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| | <html> | | <html> |
| - | <script type="text/javascript" src="https://2011.igem.org/Team:UNIPV-Pavia/Templates/jquery_js?action=raw&ctype=text/javascript"></script> | + | <h2 class="art-postheader"> Results </h2> |
| - | <script type="text/javascript"> | + | <div class="cleared"></div> |
| - |
| + | <div class="art-postcontent"> |
| - | $(document).ready(function() {
| + | |
| | | | |
| - | var nam = new Array(); | + | <!--------------------------------> |
| - | nam[1] = "Niccolò Franceschi"; | + | <!--------------------------------> |
| - | nam[2] = "Davide Bianchini"; | + | <!--------------------------------> |
| - | nam[3] = "Nicolò Politi"; | + | <!-----------MENU-----------------> |
| - | nam[4] = "Prof. Paolo Magni"; | + | <!--------------------------------> |
| - | nam[5] = "Daniele Sartori"; | + | <!--------------------------------> |
| - | nam[6] = "Edoardo Baldini"; | + | <!--------------------------------> |
| - | nam[7] = "Tommaso Goggia"; | + | <br> |
| - | nam[8] = "Viola Ghio"; | + | <a name='indice'></a> |
| - | nam[9] = "Emanuela Pasi"; | + | <div class="cleared"></div> |
| - | nam[10] = "Susanna Zucca"; | + | <div class="art-postcontent"> |
| - | nam[11] = "Federica Sampietro";
| + | <table id="toc" class="toc"> |
| - | nam[12] = "Prof. Cusella";
| + | <tr> |
| - | nam[13] = "Lorenzo Pasotti";
| + | <td><div id="toctitle"> |
| - | nam[14] = "Giuseppe Bertoni";
| + | <h2>Contents</h2> |
| - | | + | </div> |
| - | | + | <ul> |
| - | var port = new Array();
| + | <li class="toclevel-1"><a href="#assembly"><span class="tocnumber">1</span> <span class="toctext">Part assembly</span></a> |
| - | port[1] = "https://static.igem.org/mediawiki/2011/6/6f/Niccolofranceschi.jpg";
| + | <li class="toclevel-1"><a href="#characterization"><span class="tocnumber">1</span> <span class="toctext">Characterization of basic modules</span></a> |
| - | port[2] = "https://static.igem.org/mediawiki/2011/4/4f/Davidebianchini.jpg";
| + | <ul> |
| - | port[3] = "https://static.igem.org/mediawiki/2011/b/bf/Nicolopoliti.jpg";
| + | <li class="toclevel-2"><a href="#promoters"><span class="tocnumber">2.1</span> <span class="toctext">Promoter characterization</span></a></li> |
| - | port[4] = "https://static.igem.org/mediawiki/2011/8/87/Paolomagni.jpg";
| + | <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> |
| - | port[5] = "https://static.igem.org/mediawiki/2011/0/06/Danielesartori.jpg";
| + | <li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.3</span> <span class="toctext">Characterization of RBS efficiency</span></a></li> |
| - | port[6] = "https://static.igem.org/mediawiki/2011/0/04/Edoardobaldini.jpg";
| + | </ul> |
| - | port[7] = "https://static.igem.org/mediawiki/2011/8/82/Tommasogoggia.jpg";
| + | <li class="toclevel-1"><a href="#growth"><span class="tocnumber">3</span> <span class="toctext">Identification of bacterial growth parameters</span></a></li> |
| - | port[8] = "https://static.igem.org/mediawiki/2011/4/46/Violaghio.jpg";
| + | <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> |
| - | port[9] = "https://static.igem.org/mediawiki/2011/6/61/Emanuelapasi.jpg";
| + | <li class="toclevel-1"><a href="#t9002"><span class="tocnumber">5</span> <span class="toctext">Characterization of BBa_T9002 biosensor</span></a></li> |
| - | port[10] = "https://static.igem.org/mediawiki/2011/9/9e/Susannazucca.jpg";
| + | </ul></td> |
| - | port[11] = "https://static.igem.org/mediawiki/2011/f/f5/Federicasampietro.jpg";
| + | </tr> |
| - | port[12] = "https://static.igem.org/mediawiki/2011/f/f5/Federicasampietro.jpg";
| + | </table> |
| - | port[13] = "https://static.igem.org/mediawiki/2011/f/f5/Federicasampietro.jpg";
| + | </div> |
| - | port[14] = "https://static.igem.org/mediawiki/2011/f/f5/Federicasampietro.jpg";
| + | <script>if (window.showTocToggle) { var tocShowText = "show"; var tocHideText = "hide"; showTocToggle(); } </script> |
| - | | + | <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> |
| - | var des = new Array();
| + | |
| - | 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.";
| + | |
| - | 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.";
| + | |
| - | 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.";
| + | |
| - | 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.<br>For 20 years, I was a national referee of Handball. I like skiing and teaching to those students that want to learn.";
| + | |
| - | 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>I'm very fond of politics, art, reading and music.";
| + | |
| - | 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.";
| + | |
| - | 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! My name is Tommaso, and 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 know and work with other students, also from different disciplines, and learn a lot of new things that I wouldn't have considered without iGEM. Personally, I like skiing very much, bot also football and running. See you all at the jamboree!";
| + | |
| - | 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!";
| + | |
| - | 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 have an insane passion for frogs!";
| + | |
| - | des[10] = "10 10 10 10 10"; | + | |
| - | des[11] = "Undergraduate student in Molecular Biology.<br>Experience as summer student in MRC-LMB, Cambridge UK in 2009.<br>Hello! I'm 24 years old and I come from Puglia, a sunny and beautiful place in Southern Italy!
