><br />
<p align='justify'> 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. </p><br />
<p align='justify'> The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity. </p><br />
<p align='justify'> 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 />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
</table><br />
<br />
<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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------RBS---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='rbs'></a><br />
<h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
RBSs were used for the fine tuning of CTRL+E. Different experimental conditions were assayed. <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 />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>0.40</td><br />
<td class='row'>1.6814</td><br />
<td class='row'>2.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.01</td><br />
<td class='row'>ND</td><br />
<td class='row'>0.04</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.19</td><br />
<td class='row'>0.4193</td><br />
<td class='row'>0.40</td><br />
<td class='row'>0,3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br><br />
Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT, with GeneX=mRFP, AiiA or LuxI: <br><br />
<br><br />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>1.72</td><br />
<td class='row'>0.53</td><br />
<td class='row'>0.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.03</td><br />
<td class='row'>0.83</td><br />
<td class='row'>0.028</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.37</td><br />
<td class='row'>0.50</td><br />
<td class='row'>N.D.</td><br />
<td class='row'>0.3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------growth---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='growth'></a><br />
<h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'> 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 />
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> 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><br />
<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><br />
<li>1:1</li><br />
<li>1:10</li><br />
<li>1:100</li><br />
<li>1:1000</li><br />
</ul><br />
in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm. </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 />
</ul><br />
</ol><br />
<p>The results are summarized in the table and in the figure below: </p><br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><b> Culture </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><br />
<td class='row'><b> C.F.U. </b></td><br />
<td class='row'><b> Dilution Factor (10^) </b></td><br />
<td class='row'><b> N </b></td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,367950002 </td><br />
<td class='row'> 0,758004416 </td><br />
<td class='row'> 990 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 990000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,044649998 </td><br />
<td class='row'> 0,091982322 </td><br />
<td class='row'> 141 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 1410000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,004799999 </td><br />
<td class='row'> 0,009888356 </td><br />
<td class='row'> 136 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 136000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,000549998 </td><br />
<td class='row'> 0,001133037 </td><br />
<td class='row'> 20 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 200000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,54840003 </td><br />
<td class='row'> 1,129744917 </td><br />
<td class='row'> 165 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 16500000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,058200002 </td><br />
<td class='row'> 0,119896339 </td><br />
<td class='row'> 23 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 23000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,008700002 </td><br />
<td class='row'> 0,017922652 </td><br />
<td class='row'> 251 </td><br />
<td class='row'> 3 </td><br />
<td class='row'> 2510000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,000100002 </td><br />
<td class='row'> 0,000206011 </td><br />
<td class='row'> 24 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 2400000 </td><br />
</tr><br />
</table><br />
</center><br />
<br><br />
</p><br />
<center><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"><br />
</center><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 />
</p><br />
<center><br />
<table width='50%' class='data'><br />
<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><br />
<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. </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 />
<a name='HSL'></a><br />
<h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 6</sub></b></td><br />
<td class='row'>32 </td><br />
<td class='row'>0.022 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 7</sub></b></td><br />
<td class='row'>8 </td><br />
<td class='row'>0.087 </td><br />
</tr><br />
</table><br />
</center><br />
<p> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 6</sub></b></td><br />
<td class='row'>&#8734; </td><br />
<td class='row'>0 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 7</sub></b></td><br />
<td class='row'>19 </td><br />
<td class='row'>0.037 </td><br />
</tr><br />
</table><br />
</center><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; </p><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<br />
<a name='t9002'></a><br />
<h2>Characterization of BBa_T9002 biosensor</h2><br />
<div align="justify">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></div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage" width="47%" height="50%"></a></div><br />
</div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage" width="100%"></a></div><br />
</div><br />
<center><br />
<table width='50%' class='data'><br />
<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 />
<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 />
</tr><br />
</table><br />
</center><br />
<br><br />
<br><br />
<div align="justify">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.</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Results
Team:UNIPV-Pavia/Project/Results
2011-09-21T19:55:35Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<br />
<html><br />
<h2 class="art-postheader"> Results </h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"> <br />
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<a name='indice'></a><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<table id="toc" class="toc"><br />
<tr><br />
<td><div id="toctitle"><br />
<h2>Contents</h2><br />
</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><br />
</tr><br />
</table><br />
</div><br />
<script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script> <br />
<em> 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. </em> <br><br />
<br><br />
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<br />
<a name='assembly'></a><br />
<h1>Parts assembly</h1><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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<br />
<a name='characterization'></a><br />
<h1>Characterization of basic modules</h1><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------pTet and pLux-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='promoters'></a><br />
<h2>Characterization of promoters pTet and pLux</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><br />
<td class="row"><b>BioBrick code</b></td><br />
<td><b> Declared efficiency</b></td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0030 </td><br />
<td class="row"> 0,6</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0031 </td><br />
<td class="row"> 0,07</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0032 </td><br />
<td class="row"> 0,3</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0034 </td><br />
<td class="row"> 1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<div class="listcircle"><br />
<div align="justify"><br />
<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><br />
<p></p><br />
<p><br />
<ol><br />
<ul><br />
<li><b> RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li><b> RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li> <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 />
</p><br />
</li><br />
<p><br />
<li> <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.<br />
<br />
</li><br />
</p><br />
</ul><br />
</ol><br />
</p><br />
<p align='justify'> 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>. </p><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><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><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><br />
<td class='row'>4.28</td><br />
<td class='row'>0.20</td><br />
<td class='row'>1.08</td><br />
<td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>4.93</td><br />
<td class='row'>0.55</td><br />
<td class='row'>0.25</td><br />
<td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>9.49</td><br />
<td class='row'>0.02</td><br />
<td class='row'>0.47</td><br />
<td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>21.53</td><br />
<td class='row'>0.51</td><br />
<td class='row'>0.53</td><br />
<td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><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> 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><br />
<br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"> <img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><br />
<p align='justify'> The estimated parameters of the Hill curves described in the figures 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> [(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><br />
<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><br />
<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><br />
<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><br />
<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 />
<p align='justify'> The operative parameters are summarized in the table below: </p><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><br />
<td class='row'>1.53</td><br />
<td class='row'>~0</td><br />
<td class='row'>7.95</td><br />
<td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>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><br />
<td class='row'>B0032</td><br />
<td class='row'>3.16</td><br />
<td class='row'>~0</td><br />
<td class='row'>6.7</td><br />
<td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>2.73</td><br />
<td class='row'>0.23</td><br />
<td class='row'>8.96</td><br />
<td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!--------------------------------> <br />
<!--------AiiA and LuxI-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='enzymes'></a><br />
<h2>Characterization of enzymes AiiA and LuxI</h2><br />
<p align='justify'> 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 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 />
<div align="justify"><br />
<div class="thumbinner" style="width: 850px;"> <a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div><br />
</div><br />
<div align="justify"><br />
<p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p><br />
</div><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><br />
<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><br />
</table><br />
</div><br />
<br><br />
</p><br />
</p><br />
<p align='justify'> 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. </p><br />
<p align='justify'> The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity. </p><br />
<p align='justify'> 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 />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
</table><br />
<br />
<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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------RBS---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='rbs'></a><br />
<h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
RBSs were used for the fine tuning of CTRL+E. Different experimental conditions were assayed. <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 />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>0.40</td><br />
<td class='row'>1.6814</td><br />
<td class='row'>2.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.01</td><br />
<td class='row'>ND</td><br />
<td class='row'>0.04</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.19</td><br />
<td class='row'>0.4193</td><br />
<td class='row'>0.40</td><br />
<td class='row'>0,3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br><br />
Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT, with GeneX=mRFP, AiiA or LuxI: <br><br />
<br><br />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>1.72</td><br />
<td class='row'>0.53</td><br />
<td class='row'>0.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.03</td><br />
<td class='row'>0.83</td><br />
<td class='row'>0.028</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.37</td><br />
<td class='row'>0.50</td><br />
<td class='row'>N.D.</td><br />
<td class='row'>0.3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------growth---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='growth'></a><br />
<h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'> 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 />
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> 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><br />
<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><br />
<li>1:1</li><br />
<li>1:10</li><br />
<li>1:100</li><br />
<li>1:1000</li><br />
</ul><br />
in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm. </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 />
</ul><br />
</ol><br />
<p>The results are summarized in the table and in the figure below: </p><br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><b> Culture </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><br />
<td class='row'><b> C.F.U. </b></td><br />
<td class='row'><b> Dilution Factor (10^) </b></td><br />
<td class='row'><b> N </b></td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,367950002 </td><br />
<td class='row'> 0,758004416 </td><br />
<td class='row'> 990 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 990000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,044649998 </td><br />
<td class='row'> 0,091982322 </td><br />
<td class='row'> 141 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 1410000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,004799999 </td><br />
<td class='row'> 0,009888356 </td><br />
<td class='row'> 136 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 136000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,000549998 </td><br />
<td class='row'> 0,001133037 </td><br />
<td class='row'> 20 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 200000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,54840003 </td><br />
<td class='row'> 1,129744917 </td><br />
<td class='row'> 165 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 16500000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,058200002 </td><br />
<td class='row'> 0,119896339 </td><br />
<td class='row'> 23 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 23000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,008700002 </td><br />
<td class='row'> 0,017922652 </td><br />
<td class='row'> 251 </td><br />
<td class='row'> 3 </td><br />
<td class='row'> 2510000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,000100002 </td><br />
<td class='row'> 0,000206011 </td><br />
<td class='row'> 24 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 2400000 </td><br />
</tr><br />
</table><br />
</center><br />
<br><br />
</p><br />
<center><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"><br />
</center><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 />
</p><br />
<center><br />
<table width='50%' class='data'><br />
<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><br />
<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. </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 />
<a name='HSL'></a><br />
<h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 6</sub></b></td><br />
<td class='row'>32 </td><br />
<td class='row'>0.022 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 7</sub></b></td><br />
<td class='row'>8 </td><br />
<td class='row'>0.087 </td><br />
</tr><br />
</table><br />
</center><br />
<p> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 6</sub></b></td><br />
<td class='row'>&#8734; </td><br />
<td class='row'>0 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 7</sub></b></td><br />
<td class='row'>19 </td><br />
<td class='row'>0.037 </td><br />
</tr><br />
</table><br />
</center><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; </p><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------------t9002--------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='t9002'></a><br />
<h2>Characterization of BBa_T9002 biosensor</h2><br />
<div align="justify">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></div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage" width="47%" height="50%"></a></div><br />
</div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage" width="100%"></a></div><br />
</div><br />
<center><br />
<table width='50%' class='data'><br />
<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 />
<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 />
</tr><br />
</table><br />
</center><br />
<br><br />
<br><br />
<div align="justify">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.</div><br />
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</html><br />
<br />
<br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Results
Team:UNIPV-Pavia/Project/Results
2011-09-21T19:54:42Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<br />
<html><br />
<h2 class="art-postheader"> Results </h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"> <br />
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<!-----------MENU-----------------> <br />
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<br><br />
<a name='indice'></a><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<table id="toc" class="toc"><br />
<tr><br />
<td><div id="toctitle"><br />
<h2>Contents</h2><br />
</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><br />
</tr><br />
</table><br />
</div><br />
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<em> 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. </em> <br><br />
<br><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------ASSEMBLY-------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='assembly'></a><br />
<h1>Parts assembly</h1><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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------CHARACTERIZATION---------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='characterization'></a><br />
<h1>Characterization of basic modules</h1><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------pTet and pLux-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='promoters'></a><br />
<h2>Characterization of promoters pTet and pLux</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><br />
<td class="row"><b>BioBrick code</b></td><br />
<td><b> Declared efficiency</b></td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0030 </td><br />
<td class="row"> 0,6</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0031 </td><br />
<td class="row"> 0,07</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0032 </td><br />
<td class="row"> 0,3</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0034 </td><br />
<td class="row"> 1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<div class="listcircle"><br />
<div align="justify"><br />
<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><br />
<p></p><br />
<p><br />
<ol><br />
<ul><br />
<li><b> RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li><b> RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li> <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 />
</p><br />
</li><br />
<p><br />
<li> <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.<br />
<br />
</li><br />
</p><br />
</ul><br />
</ol><br />
</p><br />
<p align='justify'> 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>. </p><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><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><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><br />
<td class='row'>4.28</td><br />
<td class='row'>0.20</td><br />
<td class='row'>1.08</td><br />
<td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>4.93</td><br />
<td class='row'>0.55</td><br />
<td class='row'>0.25</td><br />
<td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>9.49</td><br />
<td class='row'>0.02</td><br />
<td class='row'>0.47</td><br />
<td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>21.53</td><br />
<td class='row'>0.51</td><br />
<td class='row'>0.53</td><br />
<td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><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> 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><br />
<br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"> <img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><br />
<p align='justify'> The estimated parameters of the Hill curves described in the figures 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> [(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><br />
<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><br />
<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><br />
<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><br />
<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 />
<p align='justify'> The operative parameters are summarized in the table below: </p><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><br />
<td class='row'>1.53</td><br />
<td class='row'>~0</td><br />
<td class='row'>7.95</td><br />
<td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>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><br />
<td class='row'>B0032</td><br />
<td class='row'>3.16</td><br />
<td class='row'>~0</td><br />
<td class='row'>6.7</td><br />
<td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>2.73</td><br />
<td class='row'>0.23</td><br />
<td class='row'>8.96</td><br />
<td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------AiiA and LuxI-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='enzymes'></a><br />
<h2>Characterization of enzymes AiiA and LuxI</h2><br />
<p align='justify'> 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 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 />
<div align="justify"><br />
<div class="thumbinner" style="width: 850px;"> <a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div><br />
</div><br />
<div align="justify"><br />
<p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p><br />
</div><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><br />
<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><br />
</table><br />
</div><br />
<br><br />
</p><br />
</p><br />
<p align='justify'> 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. </p><br />
<p align='justify'> The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity. </p><br />
<p align='justify'> 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 />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
</table><br />
<br />
<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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------RBS---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='rbs'></a><br />
<h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
RBSs were used for the fine tuning of CTRL+E. Different experimental conditions were assayed. <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 />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>0.40</td><br />
<td class='row'>1.6814</td><br />
<td class='row'>2.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.01</td><br />
<td class='row'>ND</td><br />
<td class='row'>0.04</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.19</td><br />
<td class='row'>0.4193</td><br />
<td class='row'>0.40</td><br />
<td class='row'>0,3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br><br />
Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT, with GeneX=mRFP, AiiA or LuxI: <br><br />
<br><br />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>1.72</td><br />
<td class='row'>0.53</td><br />
<td class='row'>0.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.03</td><br />
<td class='row'>0.83</td><br />
<td class='row'>0.028</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.37</td><br />
<td class='row'>0.50</td><br />
<td class='row'>N.D.</td><br />
<td class='row'>0.3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------growth---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='growth'></a><br />
<h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'> 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 />
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> 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><br />
<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><br />
<li>1:1</li><br />
<li>1:10</li><br />
<li>1:100</li><br />
<li>1:1000</li><br />
</ul><br />
in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm. </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 />
</ul><br />
</ol><br />
<p>The results are summarized in the table and in the figure below: </p><br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><b> Culture </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><br />
<td class='row'><b> C.F.U. </b></td><br />
<td class='row'><b> Dilution Factor (10^) </b></td><br />
<td class='row'><b> N </b></td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,367950002 </td><br />
<td class='row'> 0,758004416 </td><br />
<td class='row'> 990 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 990000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,044649998 </td><br />
<td class='row'> 0,091982322 </td><br />
<td class='row'> 141 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 1410000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,004799999 </td><br />
<td class='row'> 0,009888356 </td><br />
<td class='row'> 136 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 136000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,000549998 </td><br />
<td class='row'> 0,001133037 </td><br />
<td class='row'> 20 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 200000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,54840003 </td><br />
<td class='row'> 1,129744917 </td><br />
<td class='row'> 165 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 16500000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,058200002 </td><br />
<td class='row'> 0,119896339 </td><br />
<td class='row'> 23 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 23000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,008700002 </td><br />
<td class='row'> 0,017922652 </td><br />
<td class='row'> 251 </td><br />
<td class='row'> 3 </td><br />
<td class='row'> 2510000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,000100002 </td><br />
<td class='row'> 0,000206011 </td><br />
<td class='row'> 24 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 2400000 </td><br />
</tr><br />
</table><br />
</center><br />
<br><br />
</p><br />
<center><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"><br />
</center><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 />
</p><br />
<center><br />
<table width='50%' class='data'><br />
<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><br />
<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. </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 />
<a name='HSL'></a><br />
<h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 6</sub></b></td><br />
<td class='row'>32 </td><br />
<td class='row'>0.022 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 7</sub></b></td><br />
<td class='row'>8 </td><br />
<td class='row'>0.087 </td><br />
</tr><br />
</table><br />
</center><br />
<p> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 6</sub></b></td><br />
<td class='row'>&#8734; </td><br />
<td class='row'>0 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 7</sub></b></td><br />
<td class='row'>19 </td><br />
<td class='row'>0.037 </td><br />
</tr><br />
</table><br />
</center><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; </p><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------------t9002--------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='t9002'></a><br />
<h2>Characterization of BBa_T9002 biosensor</h2><br />
<div align="justify">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></div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage" width="47%" height="50%"></a></div><br />
</div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage" width="100%"></a></div><br />
</div><br />
<center><br />
<table width='50%' class='data'><br />
<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 />
<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 />
</tr><br />
</table><br />
</center><br />
<br><br />
<br><br />
<div align="justify">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.</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
</html><br />
<br />
<br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Results
Team:UNIPV-Pavia/Project/Results
2011-09-21T19:52:59Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<br />
<html><br />
<h2 class="art-postheader"> Results </h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"> <br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------MENU-----------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br><br />
<a name='indice'></a><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<table id="toc" class="toc"><br />
<tr><br />
<td><div id="toctitle"><br />
<h2>Contents</h2><br />
</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><br />
</tr><br />
</table><br />
</div><br />
<script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script> <br />
<em> 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. </em> <br><br />
<br><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------ASSEMBLY-------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='assembly'></a><br />
<h1>Parts assembly</h1><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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------CHARACTERIZATION---------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='characterization'></a><br />
<h1>Characterization of basic modules</h1><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------pTet and pLux-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='promoters'></a><br />
<h2>Characterization of promoters pTet and pLux</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><br />
<td class="row"><b>BioBrick code</b></td><br />
<td><b> Declared efficiency</b></td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0030 </td><br />
<td class="row"> 0,6</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0031 </td><br />
<td class="row"> 0,07</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0032 </td><br />
<td class="row"> 0,3</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0034 </td><br />
<td class="row"> 1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<div class="listcircle"><br />
<div align="justify"><br />
<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><br />