I like playing the violin and cooking for my iGEM friends! See you in Boston!";
| + | |
| - | des[12] = "12 12 12 12 12";
| + | |
| - | des[13] = "13 13 13 13 13";
| + | |
| - | des[14] = "14 14 14 14 14";
| + | |
| - | | + | |
| | | | |
| - | $("#tipo1").mouseenter(function() { | + | <!--------------------------------> |
| - | $("#teamnam").empty();
| + | <!--------------------------------> |
| - | $("#teamful").empty();
| + | <!--------------------------------> |
| - | $("#teamnam").append(nam[1]);
| + | <!-----------ASSEMBLY-------------> |
| - | $("#teamful").append(des[1]);
| + | <!--------------------------------> |
| - | $("#foto").attr("src", port[1]); | + | <!--------------------------------> |
| - | }); | + | <!--------------------------------> |
| - | $("#tipo2").mouseenter(function() { | + | |
| - | $("#teamnam").empty();
| + | |
| - | $("#teamful").empty();
| + | |
| - | $("#teamnam").append(nam[2]);
| + | |
| - | $("#teamful").append(des[2]);
| + | |
| - | $("#foto").attr("src", port[2]);
| + | |
| - | }); | + | |
| - | $("#tipo3").mouseenter(function() { | + | |
| - | $("#teamnam").empty();
| + | |
| - | $("#teamful").empty();
| + | |
| - | $("#teamnam").append(nam[3]);
| + | |
| - | $("#teamful").append(des[3]);
| + | |
| - | $("#foto").attr("src", port[3]);
| + | |
| - | }); | + | |
| - | $("#tipo4").mouseenter(function() {
| + | |
| - | $("#teamnam").empty();
| + | |
| - | $("#teamful").empty();
| + | |
| - | $("#teamnam").append(nam[4]);
| + | |
| - | $("#teamful").append(des[4]);
| + | |
| - | $("#foto").attr("src", port[4]);
| + | |
| - | });
| + | |
| - | $("#tipo5").mouseenter(function() {
| + | |
| - | $("#teamnam").empty();
| + | |
| - | $("#teamful").empty();
| + | |
| - | $("#teamnam").append(nam[5]);
| + | |
| - | $("#teamful").append(des[5]);
| + | |
| - | $("#foto").attr("src", port[5]);
| + | |
| - | });
| + | |
| - | $("#tipo6").mouseenter(function() {
| + | |
| - | $("#teamnam").empty();
| + | |
| - | $("#teamful").empty();
| + | |
| - | $("#teamnam").append(nam[6]);
| + | |
| - | $("#teamful").append(des[6]);
| + | |
| - | $("#foto").attr("src", port[6]);
| + | |
| - | });
| + | |
| - | $("#tipo7").mouseenter(function() {
| + | |
| - | $("#teamnam").empty();
| + | |
| - | $("#teamful").empty();
| + | |
| - | $("#teamnam").append(nam[7]);
| + | |
| - | $("#teamful").append(des[7]);
| + | |
| - | $("#foto").attr("src", port[7]);
| + | |
| - | });
| + | |
| - | $("#tipo8").mouseenter(function() {
| + | |
| - | $("#teamnam").empty();
| + | |
| - | $("#teamful").empty();
| + | |
| - | $("#teamnam").append(nam[8]);
| + | |
| - | $("#teamful").append(des[8]);
| + | |
| - | $("#foto").attr("src", port[8]);
| + | |
| - | });
| + | |
| - | $("#tipo9").mouseenter(function() {
| + | |
| - | $("#teamnam").empty();
| + | |
| - | $("#teamful").empty();
| + | |
| - | $("#teamnam").append(nam[9]);
| + | |
| - | $("#teamful").append(des[9]);
| + | |
| - | $("#foto").attr("src", port[9]);
| + | |
| - | });
| + | |
| - | $("#tipo10").mouseenter(function() {
| + | |
| - | $("#teamnam").empty();
| + | |
| - | $("#teamful").empty();
| + | |
| - | $("#teamnam").append(nam[10]);
| + | |
| - | $("#teamful").append(des[10]);
| + | |
| - | $("#foto").attr("src", port[10]);
| + | |
| - | });
| + | |
| - | $("#tipo11").mouseenter(function() {
| + | |
| - | $("#teamnam").empty();
| + | |
| - | $("#teamful").empty();
| + | |
| - | $("#teamnam").append(nam[11]);
| + | |
| - | $("#teamful").append(des[11]);
| + | |
| - | $("#foto").attr("src", port[11]);
| + | |
| - | });
| + | |
| - | | + | |
| - | | + | |
| - | | + | |
| | | | |
| - | $("#tipoT1").mouseenter(function() { | + | <a name='assembly'></a> |
| - | $("#teamnam").empty();
| + | <h1>Parts assembly</h1> |
| - | $("#teamful").empty();
| + | 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>. |
| - | $("#teamnam").append(nam[1]);
| + | <div align="right"><small><a href="#indice" title="">^top</a></small></div> |
| - | $("#teamful").append(des[1]);
| + | |
| - | $("#foto").attr("src", port[1]); | + | <!--------------------------------> |
| - | }); | + | <!--------------------------------> |
| - | $("#tipoT2").mouseenter(function() { | + | <!--------------------------------> |
| - | $("#teamnam").empty();
| + | <!-------CHARACTERIZATION---------> |
| - | $("#teamful").empty();
| + | <!--------------------------------> |
| - | $("#teamnam").append(nam[2]);
| + | <!--------------------------------> |
| - | $("#teamful").append(des[2]);
| + | <!--------------------------------> |