<p></p><br />
<p><br />
<ol><br />
<ul><br />
<li><b> RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li><b> RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li> <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 />
</p><br />
</li><br />
<p><br />
<li> <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.<br />
<br />
</li><br />
</p><br />
</ul><br />
</ol><br />
</p><br />
<p align='justify'> 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>. </p><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><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><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><br />
<td class='row'>4.28</td><br />
<td class='row'>0.20</td><br />
<td class='row'>1.08</td><br />
<td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>4.93</td><br />
<td class='row'>0.55</td><br />
<td class='row'>0.25</td><br />
<td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>9.49</td><br />
<td class='row'>0.02</td><br />
<td class='row'>0.47</td><br />
<td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>21.53</td><br />
<td class='row'>0.51</td><br />
<td class='row'>0.53</td><br />
<td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><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> 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><br />
<br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"> <img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><br />
<p align='justify'> The estimated parameters of the Hill curves described in the figures 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> [(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><br />
<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><br />
<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><br />
<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><br />
<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 />
<p align='justify'> The operative parameters are summarized in the table below: </p><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><br />
<td class='row'>1.53</td><br />
<td class='row'>~0</td><br />
<td class='row'>7.95</td><br />
<td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>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><br />
<td class='row'>B0032</td><br />
<td class='row'>3.16</td><br />
<td class='row'>~0</td><br />
<td class='row'>6.7</td><br />
<td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>2.73</td><br />
<td class='row'>0.23</td><br />
<td class='row'>8.96</td><br />
<td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------AiiA and LuxI-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='enzymes'></a><br />
<h2>Characterization of enzymes AiiA and LuxI</h2><br />
<p align='justify'> 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 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 />
<div align="justify"><br />
<div class="thumbinner" style="width: 850px;"> <a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div><br />
</div><br />
<div align="justify"><br />
<p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p><br />
</div><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><br />
<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><br />
</table><br />
</div><br />
<br><br />
</p><br />
</p><br />
<p align='justify'> 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. </p><br />
<p align='justify'> The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity. </p><br />
<p align='justify'> 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 />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
</table><br />
<br />
<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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------RBS---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='rbs'></a><br />
<h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
RBSs were used for the fine tuning of CTRL+E. Different experimental conditions were assayed. <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 />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>0.40</td><br />
<td class='row'>1.6814</td><br />
<td class='row'>2.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.01</td><br />
<td class='row'>ND</td><br />
<td class='row'>0.04</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.19</td><br />
<td class='row'>0.4193</td><br />
<td class='row'>0.40</td><br />
<td class='row'>0,3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br><br />
Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT, with GeneX=mRFP, AiiA or LuxI: <br><br />
<br><br />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>1.72</td><br />
<td class='row'>0.53</td><br />
<td class='row'>0.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.03</td><br />
<td class='row'>0.83</td><br />
<td class='row'>0.028</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.37</td><br />
<td class='row'>0.50</td><br />
<td class='row'>N.D.</td><br />
<td class='row'>0.3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------growth---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='growth'></a><br />
<h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'> 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 />
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> 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><br />
<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><br />
<li>1:1</li><br />
<li>1:10</li><br />
<li>1:100</li><br />
<li>1:1000</li><br />
</ul><br />
in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm. </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 />
</ul><br />
</ol><br />
<p>The results are summarized in the table and in the figure below: </p><br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><b> Culture </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><br />
<td class='row'><b> C.F.U. </b></td><br />
<td class='row'><b> Dilution Factor (10^) </b></td><br />
<td class='row'><b> N </b></td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,367950002 </td><br />
<td class='row'> 0,758004416 </td><br />
<td class='row'> 990 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 990000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,044649998 </td><br />
<td class='row'> 0,091982322 </td><br />
<td class='row'> 141 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 1410000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,004799999 </td><br />
<td class='row'> 0,009888356 </td><br />
<td class='row'> 136 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 136000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,000549998 </td><br />
<td class='row'> 0,001133037 </td><br />
<td class='row'> 20 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 200000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,54840003 </td><br />
<td class='row'> 1,129744917 </td><br />
<td class='row'> 165 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 16500000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,058200002 </td><br />
<td class='row'> 0,119896339 </td><br />
<td class='row'> 23 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 23000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,008700002 </td><br />
<td class='row'> 0,017922652 </td><br />
<td class='row'> 251 </td><br />
<td class='row'> 3 </td><br />
<td class='row'> 2510000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,000100002 </td><br />
<td class='row'> 0,000206011 </td><br />
<td class='row'> 24 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 2400000 </td><br />
</tr><br />
</table><br />
</center><br />
<br><br />
</p><br />
<center><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"><br />
</center><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 />
</p><br />
<center><br />
<table width='50%' class='data'><br />
<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><br />
<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. </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 />
<a name='HSL'></a><br />
<h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 6</sub></b></td><br />
<td class='row'>32 </td><br />
<td class='row'>0.022 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 7</sub></b></td><br />
<td class='row'>8 </td><br />
<td class='row'>0.087 </td><br />
</tr><br />
</table><br />
</center><br />
<p> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 6</sub></b></td><br />
<td class='row'>&#8734; </td><br />
<td class='row'>0 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 7</sub></b></td><br />
<td class='row'>19 </td><br />
<td class='row'>0.037 </td><br />
</tr><br />
</table><br />
</center><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; </p><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------------t9002--------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='t9002'></a><br />
<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'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage" width="47%" height="50%"></a></div><br />
</div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage" width="100%"></a></div><br />
</div><br />
<center><br />
<table width='50%' class='data'><br />
<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 />
<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 />
</tr><br />
</table><br />
</center><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. Hypothesizing that the inducer is 1:20 diluted (as for all of our tests), the minimum detectable HSL concentration is 3 nM.<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
</html><br />
<br />
<br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Results
Team:UNIPV-Pavia/Project/Results
2011-09-21T19:51:14Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<br />
<br />
<br />
<html><br />
<h2 class="art-postheader"> Results </h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"> <br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------MENU-----------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br><br />
<a name='indice'></a><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<table id="toc" class="toc"><br />
<tr><br />
<td><div id="toctitle"><br />
<h2>Contents</h2><br />
</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><br />
</tr><br />
</table><br />
</div><br />
<script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script> <br />
<em> 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. </em> <br><br />
<br><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------ASSEMBLY-------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='assembly'></a><br />
<h1>Parts assembly</h1><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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------CHARACTERIZATION---------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='characterization'></a><br />
<h1>Characterization of basic modules</h1><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------pTet and pLux-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='promoters'></a><br />
<h2>Characterization of promoters pTet and pLux</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><br />
<td class="row"><b>BioBrick code</b></td><br />
<td><b> Declared efficiency</b></td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0030 </td><br />
<td class="row"> 0,6</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0031 </td><br />
<td class="row"> 0,07</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0032 </td><br />
<td class="row"> 0,3</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0034 </td><br />
<td class="row"> 1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<div align="justify"><br />
<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><br />
<p></p><br />
<p><br />
<ol><br />
<ul><br />
<li><b> RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li><b> RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li> <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 />
</p><br />
</li><br />
<p><br />
<li> <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.<br />
<br />
</li><br />
</p><br />
</ul><br />
</ol><br />
</p><br />
<p align='justify'> 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>. </p><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><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><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><br />
<td class='row'>4.28</td><br />
<td class='row'>0.20</td><br />
<td class='row'>1.08</td><br />
<td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>4.93</td><br />
<td class='row'>0.55</td><br />
<td class='row'>0.25</td><br />
<td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>9.49</td><br />
<td class='row'>0.02</td><br />
<td class='row'>0.47</td><br />
<td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>21.53</td><br />
<td class='row'>0.51</td><br />
<td class='row'>0.53</td><br />
<td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><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> 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><br />
<br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"> <img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><br />
<p align='justify'> The estimated parameters of the Hill curves described in the figures 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> [(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><br />
<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><br />
<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><br />
<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><br />
<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 />
<p align='justify'> The operative parameters are summarized in the table below: </p><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><br />
<td class='row'>1.53</td><br />
<td class='row'>~0</td><br />
<td class='row'>7.95</td><br />
<td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>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><br />
<td class='row'>B0032</td><br />
<td class='row'>3.16</td><br />
<td class='row'>~0</td><br />
<td class='row'>6.7</td><br />
<td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>2.73</td><br />
<td class='row'>0.23</td><br />
<td class='row'>8.96</td><br />
<td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------AiiA and LuxI-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='enzymes'></a><br />
<h2>Characterization of enzymes AiiA and LuxI</h2><br />
<p align='justify'> 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 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 />
<div align="justify"><br />
<div class="thumbinner" style="width: 850px;"> <a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div><br />
</div><br />
<div align="justify"><br />
<p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p><br />
</div><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><br />
<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><br />
</table><br />
</div><br />
<br><br />
</p><br />
</p><br />
<p align='justify'> 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. </p><br />
<p align='justify'> The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity. </p><br />
<p align='justify'> 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 />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
</table><br />
<br />
<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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------RBS---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='rbs'></a><br />
<h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
RBSs were used for the fine tuning of CTRL+E. Different experimental conditions were assayed. <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 />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>0.40</td><br />
<td class='row'>1.6814</td><br />
<td class='row'>2.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.01</td><br />
<td class='row'>ND</td><br />
<td class='row'>0.04</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.19</td><br />
<td class='row'>0.4193</td><br />
<td class='row'>0.40</td><br />
<td class='row'>0,3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br><br />
Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT, with GeneX=mRFP, AiiA or LuxI: <br><br />
<br><br />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>1.72</td><br />
<td class='row'>0.53</td><br />
<td class='row'>0.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.03</td><br />
<td class='row'>0.83</td><br />
<td class='row'>0.028</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.37</td><br />
<td class='row'>0.50</td><br />
<td class='row'>N.D.</td><br />
<td class='row'>0.3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------growth---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='growth'></a><br />
<h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'> 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 />
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> 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><br />
<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><br />
<li>1:1</li><br />
<li>1:10</li><br />
<li>1:100</li><br />
<li>1:1000</li><br />
</ul><br />
in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm. </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 />
</ul><br />
</ol><br />
<p>The results are summarized in the table and in the figure below: </p><br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><b> Culture </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><br />
<td class='row'><b> C.F.U. </b></td><br />
<td class='row'><b> Dilution Factor (10^) </b></td><br />
<td class='row'><b> N </b></td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,367950002 </td><br />
<td class='row'> 0,758004416 </td><br />
<td class='row'> 990 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 990000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,044649998 </td><br />
<td class='row'> 0,091982322 </td><br />
<td class='row'> 141 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 1410000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,004799999 </td><br />
<td class='row'> 0,009888356 </td><br />
<td class='row'> 136 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 136000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,000549998 </td><br />
<td class='row'> 0,001133037 </td><br />
<td class='row'> 20 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 200000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,54840003 </td><br />
<td class='row'> 1,129744917 </td><br />
<td class='row'> 165 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 16500000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,058200002 </td><br />
<td class='row'> 0,119896339 </td><br />
<td class='row'> 23 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 23000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,008700002 </td><br />
<td class='row'> 0,017922652 </td><br />
<td class='row'> 251 </td><br />
<td class='row'> 3 </td><br />
<td class='row'> 2510000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,000100002 </td><br />
<td class='row'> 0,000206011 </td><br />
<td class='row'> 24 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 2400000 </td><br />
</tr><br />
</table><br />
</center><br />
<br><br />
</p><br />
<center><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"><br />
</center><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 />
</p><br />
<center><br />
<table width='50%' class='data'><br />
<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><br />
<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. </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 />
<a name='HSL'></a><br />
<h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 6</sub></b></td><br />
<td class='row'>32 </td><br />
<td class='row'>0.022 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 7</sub></b></td><br />
<td class='row'>8 </td><br />
<td class='row'>0.087 </td><br />
</tr><br />
</table><br />
</center><br />
<p> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 6</sub></b></td><br />
<td class='row'>&#8734; </td><br />
<td class='row'>0 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 7</sub></b></td><br />
<td class='row'>19 </td><br />
<td class='row'>0.037 </td><br />
</tr><br />
</table><br />
</center><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; </p><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<br />
<a name='t9002'></a><br />
<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'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage" width="47%" height="50%"></a></div><br />
</div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage" width="100%"></a></div><br />
</div><br />
<center><br />
<table width='50%' class='data'><br />
<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 />
<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 />
</tr><br />
</table><br />
</center><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. Hypothesizing that the inducer is 1:20 diluted (as for all of our tests), the minimum detectable HSL concentration is 3 nM.<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Results
Team:UNIPV-Pavia/Project/Results
2011-09-21T19:50:50Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
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<h2 class="art-postheader"> Results </h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"> <br />
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<table id="toc" class="toc"><br />
<tr><br />
<td><div id="toctitle"><br />
<h2>Contents</h2><br />
</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><br />
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</table><br />
</div><br />
<script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script> <br />
<em> 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. </em> <br><br />
<br><br />
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<br />
<a name='assembly'></a><br />
<h1>Parts assembly</h1><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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<br />
<a name='characterization'></a><br />
<h1>Characterization of basic modules</h1><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------pTet and pLux-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='promoters'></a><br />
<h2>Characterization of promoters pTet and pLux</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><br />
<td class="row"><b>BioBrick code</b></td><br />
<td><b> Declared efficiency</b></td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0030 </td><br />
<td class="row"> 0,6</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0031 </td><br />
<td class="row"> 0,07</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0032 </td><br />
<td class="row"> 0,3</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0034 </td><br />
<td class="row"> 1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<div align="justify"><br />
<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><br />
<p></p><br />
<p><br />
<ol><br />
<ul><br />
<li><b> RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li><b> RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li> <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 />
</p><br />
</li><br />
<p><br />
<li> <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.<br />
<br />
</li><br />
</p><br />
</ul><br />
</ol><br />
</p><br />
<p align='justify'> 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>. </p><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><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><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><br />
<td class='row'>4.28</td><br />
<td class='row'>0.20</td><br />
<td class='row'>1.08</td><br />
<td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>4.93</td><br />
<td class='row'>0.55</td><br />
<td class='row'>0.25</td><br />
<td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>9.49</td><br />
<td class='row'>0.02</td><br />
<td class='row'>0.47</td><br />
<td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>21.53</td><br />
<td class='row'>0.51</td><br />
<td class='row'>0.53</td><br />
<td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><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> 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><br />
<br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"> <img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><br />
<p align='justify'> The estimated parameters of the Hill curves described in the figures 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> [(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><br />
<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><br />
<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><br />
<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><br />
<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 />
<p align='justify'> The operative parameters are summarized in the table below: </p><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><br />
<td class='row'>1.53</td><br />
<td class='row'>~0</td><br />
<td class='row'>7.95</td><br />
<td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>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><br />
<td class='row'>B0032</td><br />
<td class='row'>3.16</td><br />
<td class='row'>~0</td><br />
<td class='row'>6.7</td><br />
<td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>2.73</td><br />
<td class='row'>0.23</td><br />
<td class='row'>8.96</td><br />
<td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------AiiA and LuxI-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='enzymes'></a><br />
<h2>Characterization of enzymes AiiA and LuxI</h2><br />
<p align='justify'> 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 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 />
<div align="justify"><br />
<div class="thumbinner" style="width: 850px;"> <a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div><br />
</div><br />
<div align="justify"><br />
<p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p><br />
</div><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><br />
<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><br />
</table><br />
</div><br />
<br><br />
</p><br />
</p><br />
<p align='justify'> 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. </p><br />
<p align='justify'> The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity. </p><br />
<p align='justify'> 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 />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
</table><br />
<br />
<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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------RBS---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='rbs'></a><br />
<h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
RBSs were used for the fine tuning of CTRL+E. Different experimental conditions were assayed. <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 />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>0.40</td><br />
<td class='row'>1.6814</td><br />
<td class='row'>2.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.01</td><br />
<td class='row'>ND</td><br />
<td class='row'>0.04</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.19</td><br />
<td class='row'>0.4193</td><br />
<td class='row'>0.40</td><br />
<td class='row'>0,3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br><br />
Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT, with GeneX=mRFP, AiiA or LuxI: <br><br />
<br><br />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>1.72</td><br />
<td class='row'>0.53</td><br />
<td class='row'>0.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.03</td><br />
<td class='row'>0.83</td><br />
<td class='row'>0.028</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.37</td><br />
<td class='row'>0.50</td><br />
<td class='row'>N.D.</td><br />
<td class='row'>0.3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------growth---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='growth'></a><br />
<h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'> 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 />
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> 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><br />
<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><br />
<li>1:1</li><br />
<li>1:10</li><br />
<li>1:100</li><br />
<li>1:1000</li><br />
</ul><br />
in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm. </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 />
</ul><br />
</ol><br />
<p>The results are summarized in the table and in the figure below: </p><br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><b> Culture </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><br />
<td class='row'><b> C.F.U. </b></td><br />
<td class='row'><b> Dilution Factor (10^) </b></td><br />
<td class='row'><b> N </b></td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,367950002 </td><br />
<td class='row'> 0,758004416 </td><br />
<td class='row'> 990 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 990000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,044649998 </td><br />
<td class='row'> 0,091982322 </td><br />
<td class='row'> 141 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 1410000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,004799999 </td><br />
<td class='row'> 0,009888356 </td><br />
<td class='row'> 136 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 136000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,000549998 </td><br />
<td class='row'> 0,001133037 </td><br />
<td class='row'> 20 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 200000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,54840003 </td><br />
<td class='row'> 1,129744917 </td><br />
<td class='row'> 165 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 16500000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,058200002 </td><br />
<td class='row'> 0,119896339 </td><br />
<td class='row'> 23 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 23000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,008700002 </td><br />
<td class='row'> 0,017922652 </td><br />
<td class='row'> 251 </td><br />
<td class='row'> 3 </td><br />
<td class='row'> 2510000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,000100002 </td><br />
<td class='row'> 0,000206011 </td><br />
<td class='row'> 24 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 2400000 </td><br />
</tr><br />
</table><br />
</center><br />
<br><br />
</p><br />
<center><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"><br />
</center><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 />
</p><br />
<center><br />
<table width='50%' class='data'><br />
<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><br />
<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. </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 />
<a name='HSL'></a><br />
<h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 6</sub></b></td><br />
<td class='row'>32 </td><br />
<td class='row'>0.022 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 7</sub></b></td><br />
<td class='row'>8 </td><br />
<td class='row'>0.087 </td><br />
</tr><br />
</table><br />
</center><br />
<p> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 6</sub></b></td><br />
<td class='row'>&#8734; </td><br />
<td class='row'>0 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 7</sub></b></td><br />
<td class='row'>19 </td><br />