| - | $("#foto").attr("src", port[2]);
| + | |
| - | });
| + | <a name='characterization'></a> |
| - | $("#tipoT3").mouseenter(function() {
| + | <h1>Characterization of basic modules</h1> |
| - | $("#teamnam").empty();
| + | |
| - | $("#teamful").empty();
| + | <!--------------------------------> |
| - | $("#teamnam").append(nam[3]);
| + | <!--------------------------------> |
| - | $("#teamful").append(des[3]);
| + | <!--------------------------------> |
| - | $("#foto").attr("src", port[3]);
| + | <!--------pTet and pLux-----------> |
| - | }); | + | <!--------------------------------> |
| - | $("#tipoT4").mouseenter(function() { | + | <!--------------------------------> |
| - | $("#teamnam").empty();
| + | <!--------------------------------> |
| - | $("#teamful").empty();
| + | |
| - | $("#teamnam").append(nam[4]);
| + | <a name='promoters'></a> |
| - | $("#teamful").append(des[4]);
| + | <h2>Characterization of promoters pTet and pLux</h2> |
| - | $("#foto").attr("src", port[4]);
| + | <p align='justify'> |
| - | });
| + | <p>Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection.</p> |
| - | $("#tipoT5").mouseenter(function() {
| + | <p>The assembled RBSs are:</p> |
| - | $("#teamnam").empty();
| + | <br> |
| - | $("#teamful").empty();
| + | <div align='center'> |
| - | $("#teamnam").append(nam[5]);
| + | <table class='data'> |
| - | $("#teamful").append(des[5]);
| + | <tr> |
| - | $("#foto").attr("src", port[5]);
| + | <td class="row"><b>BioBrick code</b></td> |
| - | });
| + | <td><b> Declared efficiency</b></td> |
| - | $("#tipoT6").mouseenter(function() {
| + | </tr> |
| - | $("#teamnam").empty();
| + | <tr> |
| - | $("#teamful").empty();
| + | <td class="row">BBa_B0030 </td> |
| - | $("#teamnam").append(nam[6]);
| + | <td class="row"> 0,6</td> |
| - | $("#teamful").append(des[6]);
| + | </tr> |
| - | $("#foto").attr("src", port[6]);
| + | <tr> |
| - | });
| + | <td class="row">BBa_B0031 </td> |
| - | $("#tipoT7").mouseenter(function() {
| + | <td class="row"> 0,07</td> |
| - | $("#teamnam").empty();
| + | </tr> |
| - | $("#teamful").empty();
| + | <tr> |
| - | $("#teamnam").append(nam[7]);
| + | <td class="row">BBa_B0032 </td> |
| - | $("#teamful").append(des[7]);
| + | <td class="row"> 0,3</td> |
| - | $("#foto").attr("src", port[7]);
| + | </tr> |
| - | });
| + | <tr> |
| - | $("#tipoT8").mouseenter(function() {
| + | <td class="row">BBa_B0034 </td> |
| - | $("#teamnam").empty();
| + | <td class="row"> 1</td> |
| - | $("#teamful").empty();
| + | </tr> |
| - | $("#teamnam").append(nam[8]);
| + | </table> |
| - | $("#teamful").append(des[8]);
| + | </div> |
| - | $("#foto").attr("src", port[8]);
| + | <br> |
| - | }); | + | <div align="justify"> |
| - | $("#tipoT9").mouseenter(function() { | + | <p>For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.</p> |
| - | $("#teamnam").empty();
| + | <p>The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. |
| - | $("#teamful").empty();
| + | 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> |
| - | $("#teamnam").append(nam[9]);
| + | <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> |
| - | $("#teamful").append(des[9]);
| + | </p> |
| - | $("#foto").attr("src", port[9]);
| + | <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> |
| - | $("#tipoT10").mouseenter(function() { | + | <p> |
| - | $("#teamnam").empty();
| + | <ol> |
| - | $("#teamful").empty();
| + | <ul> |
| - | $("#teamnam").append(nam[10]);
| + | <li><b> RPU<sub>max</sub></b> is equal to the α and represents the maximum promoter activity |
| - | $("#teamful").append(des[10]);
| + | </p> |
| - | $("#foto").attr("src", port[10]);
| + | </li> |
| - | }); | + | <p> |
| - | $("#tipoT11").mouseenter(function() { | + | <li><b> RPU<sub>min</sub></b> is equal to the α * δ represents the minimum promoter activity |
| - | $("#teamnam").empty();
| + | </p> |
| - | $("#teamful").empty();
| + | </li> |
| - | $("#teamnam").append(nam[11]);
| + | <p> |
| - | $("#teamful").append(des[11]);
| + | <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 |
| - | $("#foto").attr("src", port[11]);
| + | </p> |
| - | });
| + | </li> |
| - | $("#tipoT12").mouseenter(function() {
| + | <p> |
| - | $("#teamnam").empty();
| + | <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. |
| - | $("#teamful").empty();
| + | |
| - | $("#teamnam").append(nam[12]);
| + | </li> |
| - | $("#teamful").append(des[12]);
| + | </p> |
| - | $("#foto").attr("src", port[12]);
| + | </ul> |
| - | }); | + | </ol> |