<td class='row'>0.037 </td><br />
</tr><br />
</table><br />
</center><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; </p><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------------t9002--------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='t9002'></a><br />
<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'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage" width="47%" height="50%"></a></div><br />
</div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage" width="100%"></a></div><br />
</div><br />
<center><br />
<table width='50%' class='data'><br />
<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 />
<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 />
</tr><br />
</table><br />
</center><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. 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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<br><br />
<br><br />
<br><br />
<br />
</html><br />
<br />
<br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Motivation2
Team:UNIPV-Pavia/Project/Motivation2
2011-09-21T19:50:38Z
<p>Edoardo Baldini: Blanked the page</p>
<hr />
<div></div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Test
Team:UNIPV-Pavia/Test
2011-09-21T19:49:37Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<br />
<br />
<br />
<html><br />
<h2 class="art-postheader"> Results </h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"> <br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------MENU-----------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br><br />
<a name='indice'></a><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<table id="toc" class="toc"><br />
<tr><br />
<td><div id="toctitle"><br />
<h2>Contents</h2><br />
</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><br />
</tr><br />
</table><br />
</div><br />
<script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script> <br />
<em> 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. </em> <br><br />
<br><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------ASSEMBLY-------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='assembly'></a><br />
<h1>Parts assembly</h1><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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------CHARACTERIZATION---------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='characterization'></a><br />
<h1>Characterization of basic modules</h1><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------pTet and pLux-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='promoters'></a><br />
<h2>Characterization of promoters pTet and pLux</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><br />
<td class="row"><b>BioBrick code</b></td><br />
<td><b> Declared efficiency</b></td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0030 </td><br />
<td class="row"> 0,6</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0031 </td><br />
<td class="row"> 0,07</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0032 </td><br />
<td class="row"> 0,3</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0034 </td><br />
<td class="row"> 1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<div align="justify"><br />
<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><br />
<p></p><br />
<p><br />
<ol><br />
<ul><br />
<li><b> RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li><b> RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li> <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 />
</p><br />
</li><br />
<p><br />
<li> <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.<br />
<br />
</li><br />
</p><br />
</ul><br />
</ol><br />
</p><br />
<p align='justify'> 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>. </p><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><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><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><br />
<td class='row'>4.28</td><br />
<td class='row'>0.20</td><br />
<td class='row'>1.08</td><br />
<td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>4.93</td><br />
<td class='row'>0.55</td><br />
<td class='row'>0.25</td><br />
<td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>9.49</td><br />
<td class='row'>0.02</td><br />
<td class='row'>0.47</td><br />
<td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>21.53</td><br />
<td class='row'>0.51</td><br />
<td class='row'>0.53</td><br />
<td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><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> 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><br />
<br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"> <img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><br />
<p align='justify'> The estimated parameters of the Hill curves described in the figures 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> [(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><br />
<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><br />
<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><br />
<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><br />
<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 />
<p align='justify'> The operative parameters are summarized in the table below: </p><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><br />
<td class='row'>1.53</td><br />
<td class='row'>~0</td><br />
<td class='row'>7.95</td><br />
<td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>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><br />
<td class='row'>B0032</td><br />
<td class='row'>3.16</td><br />
<td class='row'>~0</td><br />
<td class='row'>6.7</td><br />
<td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>2.73</td><br />
<td class='row'>0.23</td><br />
<td class='row'>8.96</td><br />
<td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------AiiA and LuxI-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='enzymes'></a><br />
<h2>Characterization of enzymes AiiA and LuxI</h2><br />
<p align='justify'> 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 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 />
<div align="justify"><br />
<div class="thumbinner" style="width: 850px;"> <a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div><br />
</div><br />
<div align="justify"><br />
<p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p><br />
</div><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><br />
<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><br />
</table><br />
</div><br />
<br><br />
</p><br />
</p><br />
<p align='justify'> 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. </p><br />
<p align='justify'> The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity. </p><br />
<p align='justify'> 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 />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
</table><br />
<br />
<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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------RBS---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='rbs'></a><br />
<h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
RBSs were used for the fine tuning of CTRL+E. Different experimental conditions were assayed. <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 />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>0.40</td><br />
<td class='row'>1.6814</td><br />
<td class='row'>2.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.01</td><br />
<td class='row'>ND</td><br />
<td class='row'>0.04</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.19</td><br />
<td class='row'>0.4193</td><br />
<td class='row'>0.40</td><br />
<td class='row'>0,3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br><br />
Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT, with GeneX=mRFP, AiiA or LuxI: <br><br />
<br><br />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>1.72</td><br />
<td class='row'>0.53</td><br />
<td class='row'>0.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.03</td><br />
<td class='row'>0.83</td><br />
<td class='row'>0.028</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.37</td><br />
<td class='row'>0.50</td><br />
<td class='row'>N.D.</td><br />
<td class='row'>0.3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------growth---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='growth'></a><br />
<h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'> 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 />
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> 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><br />
<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><br />
<li>1:1</li><br />
<li>1:10</li><br />
<li>1:100</li><br />
<li>1:1000</li><br />
</ul><br />
in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm. </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 />
</ul><br />
</ol><br />
<p>The results are summarized in the table and in the figure below: </p><br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><b> Culture </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><br />
<td class='row'><b> C.F.U. </b></td><br />
<td class='row'><b> Dilution Factor (10^) </b></td><br />
<td class='row'><b> N </b></td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,367950002 </td><br />
<td class='row'> 0,758004416 </td><br />
<td class='row'> 990 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 990000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,044649998 </td><br />
<td class='row'> 0,091982322 </td><br />
<td class='row'> 141 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 1410000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,004799999 </td><br />
<td class='row'> 0,009888356 </td><br />
<td class='row'> 136 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 136000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,000549998 </td><br />
<td class='row'> 0,001133037 </td><br />
<td class='row'> 20 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 200000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,54840003 </td><br />
<td class='row'> 1,129744917 </td><br />
<td class='row'> 165 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 16500000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,058200002 </td><br />
<td class='row'> 0,119896339 </td><br />
<td class='row'> 23 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 23000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,008700002 </td><br />
<td class='row'> 0,017922652 </td><br />
<td class='row'> 251 </td><br />
<td class='row'> 3 </td><br />
<td class='row'> 2510000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,000100002 </td><br />
<td class='row'> 0,000206011 </td><br />
<td class='row'> 24 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 2400000 </td><br />
</tr><br />
</table><br />
</center><br />
<br><br />
</p><br />
<center><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"><br />
</center><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 />
</p><br />
<center><br />
<table width='50%' class='data'><br />
<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><br />
<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. </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 />
<a name='HSL'></a><br />
<h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 6</sub></b></td><br />
<td class='row'>32 </td><br />
<td class='row'>0.022 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 7</sub></b></td><br />
<td class='row'>8 </td><br />
<td class='row'>0.087 </td><br />
</tr><br />
</table><br />
</center><br />
<p> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 6</sub></b></td><br />
<td class='row'>&#8734; </td><br />
<td class='row'>0 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 7</sub></b></td><br />
<td class='row'>19 </td><br />
<td class='row'>0.037 </td><br />
</tr><br />
</table><br />
</center><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; </p><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------------t9002--------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='t9002'></a><br />
<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'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage" width="47%" height="50%"></a></div><br />
</div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage" width="100%"></a></div><br />
</div><br />
<center><br />
<table width='50%' class='data'><br />
<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 />
<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 />
</tr><br />
</table><br />
</center><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. Hypothesizing that the inducer is 1:20 diluted (as for all of our tests), the minimum detectable HSL concentration is 3 nM.<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br><br />
<br><br />
<br><br />
<br />
</html><br />
<br />
<br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Motivation2
Team:UNIPV-Pavia/Project/Motivation2
2011-09-21T19:47:36Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<html><br />
<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><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 />
<!-------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 />
<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 />
<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 />
<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 />
<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><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 />
<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 />
<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 />
<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 />
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 />
<br />
<br />
</html><br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Test
Team:UNIPV-Pavia/Test
2011-09-21T19:47:32Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<br />
<br />
<br />
<html><br />
<h2 class="art-postheader"> Results </h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"> <br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------MENU-----------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br><br />
<a name='indice'></a><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<table id="toc" class="toc"><br />
<tr><br />
<td><div id="toctitle"><br />
<h2>Contents</h2><br />
</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><br />
</tr><br />
</table><br />
</div><br />
<script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script> <br />
<em> 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. </em> <br><br />
<br><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------ASSEMBLY-------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='assembly'></a><br />
<h1>Parts assembly</h1><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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------CHARACTERIZATION---------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='characterization'></a><br />
<h1>Characterization of basic modules</h1><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------pTet and pLux-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='promoters'></a><br />
<h2>Characterization of promoters pTet and pLux</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><br />
<td class="row"><b>BioBrick code</b></td><br />
<td><b> Declared efficiency</b></td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0030 </td><br />
<td class="row"> 0,6</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0031 </td><br />
<td class="row"> 0,07</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0032 </td><br />
<td class="row"> 0,3</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0034 </td><br />
<td class="row"> 1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<div align="justify"><br />
<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><br />
<p></p><br />
<p><br />
<ol><br />
<ul><br />
<li><b> RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li><b> RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li> <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 />
</p><br />
</li><br />
<p><br />
<li> <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.<br />
<br />
</li><br />
</p><br />
</ul><br />
</ol><br />
</p><br />
<p align='justify'> 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>. </p><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><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><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><br />
<td class='row'>4.28</td><br />
<td class='row'>0.20</td><br />
<td class='row'>1.08</td><br />
<td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>4.93</td><br />
<td class='row'>0.55</td><br />
<td class='row'>0.25</td><br />
<td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>9.49</td><br />
<td class='row'>0.02</td><br />
<td class='row'>0.47</td><br />
<td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>21.53</td><br />
<td class='row'>0.51</td><br />
<td class='row'>0.53</td><br />
<td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><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> 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><br />
<br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"> <img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><br />
<p align='justify'> The estimated parameters of the Hill curves described in the figures 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> [(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><br />
<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><br />
<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><br />
<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><br />
<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 />
<p align='justify'> The operative parameters are summarized in the table below: </p><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><br />
<td class='row'>1.53</td><br />
<td class='row'>~0</td><br />
<td class='row'>7.95</td><br />
<td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>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><br />
<td class='row'>B0032</td><br />
<td class='row'>3.16</td><br />
<td class='row'>~0</td><br />
<td class='row'>6.7</td><br />
<td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>2.73</td><br />
<td class='row'>0.23</td><br />
<td class='row'>8.96</td><br />
<td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------AiiA and LuxI-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='enzymes'></a><br />
<h2>Characterization of enzymes AiiA and LuxI</h2><br />
<p align='justify'> 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 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 />
<div align="justify"><br />
<div class="thumbinner" style="width: 850px;"> <a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div><br />
</div><br />
<div align="justify"><br />
<p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p><br />
</div><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><br />
<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><br />
</table><br />
</div><br />
<br><br />
</p><br />
</p><br />
<p align='justify'> 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. </p><br />
<p align='justify'> The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity. </p><br />
<p align='justify'> 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 />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
</table><br />
<br />
<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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------RBS---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='rbs'></a><br />
<h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
RBSs were used for the fine tuning of CTRL+E. Different experimental conditions were assayed. <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 />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>0.40</td><br />
<td class='row'>1.6814</td><br />
<td class='row'>2.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.01</td><br />
<td class='row'>ND</td><br />
<td class='row'>0.04</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.19</td><br />
<td class='row'>0.4193</td><br />
<td class='row'>0.40</td><br />
<td class='row'>0,3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br><br />
Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT, with GeneX=mRFP, AiiA or LuxI: <br><br />
<br><br />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>1.72</td><br />
<td class='row'>0.53</td><br />
<td class='row'>0.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.03</td><br />
<td class='row'>0.83</td><br />
<td class='row'>0.028</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.37</td><br />
<td class='row'>0.50</td><br />
<td class='row'>N.D.</td><br />
<td class='row'>0.3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------growth---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='growth'></a><br />
<h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'> 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 />
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> 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><br />
<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><br />
<li>1:1</li><br />
<li>1:10</li><br />
<li>1:100</li><br />
<li>1:1000</li><br />
</ul><br />
in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm. </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 />
</ul><br />
</ol><br />
<p>The results are summarized in the table and in the figure below: </p><br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><b> Culture </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><br />
<td class='row'><b> C.F.U. </b></td><br />
<td class='row'><b> Dilution Factor (10^) </b></td><br />
<td class='row'><b> N </b></td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,367950002 </td><br />
<td class='row'> 0,758004416 </td><br />
<td class='row'> 990 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 990000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,044649998 </td><br />
<td class='row'> 0,091982322 </td><br />
<td class='row'> 141 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 1410000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,004799999 </td><br />
<td class='row'> 0,009888356 </td><br />
<td class='row'> 136 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 136000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,000549998 </td><br />
<td class='row'> 0,001133037 </td><br />
<td class='row'> 20 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 200000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,54840003 </td><br />
<td class='row'> 1,129744917 </td><br />
<td class='row'> 165 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 16500000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,058200002 </td><br />
<td class='row'> 0,119896339 </td><br />
<td class='row'> 23 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 23000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,008700002 </td><br />
<td class='row'> 0,017922652 </td><br />
<td class='row'> 251 </td><br />
<td class='row'> 3 </td><br />
<td class='row'> 2510000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,000100002 </td><br />
<td class='row'> 0,000206011 </td><br />
<td class='row'> 24 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 2400000 </td><br />
</tr><br />
</table><br />
</center><br />
<br><br />
</p><br />
<center><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"><br />
</center><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 />
</p><br />
<center><br />
<table width='50%' class='data'><br />
<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><br />
<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. </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 />
<a name='HSL'></a><br />
<h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 6</sub></b></td><br />
<td class='row'>32 </td><br />
<td class='row'>0.022 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 7</sub></b></td><br />
<td class='row'>8 </td><br />
<td class='row'>0.087 </td><br />
</tr><br />
</table><br />
</center><br />
<p> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 6</sub></b></td><br />
<td class='row'>&#8734; </td><br />
<td class='row'>0 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 7</sub></b></td><br />
<td class='row'>19 </td><br />
<td class='row'>0.037 </td><br />
</tr><br />
</table><br />
</center><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; </p><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------------t9002--------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='t9002'></a><br />
<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'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage" width="47%" height="50%"></a></div><br />
</div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage" width="100%"></a></div><br />
</div><br />
<center><br />
<table width='50%' class='data'><br />
<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 />
<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 />
</tr><br />
</table><br />
</center><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. Hypothesizing that the inducer is 1:20 diluted (as for all of our tests), the minimum detectable HSL concentration is 3 nM.<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
<br />
</html><br />
<br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Motivation2
Team:UNIPV-Pavia/Project/Motivation2
2011-09-21T19:47:18Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<html><br />
<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><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 />
<!-------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 />
<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 />
<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 />
<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 />
<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><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 />
<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 />
<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 />
<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 />
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 />
<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 />
<br />
</html><br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Motivation2
Team:UNIPV-Pavia/Project/Motivation2
2011-09-21T19:46:52Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<html><br />
<br />
<h2 class="art-postheader"><br />
Results<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<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 />
<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 />
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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 />
<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 />
<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 />
<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 />
<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><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 />
<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 />
<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 />
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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 />
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 />
</html><br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Motivation2
Team:UNIPV-Pavia/Project/Motivation2
2011-09-21T19:46:16Z
<p>Edoardo Baldini: </p>
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<h2 class="art-postheader"><br />
Results<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 />
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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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<!-----------ASSEMBLY-------------><br />
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<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 />
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<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
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<!-------CHARACTERIZATION---------><br />
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<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 />
<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 />
<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 />
<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 />
<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><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 />
<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 />
</html><br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Motivation2
Team:UNIPV-Pavia/Project/Motivation2
2011-09-21T19:45:46Z
<p>Edoardo Baldini: </p>
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Results<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 />
<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 />
<!-------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 />
<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 />
<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 />
<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 />
<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><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 />
</html><br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Motivation2
Team:UNIPV-Pavia/Project/Motivation2
2011-09-21T19:45:22Z
<p>Edoardo Baldini: </p>
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<div>{{main}}<br />
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<h2 class="art-postheader"><br />
Results<br />
</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="#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 />
<!-------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 />
<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 />
<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 />
<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 />
<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><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 />
<br />
</html><br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Test
Team:UNIPV-Pavia/Test
2011-09-21T19:44:11Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
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<h2 class="art-postheader"> Results </h2><br />
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<div class="art-postcontent"> <br />
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<a name='indice'></a><br />
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<div class="art-postcontent"><br />
<table id="toc" class="toc"><br />
<tr><br />
<td><div id="toctitle"><br />
<h2>Contents</h2><br />
</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><br />
</tr><br />
</table><br />
</div><br />
<script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script> <br />
<em> 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. </em> <br><br />
<br><br />
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<!-----------ASSEMBLY-------------> <br />
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<br />
<a name='assembly'></a><br />
<h1>Parts assembly</h1><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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
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<!-------CHARACTERIZATION---------> <br />
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<br />
<a name='characterization'></a><br />