| - | $("#tipoT13").mouseenter(function() { | + | </p> |
| - | $("#teamnam").empty();
| + | <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> |
| - | $("#teamful").empty();
| + | <table class='data' width='100%'> |
| - | $("#teamnam").append(nam[13]);
| + | <tr> |
| - | $("#teamful").append(des[13]);
| + | <td class="row"><b>RBS</b></td> |
| - | $("#foto").attr("src", port[13]);
| + | <td class="row"><b>α<sub>p<sub>Lux</sub></sub> [(AUr/min)/cell]</b></td> |
| - | });
| + | <td class="row"><b>δ<sub>p<sub>Lux</sub></sub> [-]</b></td> |
| - | $("#tipoT14").mouseenter(function() {
| + | <td class="row"><b>η<sub>p<sub>Lux</sub></sub> [-]</b></td> |
| - | $("#teamnam").empty();
| + | <td class="row"><b>k<sub>p<sub>Lux</sub></sub> [ng/ml]</b></td> |
| - | $("#teamful").empty();
| + | </tr> |
| - | $("#teamnam").append(nam[14]);
| + | <tr> |
| - | $("#teamful").append(des[14]);
| + | <td class="row">BBa_B0030</td> |
| - | $("#foto").attr("src", port[14]);
| + | <td class="row">438 [10]</td> |
| - | }); | + | <td class="row">0.05 [>100]</td> |
| - | | + | <td class="row">2 [47]</td> |
| - | | + | <td class="row">1.88 [27]</td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td class="row">BBa_B0031</td> |
| | + | <td class="row">9.8 [7]</td> |
| | + | <td class="row">0.11 [57]</td> |
| | + | <td class="row">1.2 [29]</td> |
| | + | <td class="row">1.5 [26]</td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td class="row">BBa_B0032</td> |
| | + | <td class="row">206 [3]</td> |
| | + | <td class="row">0 [>>100]</td> |
| | + | <td class="row">1.36 [10]</td> |
| | + | <td class="row">1.87 [9]</td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td class="row">BBa_B0034</td> |
| | + | <td class="row">1105 [6]</td> |
| | + | <td class="row">0.02 [>100]</td> |
| | + | <td class="row">1.33 [19]</td> |
| | + | <td class="row">2.34 [18]</td> |
| | + | </tr> |
| | + | </table> |
| | + | <div align="center">Data are provided as average [CV%].</div> |
| | + | <br> |
| | + | <p>The operative parameters are summarized in the table below:</p> |
| | + | </div> |
| | + | <table align='center' class='data' width='100%'> |
| | + | <tr> |
| | + | <td class='row'><b>RBS</b></td> |
| | + | <td class='row'><b>RPU<sub>max</sub></b></td> |
| | + | <td class='row'><b>RPU<sub>min</sub></b></td> |
| | + | <td class='row'><b>Switch point [nM]</b></td> |
| | + | <td class='row'><b>Linear boundaries [MIN; MAX] [nM]</b></td> |
| | + | </tr> |
| | + | <tr> |
| | + | <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> |
| | + | </tr> |
| | + | <tr> |
| | + | <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> |
| | + | </tr> |
| | + | <tr> |
| | + | <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> |
| | + | </tr> |
| | + | <tr> |
| | + | <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> |
| | + | </tr> |
| | + | </table> |
| | + | <table width='100%'> |
| | + | <tr> |
| | + | <td width='50%'><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></td> |
| | + | <td width='50%'><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></td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td width='50%'><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></td> |
| | + | <td width='50%'><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></td> |
| | + | </tr> |
| | + | </table> |
| | + | <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> |
| | + | <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> |
| | | | |
| | + | <center> |
| | + | <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> |
| | + | </center> |
| | + | <table width='100%'> |
| | + | <tr> |
| | + | <td width='50%'><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></td> |
| | + | <td width='50%'><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></td> |
| | + | </tr> |
| | + | </table> |
| | + | <p align='justify'> The estimated parameters of the Hill curves described in the figures are summarized in the table below: </p> |
| | + | <br> |
| | + | <table class='data' width='100%' title='parameter value'> |
| | + | <tr> |
| | + | <td class="row"><b>RBS</b></td> |
| | + | <td class="row"><b>α<sub>p<sub>Tet</sub></sub> [(AUr/min)/cell]</b></td> |
| | + | <td class="row"><b>δ<sub>p<sub>Tet</sub></sub> [-]</b></td> |
| | + | <td class="row"><b>η<sub>p<sub>Tet</sub></sub> [-]</b></td> |
| | + | <td class="row"><b>k<sub>p<sub>Tet</sub></sub> [nM]</b></td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td class="row">BBa_B0030</td> |