<h1>Characterization of basic modules</h1><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------pTet and pLux-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='promoters'></a><br />
<h2>Characterization of promoters pTet and pLux</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><br />
<td class="row"><b>BioBrick code</b></td><br />
<td><b> Declared efficiency</b></td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0030 </td><br />
<td class="row"> 0,6</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0031 </td><br />
<td class="row"> 0,07</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0032 </td><br />
<td class="row"> 0,3</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0034 </td><br />
<td class="row"> 1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<div align="justify"><br />
<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><br />
<p></p><br />
<p><br />
<ol><br />
<ul><br />
<li><b> RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li><b> RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li> <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 />
</p><br />
</li><br />
<p><br />
<li> <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.<br />
<br />
</li><br />
</p><br />
</ul><br />
</ol><br />
</p><br />
<p align='justify'> 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>. </p><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><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><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><br />
<td class='row'>4.28</td><br />
<td class='row'>0.20</td><br />
<td class='row'>1.08</td><br />
<td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>4.93</td><br />
<td class='row'>0.55</td><br />
<td class='row'>0.25</td><br />
<td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>9.49</td><br />
<td class='row'>0.02</td><br />
<td class='row'>0.47</td><br />
<td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>21.53</td><br />
<td class='row'>0.51</td><br />
<td class='row'>0.53</td><br />
<td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><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> 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><br />
<br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"> <img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><br />
<p align='justify'> The estimated parameters of the Hill curves described in the figures 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> [(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><br />
<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><br />
<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><br />
<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><br />
<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 />
<p align='justify'> The operative parameters are summarized in the table below: </p><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><br />
<td class='row'>1.53</td><br />
<td class='row'>~0</td><br />
<td class='row'>7.95</td><br />
<td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>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><br />
<td class='row'>B0032</td><br />
<td class='row'>3.16</td><br />
<td class='row'>~0</td><br />
<td class='row'>6.7</td><br />
<td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>2.73</td><br />
<td class='row'>0.23</td><br />
<td class='row'>8.96</td><br />
<td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------AiiA and LuxI-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='enzymes'></a><br />
<h2>Characterization of enzymes AiiA and LuxI</h2><br />
<p align='justify'> 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 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 />
<div align="justify"><br />
<div class="thumbinner" style="width: 850px;"> <a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div><br />
</div><br />
<div align="justify"><br />
<p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p><br />
</div><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><br />
<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><br />
</table><br />
</div><br />
<br><br />
</p><br />
</p><br />
<p align='justify'> 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. </p><br />
<p align='justify'> The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity. </p><br />
<p align='justify'> 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 />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
</table><br />
<br />
<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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------RBS---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='rbs'></a><br />
<h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
RBSs were used for the fine tuning of CTRL+E. Different experimental conditions were assayed. <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 />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>0.40</td><br />
<td class='row'>1.6814</td><br />
<td class='row'>2.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.01</td><br />
<td class='row'>ND</td><br />
<td class='row'>0.04</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.19</td><br />
<td class='row'>0.4193</td><br />
<td class='row'>0.40</td><br />
<td class='row'>0,3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br><br />
Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT, with GeneX=mRFP, AiiA or LuxI: <br><br />
<br><br />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>1.72</td><br />
<td class='row'>0.53</td><br />
<td class='row'>0.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.03</td><br />
<td class='row'>0.83</td><br />
<td class='row'>0.028</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.37</td><br />
<td class='row'>0.50</td><br />
<td class='row'>N.D.</td><br />
<td class='row'>0.3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------growth---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='growth'></a><br />
<h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'> 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 />
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> 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><br />
<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><br />
<li>1:1</li><br />
<li>1:10</li><br />
<li>1:100</li><br />
<li>1:1000</li><br />
</ul><br />
in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm. </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 />
</ul><br />
</ol><br />
<p>The results are summarized in the table and in the figure below: </p><br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><b> Culture </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><br />
<td class='row'><b> C.F.U. </b></td><br />
<td class='row'><b> Dilution Factor (10^) </b></td><br />
<td class='row'><b> N </b></td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,367950002 </td><br />
<td class='row'> 0,758004416 </td><br />
<td class='row'> 990 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 990000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,044649998 </td><br />
<td class='row'> 0,091982322 </td><br />
<td class='row'> 141 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 1410000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,004799999 </td><br />
<td class='row'> 0,009888356 </td><br />
<td class='row'> 136 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 136000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,000549998 </td><br />
<td class='row'> 0,001133037 </td><br />
<td class='row'> 20 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 200000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,54840003 </td><br />
<td class='row'> 1,129744917 </td><br />
<td class='row'> 165 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 16500000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,058200002 </td><br />
<td class='row'> 0,119896339 </td><br />
<td class='row'> 23 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 23000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,008700002 </td><br />
<td class='row'> 0,017922652 </td><br />
<td class='row'> 251 </td><br />
<td class='row'> 3 </td><br />
<td class='row'> 2510000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,000100002 </td><br />
<td class='row'> 0,000206011 </td><br />
<td class='row'> 24 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 2400000 </td><br />
</tr><br />
</table><br />
</center><br />
<br><br />
</p><br />
<center><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"><br />
</center><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 />
</p><br />
<center><br />
<table width='50%' class='data'><br />
<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><br />
<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. </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 />
<a name='HSL'></a><br />
<h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 6</sub></b></td><br />
<td class='row'>32 </td><br />
<td class='row'>0.022 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 7</sub></b></td><br />
<td class='row'>8 </td><br />
<td class='row'>0.087 </td><br />
</tr><br />
</table><br />
</center><br />
<p> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 6</sub></b></td><br />
<td class='row'>&#8734; </td><br />
<td class='row'>0 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 7</sub></b></td><br />
<td class='row'>19 </td><br />
<td class='row'>0.037 </td><br />
</tr><br />
</table><br />
</center><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; </p><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------------t9002--------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='t9002'></a><br />
<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'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage" width="47%" height="50%"></a></div><br />
</div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage" width="100%"></a></div><br />
</div><br />
<center><br />
<table width='50%' class='data'><br />
<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 />
<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 />
</tr><br />
</table><br />
</center><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. Hypothesizing that the inducer is 1:20 diluted (as for all of our tests), the minimum detectable HSL concentration is 3 nM.<br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<br />
</div><br />
</html><br />
<br />
<br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Motivation2
Team:UNIPV-Pavia/Project/Motivation2
2011-09-21T19:44:10Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<html><br />
<br />
<h2 class="art-postheader"><br />
Results<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<br />
<br />
<!--------------------------------><br />
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<!-----------MENU-----------------><br />
<!--------------------------------><br />
<!--------------------------------><br />
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<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 />
<!-------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 />
<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 />
<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 />
<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 />
<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 />
<br />
<br />
<br />
<br />
</html><br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Motivation2
Team:UNIPV-Pavia/Project/Motivation2
2011-09-21T19:42:56Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<html><br />
<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><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 />
<!-------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 />
<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 />
<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 />
<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 />
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<br />
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</html><br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Motivation2
Team:UNIPV-Pavia/Project/Motivation2
2011-09-21T19:42:34Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<html><br />
<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><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 />
<!-------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 />
<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 />
<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 />
<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 />
<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 />
</html><br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Motivation2
Team:UNIPV-Pavia/Project/Motivation2
2011-09-21T19:41:40Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<html><br />
<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><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 />
<!-------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 />
<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 />
<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 />
<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 />
</html><br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Motivation2
Team:UNIPV-Pavia/Project/Motivation2
2011-09-21T19:41:15Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<html><br />
<br />
<h2 class="art-postheader"><br />
Results<br />
</h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<br />
<br />
<br />
<!--------------------------------><br />
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<!-----------MENU-----------------><br />
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<!--------------------------------><br />
<!--------------------------------><br />
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<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 />
<!-------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 />
<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 />
<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 />
<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 />
</html><br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Motivation2
Team:UNIPV-Pavia/Project/Motivation2
2011-09-21T19:40:50Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<html><br />
<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><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 />
<!-------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 />
<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 />
<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 />
</html><br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Motivation2
Team:UNIPV-Pavia/Project/Motivation2
2011-09-21T19:39:55Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<html><br />
<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><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 />
<!-------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 />
<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 />
<br />
<br />
<br />
<br />
</html><br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Test
Team:UNIPV-Pavia/Test
2011-09-21T19:39:46Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<br />
<br />
<br />
<html><br />
<h2 class="art-postheader"> Results </h2><br />
<div class="cleared"></div><br />
<div class="art-postcontent"> <br />
<br />
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<br><br />
<a name='indice'></a><br />
<div class="cleared"></div><br />
<div class="art-postcontent"><br />
<table id="toc" class="toc"><br />
<tr><br />
<td><div id="toctitle"><br />
<h2>Contents</h2><br />
</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><br />
</tr><br />
</table><br />