| | + | <td class="row">230.67 [3.7]</td> |
| | + | <td class="row">0.028 [91.61]</td> |
| | + | <td class="row">4.61 [23.73]</td> |
| | + | <td class="row">8.75 [4.16]</td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td class="row">BBa_B0031</td> |
| | + | <td class="row">ND</td> |
| | + | <td class="row">ND</td> |
| | + | <td class="row">ND</td> |
| | + | <td class="row">ND</td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td class="row">BBa_B0032</td> |
| | + | <td class="row">55.77 [12]</td> |
| | + | <td class="row">1.53E-11 [>>100]</td> |
| | + | <td class="row">4.98 [57.62]</td> |
| | + | <td class="row">7.26 [14.98]</td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td class="row">BBa_B0034</td> |
| | + | <td class="row">120 [5.95]</td> |
| | + | <td class="row">0.085 [40.6]</td> |
| | + | <td class="row">24.85 [47.6]</td> |
| | + | <td class="row">9 [5.43]</td> |
| | + | </tr> |
| | + | </table> |
| | + | Data are provided as average [CV%] <br> |
| | + | <br> |
| | + | <p align='justify'> The operative parameters are summarized in the table below: </p> |
| | + | <table align='center' class='data' width='100%'> |
| | + | <tr> |
| | + | <td class='row'><b>RBS</b></td> |
| | + | <td class='row'><b>RPU<sub>max</sub></b></td> |
| | + | <td class='row'><b>RPU<sub>min</sub></b></td> |
| | + | <td class='row'><b>Switch point [ng/ml]</b></td> |
| | + | <td class='row'><b>Linear boundaries [MIN; MAX] [ng/ml]</b></td> |
| | + | </tr> |
| | + | <tr> |
| | + | <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> |
| | + | </tr> |
| | + | <tr> |
| | + | <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> |
| | + | </tr> |
| | + | <tr> |
| | + | <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> |
| | + | </tr> |
| | + | <tr> |
| | + | <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> |
| | + | </tr> |
| | + | </table> |
| | + | <div align="right"><small><a href="#indice" title="">^top</a></small></div> |
| | | | |
| | + | <!--------------------------------> |
| | + | <!--------------------------------> |
| | + | <!--------------------------------> |
| | + | <!--------AiiA and LuxI-----------> |
| | + | <!--------------------------------> |
| | + | <!--------------------------------> |
| | + | <!--------------------------------> |
| | | | |
| | + | <a name='enzymes'></a> |
| | + | <h2>Characterization of enzymes AiiA and LuxI</h2> |
| | + | <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> |
| | + | The parameters V<sub>max</sub>, k<sub>M,LuxI</sub> and α<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. |
| | + | <div align="justify"> |
| | + | <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> |
| | + | </div> |
| | + | <div align="justify"> |
| | + | <p>The estimated parameters for the enzymatic activity of LuxI are reported in the table below:</p> |
| | + | </div> |
| | + | <div align=center"> |
| | + | <table align="center" class='data'> |
| | + | <tr> |
| | + | <td class='row'><b>V<sub>max</sub></b></td> |
| | + | <td class='row'><b>k<sub>M,LuxI</sub></b></td> |
| | + | <td class='row'><b>α<sub>B0030</sub></b></td> |
| | + | <td class='row'><b>α<sub>B0031</sub></b></td> |
| | + | <td class='row'><b>α<sub>B0032</sub></b></td> |
| | + | <td class='row'><b>α<sub>B0034</sub></b></td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td class='row'>3.56*10<sup>-9</sup></td> |
| | + | <td class='row'>6.87*10<sup>3</sup></td> |
| | + | <td class='row'> 87 </td> |
| | + | <td class='row'>8.5</td> |
| | + | <td class='row'>ND</td> |
| | + | <td class='row'> 252 </td> |
| | + | </tr> |
| | + | </table> |
| | + | </div> |
| | + | <br> |
| | + | </p> |
| | + | </p> |
| | + | <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. |
| | + | 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> |
| | + | <p align='justify'> The AiiA enzyme activity has been characterized under the regulation of p<sub>tet</sub> promoter, assaying its enzymatic activity. </p> |
| | + | <p align='justify'> The parameters k<sub>cat</sub>, k<sub>M,AiiA</sub> and α<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. |
| | + | Unfortunately, their estimation revealed difficult.<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. |
| | + | <table width='100%'> |
| | + | <tr> |
| | + | <td width='50%'><div style="text-align:justify"> |
| | + | <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> |
| | + | </div></td> |
| | + | <td width='50%'><div style="text-align:justify"> |
| | + | <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> |
| | + | </div></td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td width='50%'><div style="text-align:justify"> |
| | + | <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> |
| | + | </div></td> |
| | + | <td width='50%'><div style="text-align:justify"> |
| | + | <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> |
| | + | </div></td> |
| | + | </tr> |
| | + | </table> |
| | | | |
| | | | |
| - | });
| + | 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. |
| - | </script>
| + | <div align="right"><small><a href="#indice" title="">^top</a></small></div> |
| - | <style type="text/css"> | + | |
| - | #teamcon { | + | <!--------------------------------> |
| - | width:756px;
| + | <!--------------------------------> |
| - | height:1200px;
| + | <!--------------------------------> |
| - | position:relative;
| + | <!--------------RBS---------------> |
| - | z-index:0;
| + | <!--------------------------------> |
| - | }
| + | <!--------------------------------> |
| - | #teamfoto {
| + | <!--------------------------------> |
| - | width:750px;
| + | |
| - | position:relative;
| + | <a name='rbs'></a> |
| - | z-index:1;
| + | <h2>Characterization of the efficiency of RBSs from the community collection</h2> |
| - | }
| + | RBSs were used for the fine tuning of CTRL+E. Different experimental conditions were assayed. <br> |
| - | #xxx {
| + | <br> |
| - | width:100px;
| + | Estimated efficiencies in pSB4C5 plasmid with -RBSx-mRFP-TT coding sequence under the control of the specified promoter: <br> |
| - | height:100px;
| + | <br> |
| - | position:absolute;
| + | <div align="center"> |
| - | left:6px;
| + | <table class='data' width='70%'> |
| - | top:30px;
| + | <tr> |
| - | background-color:#123456;
| + | <td class='row'><b>RBS</b></td> |
| - | color:#ffffff;
| + | <td class='row'><b>eff<sub>p<sub>Lux</sub></sub></b></td> |
| - | z-index:0;
| + | <td class='row'><b>eff<sub>p<sub>Tet</sub></sub></b></td> |
| - | }
| + | <td class='row'><b>eff<sub>J23101</sub></b></td> |
| - | #teambox {
| + | <td class='row'><b>Declared efficiency</b></td> |
| - | width:756px;
| + | </tr> |
| - | height:300px;
| + | <tr> |
| - | position:absolute;
| + | <td class='row'>B0030</td> |
| - | left:-1px;
| + | <td class='row'>0.40</td> |
| - | top:502px;
| + | <td class='row'>1.6814</td> |
| - | background-color:#889399;
| + | <td class='row'>2.45</td> |
| - | color:#ffffff;
| + | <td class='row'>0,6</td> |
| - | z-index:0;
| + | </tr> |
| - | }
| + | <tr> |
| - | #teamnam {
| + | <td class='row'>B0031</td> |
| - | width:500px;
| + | <td class='row'>0.01</td> |
| - | position:relative;
| + | <td class='row'>ND</td> |
| - | left:250px;
| + | <td class='row'>0.04</td> |
| - | top:40px;
| + | <td class='row'>0,07</td> |
| - | font-weight:bold;
| + | </tr> |
| - | z-index:0;
| + | <tr> |
| - | }
| + | <td class='row'>B0032</td> |
| - | #teamful {
| + | <td class='row'>0.19</td> |
| - | width:500px;
| + | <td class='row'>0.4193</td> |
| - | position:relative;
| + | <td class='row'>0.40</td> |
| - | left:249px;
| + | <td class='row'>0,3</td> |
| - | top:63px;
| + | </tr> |
| - | z-index:0;
| + | <tr> |
| - | }
| + | <td class='row'>B0034</td> |
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| - | font-weight: bold;
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| + | |
| - | </style> | + | |
| - | <h2 class="art-postheader">The Team</h2>
| + | |
| - | | + | |
| - | <div id="teamcon"> <img src="https://static.igem.org/mediawiki/2011/1/1f/UNIPV-team.jpg" align="left" id="teamfoto"> | + | |
| - | <div id="tipo1"> </div>
| + | |
| - | <div id="tipo2"> </div>
| + | |
| - | <div id="tipo3"> </div>
| + | |
| - | <div id="tipo4"> </div>
| + | |
| - | <div id="tipo5"> </div>
| + | |
| - | <div id="tipo6"> </div>
| + | |
| - | <div id="tipo7"> </div>
| + | |
| - | <div id="tipo8"> </div>
| + | |
| - | <div id="tipo9"> </div>
| + | |
| - | <div id="tipo10"> </div>
| + | |
| - | <div id="tipo11"> </div>
| + | |
| - | <div id="teambox">
| + | |
| - | <div id="xxx"><img src="https://static.igem.org/mediawiki/2011/8/8e/UNIPV_iGEMlogo.png" width='195' id="foto"></div>
| + | |
| - | <div id="teamnam"> </div>
| + | |
| - | <div id="teamful"> </div>
| + | |
| | </div> | | </div> |
| - | <div id="instructors">Instructors</div> | + | <br> |
| - | <div id="advisors">Advisors</div> | + | <br> |
| - | <div id="students">Students</div>
| + | Estimated efficiencies in pSB4C5 plasmid with pTet-RBSx-GeneX-TT, with GeneX=mRFP, AiiA or LuxI: <br> |