</div><br />
<script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script> <br />
<em> 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. </em> <br><br />
<br><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------ASSEMBLY-------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='assembly'></a><br />
<h1>Parts assembly</h1><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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------CHARACTERIZATION---------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='characterization'></a><br />
<h1>Characterization of basic modules</h1><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------pTet and pLux-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='promoters'></a><br />
<h2>Characterization of promoters pTet and pLux</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><br />
<td class="row"><b>BioBrick code</b></td><br />
<td><b> Declared efficiency</b></td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0030 </td><br />
<td class="row"> 0,6</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0031 </td><br />
<td class="row"> 0,07</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0032 </td><br />
<td class="row"> 0,3</td><br />
</tr><br />
<tr><br />
<td class="row">BBa_B0034 </td><br />
<td class="row"> 1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<div align="justify"><br />
<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><br />
<p></p><br />
<p><br />
<ol><br />
<ul><br />
<li><b> RPU<sub>max</sub></b> is equal to the &alpha; and represents the maximum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li><b> RPU<sub>min</sub></b> is equal to the &alpha; * &delta; represents the minimum promoter activity<br />
</p><br />
</li><br />
<p><br />
<li> <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 />
</p><br />
</li><br />
<p><br />
<li> <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.<br />
<br />
</li><br />
</p><br />
</ul><br />
</ol><br />
</p><br />
<p align='justify'> 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>. </p><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><br />
<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><br />
<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><br />
<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><br />
<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><br />
</div><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><br />
<td class='row'>4.28</td><br />
<td class='row'>0.20</td><br />
<td class='row'>1.08</td><br />
<td class='row'>[0.36; 3.27]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>4.93</td><br />
<td class='row'>0.55</td><br />
<td class='row'>0.25</td><br />
<td class='row'>[0.03; 2.30]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>9.49</td><br />
<td class='row'>0.02</td><br />
<td class='row'>0.47</td><br />
<td class='row'>[0.07; 3.07]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>21.53</td><br />
<td class='row'>0.51</td><br />
<td class='row'>0.53</td><br />
<td class='row'>[0.08; 3.77]</td><br />
</tr><br />
</table><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><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> 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><br />
<br />
<center><br />
<a href="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="image"> <img alt="" src="https://static.igem.org/mediawiki/2011/a/af/E32_RPU_80.jpg" class="thumbimage" width="50%"></a><br />
</center><br />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<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><br />
</div></td><br />
</tr><br />
</table><br />
<p align='justify'> The estimated parameters of the Hill curves described in the figures 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> [(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><br />
<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><br />
<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><br />
<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><br />
<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 />
<p align='justify'> The operative parameters are summarized in the table below: </p><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><br />
<td class='row'>1.53</td><br />
<td class='row'>~0</td><br />
<td class='row'>7.95</td><br />
<td class='row'>[4.66;11.99]</td><br />
</tr><br />
<tr><br />
<td class='row'>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><br />
<td class='row'>B0032</td><br />
<td class='row'>3.16</td><br />
<td class='row'>~0</td><br />
<td class='row'>6.7</td><br />
<td class='row'>[4.45;10.05]</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>2.73</td><br />
<td class='row'>0.23</td><br />
<td class='row'>8.96</td><br />
<td class='row'>[8.27;9.71]</td><br />
</tr><br />
</table><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------AiiA and LuxI-----------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='enzymes'></a><br />
<h2>Characterization of enzymes AiiA and LuxI</h2><br />
<p align='justify'> 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 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 />
<div align="justify"><br />
<div class="thumbinner" style="width: 850px;"> <a href="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/40/Luxi_prod.jpg" class="thumbimage" width="85%" height="50%"></a></div><br />
</div><br />
<div align="justify"><br />
<p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p><br />
</div><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><br />
<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><br />
</table><br />
</div><br />
<br><br />
</p><br />
</p><br />
<p align='justify'> 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. </p><br />
<p align='justify'> The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity. </p><br />
<p align='justify'> 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 />
<table width='100%'><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/69/Aiia_LC_B0030.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/6/63/Aiia_LC_B0031.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
<tr><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/f/f5/Aiia_LC_B0032.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
<td width='50%'><div style="text-align:justify"><br />
<div class="thumbinner" width='100%'> <a href="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg"> <img alt="" src="https://static.igem.org/mediawiki/2011/8/8d/Aiia_LC_B0034.jpg" class="thumbimage" width="100%"></a></div><br />
</div></td><br />
</tr><br />
</table><br />
<br />
<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 />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------RBS---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='rbs'></a><br />
<h2>Characterization of the efficiency of RBSs from the community collection</h2><br />
RBSs were used for the fine tuning of CTRL+E. Different experimental conditions were assayed. <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 />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>0.40</td><br />
<td class='row'>1.6814</td><br />
<td class='row'>2.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.01</td><br />
<td class='row'>ND</td><br />
<td class='row'>0.04</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.19</td><br />
<td class='row'>0.4193</td><br />
<td class='row'>0.40</td><br />
<td class='row'>0,3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<br><br />
<br><br />
Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT, with GeneX=mRFP, AiiA or LuxI: <br><br />
<br><br />
<div align="center"><br />
<table class='data' width='70%'><br />
<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><br />
<tr><br />
<td class='row'>B0030</td><br />
<td class='row'>1.72</td><br />
<td class='row'>0.53</td><br />
<td class='row'>0.45</td><br />
<td class='row'>0,6</td><br />
</tr><br />
<tr><br />
<td class='row'>B0031</td><br />
<td class='row'>0.03</td><br />
<td class='row'>0.83</td><br />
<td class='row'>0.028</td><br />
<td class='row'>0,07</td><br />
</tr><br />
<tr><br />
<td class='row'>B0032</td><br />
<td class='row'>0.37</td><br />
<td class='row'>0.50</td><br />
<td class='row'>N.D.</td><br />
<td class='row'>0.3</td><br />
</tr><br />
<tr><br />
<td class='row'>B0034</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
<td class='row'>1</td><br />
</tr><br />
</table><br />
</div><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-----------growth---------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='growth'></a><br />
<h2>Identification of bacterial growth parameters</h2><br />
<br />
<br />
<p align='justify'> 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 />
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> 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><br />
<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><br />
<li>1:1</li><br />
<li>1:10</li><br />
<li>1:100</li><br />
<li>1:1000</li><br />
</ul><br />
in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm. </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 />
</ul><br />
</ol><br />
<p>The results are summarized in the table and in the figure below: </p><br />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'><b> Culture </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> TECAN </b></td><br />
<td class='row'><b> O.D.<sub>600</sub> Spectrophotometer </b></td><br />
<td class='row'><b> C.F.U. </b></td><br />
<td class='row'><b> Dilution Factor (10^) </b></td><br />
<td class='row'><b> N </b></td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,367950002 </td><br />
<td class='row'> 0,758004416 </td><br />
<td class='row'> 990 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 990000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,044649998 </td><br />
<td class='row'> 0,091982322 </td><br />
<td class='row'> 141 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 1410000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,004799999 </td><br />
<td class='row'> 0,009888356 </td><br />
<td class='row'> 136 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 136000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C1 </td><br />
<td class='row'> 0,000549998 </td><br />
<td class='row'> 0,001133037 </td><br />
<td class='row'> 20 </td><br />
<td class='row'> 6 </td><br />
<td class='row'> 200000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,54840003 </td><br />
<td class='row'> 1,129744917 </td><br />
<td class='row'> 165 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 16500000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,058200002 </td><br />
<td class='row'> 0,119896339 </td><br />
<td class='row'> 23 </td><br />
<td class='row'> 5 </td><br />
<td class='row'> 23000000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,008700002 </td><br />
<td class='row'> 0,017922652 </td><br />
<td class='row'> 251 </td><br />
<td class='row'> 3 </td><br />
<td class='row'> 2510000 </td><br />
</tr><br />
<tr><br />
<td class='row'> C2 </td><br />
<td class='row'> 0,000100002 </td><br />
<td class='row'> 0,000206011 </td><br />
<td class='row'> 24 </td><br />
<td class='row'> 4 </td><br />
<td class='row'> 2400000 </td><br />
</tr><br />
</table><br />
</center><br />
<br><br />
</p><br />
<center><br />
<img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"><br />
</center><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 />
</p><br />
<center><br />
<table width='50%' class='data'><br />
<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><br />
<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. </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 />
<a name='HSL'></a><br />
<h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2><br />
<p align='justify'> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 6</sub></b></td><br />
<td class='row'>32 </td><br />
<td class='row'>0.022 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>M9<sub>pH 7</sub></b></td><br />
<td class='row'>8 </td><br />
<td class='row'>0.087 </td><br />
</tr><br />
</table><br />
</center><br />
<p> 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 />
<center><br />
<table class='data'><br />
<tr><br />
<td class='row'></td><br />
<td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td><br />
<td class='row'><b>&gamma;<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 6</sub></b></td><br />
<td class='row'>&#8734; </td><br />
<td class='row'>0 </td><br />
</tr><br />
<tr><br />
<td class='row'><b>Culture<sub>pH 7</sub></b></td><br />
<td class='row'>19 </td><br />
<td class='row'>0.037 </td><br />
</tr><br />
</table><br />
</center><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; </p><br />
</p><br />
<div align="right"><small><a href="#indice" title="">^top</a></small></div><br />
<br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!-------------t9002--------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<!--------------------------------> <br />
<br />
<a name='t9002'></a><br />
<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'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/5/50/Activation_T9002.jpg" class="thumbimage" width="47%" height="50%"></a></div><br />
</div><br />
<br><br />
<div style='text-align:center'><br />
<div class="thumbinner" style="width:100%;"> <img alt="" src="https://static.igem.org/mediawiki/2011/4/4e/T9002_activation.jpg" class="thumbimage" width="100%"></a></div><br />
</div><br />
<center><br />
<table width='50%' class='data'><br />
<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 />
<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 />
</tr><br />
</table><br />
</center><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. 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>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Motivation2
Team:UNIPV-Pavia/Project/Motivation2
2011-09-21T19:39:35Z
<p>Edoardo Baldini: </p>
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Results<br />
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<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 />
<!-------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 />
<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 />
<br />
<br />
<br />
</html><br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Motivation2
Team:UNIPV-Pavia/Project/Motivation2
2011-09-21T19:39:05Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<html><br />
<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><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 />
<!-------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 />
<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 />
<br />
<br />
</html><br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Motivation2
Team:UNIPV-Pavia/Project/Motivation2
2011-09-21T19:38:11Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<html><br />
<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><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 />
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<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 />
<!-------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 />
<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 />
</html><br />
{{end}}</div>
Edoardo Baldini
http://2011.igem.org/Team:UNIPV-Pavia/Project/Motivation2
Team:UNIPV-Pavia/Project/Motivation2
2011-09-21T19:37:43Z
<p>Edoardo Baldini: </p>
<hr />
<div>{{main}}<br />
<html><br />
<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 />
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<!--------------------------------><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 />
<!-------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 />
<br />
</html><br />
{{end}}</div>
Edoardo Baldini