| - | <div id="tipoT1">Niccolò Franceschi</div>
| + | <br> |
| - | <div id="tipoT2">Davide Bianchini</div>
| + | <div align="center"> |
| - | <div id="tipoT3">Nicolò Politi</div>
| + | <table class='data' width='70%'> |
| - | <div id="tipoT4">Prof. Paolo Magni</div>
| + | <tr> |
| - | <div id="tipoT5">Daniele Sartori</div>
| + | <td class='row'><b>RBS</b></td> |
| - | <div id="tipoT6">Edoardo Baldini</div>
| + | <td class='row'><b>eff<sub>mRFP</sub></b></td> |
| - | <div id="tipoT7">Tommaso Goggia</div>
| + | <td class='row'><b>eff<sub>AiiA</sub></b></td> |
| - | <div id="tipoT8">Viola Ghio</div>
| + | <td class='row'><b>eff<sub>LuxI</sub></b></td> |
| - | <div id="tipoT9">Emanuela Pasi</div>
| + | <td class='row'><b>Declared efficiency</b></td> |
| - | <div id="tipoT10">Susanna Zucca</div>
| + | </tr> |
| - | <div id="tipoT11">Federica Sampietro</div>
| + | <tr> |
| - | <div id="tipoT12">Prof. Maria Gabriella Cusella</div>
| + | <td class='row'>B0030</td> |
| - | <div id="tipoT13">Lorenzo Pasotti</div>
| + | <td class='row'>1.72</td> |
| - | <div id="tipoT14">Giuseppe Bertoni</div> | + | <td class='row'>0.53</td> |
| - | </div> | + | <td class='row'>0.45</td> |
| - | </html> | + | <td class='row'>0,6</td> |
| | + | </tr> |
| | + | <tr> |
| | + | <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> |
| | + | </tr> |
| | + | <tr> |
| | + | <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> |
| | + | </tr> |
| | + | <tr> |
| | + | <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> |
| | + | </tr> |
| | + | </table> |
| | + | </div> |
| | + | <div align="right"><small><a href="#indice" title="">^top</a></small></div> |
| | + | |
| | + | <!--------------------------------> |
| | + | <!--------------------------------> |
| | + | <!--------------------------------> |
| | + | <!-----------growth---------------> |
| | + | <!--------------------------------> |
| | + | <!--------------------------------> |
| | + | <!--------------------------------> |
| | + | |
| | + | <a name='growth'></a> |
| | + | <h2>Identification of bacterial growth parameters</h2> |
| | | | |
| | + | |
| | + | <p align='justify'> The bacterial growth curve has been modelled as a logistic curve and is represented by the following equation: <br> |
| | + | <br> |
| | + | dN/dt=N*μ*(N<sub>max</sub>-N)/N<sub>max</sub><br> |
| | + | N(0)=n<sub>0</sub> <br> |
| | + | <br> |
| | + | where μ 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. |
| | + | <ol> |
| | + | <ul> |
| | + | <li>Two cultures C1 and C2 (MGZ1 cells) were grown in 1ml M9 medium till saturation (ON liquid culture, 37°C, 220 rpm).</li> |
| | + | <li>Next morning, both C1 and C2 were diluted in M9 medium with a final volume of 1ml with the following dilution factors: |
| | + | <ul> |
| | + | <li>1:1</li> |
| | + | <li>1:10</li> |
| | + | <li>1:100</li> |
| | + | <li>1:1000</li> |
| | + | </ul> |
| | + | in fresh M9 medium. These cultures were grown for further 1 hour at 37°C, 220 rpm. </li> |
| | + | <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> |
| | + | <em>NB: from now on, cultures must be placed in ice to stop cell growth.</em></li> |
| | + | <li>At the same time, proper dilution of the cultures were plated on LB agar plates.<br> |
| | + | <em>NB: All the dilutions are performed moving 100 μl of culture in previously ice-chilled 900 μl fresch M9. 100 μl of the final dilution are plated (It still represents a 1:10 dilution!)</em></li> |
| | + | <li>Plates were grown overnight and next morning C.F.U. were counted.</li> |
| | + | <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> |
| | + | <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> |
| | + | </ul> |
| | + | </ol> |
| | + | <p>The results are summarized in the table and in the figure below: </p> |
| | + | <center> |
| | + | <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> |
| | + | <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> |
| | + | <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> |
| | + | <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> |
| | + | <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> |
| | + | <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> |
| | + | <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> |
| | + | <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> |
| | + | <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> |
| | + | </table> |
| | + | </center> |
| | + | <br> |
| | + | </p> |
| | + | <center> |
| | + | <img alt="" src="https://static.igem.org/mediawiki/2011/3/35/UNIPV_ODvsCFU.png" width="90%"> |
| | + | </center> |
| | + | <p>The estimation of μ 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> |
| | + | The estimated parameters are summarized in the table below: <br> |
| | + | </p> |
| | + | <center> |
| | + | <table width='50%' class='data'> |
| | + | <tr> |
| | + | <td class='row'><b>N<sub>max</sub> [cell number]</b></td> |
| | + | <td class='row'><b>μ [min<sup>-1</sup>]</b></td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td class='row'>1*10<sup>9</sup></td> |
| | + | <td class='row'>0.004925</td> |
| | + | </tr> |
| | + | </table> |
| | + | </center> |
| | + | <p> </p> |
| | + | <p>The reported value of μ corresponds to a doubling time of 142 minutes. </p> |
| | + | <div align="right"><small><a href="#indice" title="">^top</a></small></div> |
| | + | |
| | + | <!--------------------------------> |
| | + | <!--------------------------------> |
| | + | <!--------------------------------> |
| | + | <!--------------HSL---------------> |
| | + | <!--------------------------------> |
| | + | <!--------------------------------> |
| | + | <!--------------------------------> |
| | + | |
| | + | <a name='HSL'></a> |
| | + | <h2>Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</h2> |
| | + | <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> |
| | + | Two different M9 media were prepared, one with the nominal pH (7.0) and one with pH=6.0.<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> |
| | + | <ul> |
| | + | <li>t=0 h;</li> |
| | + | <li>t=1 h;</li> |
| | + | <li>t=2 h;</li> |
| | + | <li>t=4 h;</li> |
| | + | <li>t=8 h;</li> |
| | + | </ul> |
| | + | <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> |
| | + | <center> |
| | + | <table class='data'> |
| | + | <tr> |
| | + | <td class='row'></td> |
| | + | <td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td> |
| | + | <td class='row'><b>γ<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td class='row'><b>M9<sub>pH 6</sub></b></td> |
| | + | <td class='row'>32 </td> |
| | + | <td class='row'>0.022 </td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td class='row'><b>M9<sub>pH 7</sub></b></td> |
| | + | <td class='row'>8 </td> |
| | + | <td class='row'>0.087 </td> |
| | + | </tr> |
| | + | </table> |
| | + | </center> |
| | + | <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> |
| | + | <center> |
| | + | <table class='data'> |
| | + | <tr> |
| | + | <td class='row'></td> |
| | + | <td class='row'><b>t<sub>1/2</sub><sup>*</sup> [h]</b></td> |
| | + | <td class='row'><b>γ<sub>HSL</sub><sup>**</sup> [h<sup>-1</sup>]</b></td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td class='row'><b>Culture<sub>pH 6</sub></b></td> |
| | + | <td class='row'>∞ </td> |
| | + | <td class='row'>0 </td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td class='row'><b>Culture<sub>pH 7</sub></b></td> |
| | + | <td class='row'>19 </td> |
| | + | <td class='row'>0.037 </td> |
| | + | </tr> |
| | + | </table> |
| | + | </center> |
| | + | <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> |
| | + | Thus in the simulations we set γ<sub>HSL</sub>=0; </p> |
| | + | </p> |
| | + | <div align="right"><small><a href="#indice" title="">^top</a></small></div> |
| | + | |
| | + | <!--------------------------------> |
| | + | <!--------------------------------> |
| | + | <!--------------------------------> |
| | + | <!-------------t9002--------------> |
| | + | <!--------------------------------> |
| | + | <!--------------------------------> |
| | + | <!--------------------------------> |
| | + | |
| | + | <a name='t9002'></a> |
| | + | <h2>Characterization of BBa_T9002 biosensor</h2> |
| | + | 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> |
| | + | <div style='text-align:center'> |
| | + | <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> |
| | + | </div> |
| | + | <br> |
| | + | <div style='text-align:center'> |
| | + | <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> |
| | + | </div> |
| | + | <center> |
| | + | <table width='50%' class='data'> |
| | + | <tr> |
| | + | <td class='row'><b>Minimum [S<sub>cell</sub>]</b></td> |
| | + | <td class='row'><b>Maximum [S<sub>cell</sub>]</b></td> |
| | + | <td class='row'><b>Switch point [nM]</b></td> |
| | + | <td class='row'><b>Lower boundary of linearity [nM]</b></td> |
| | + | <td class='row'><b>Upper boundary of linearity [nM]</b></td> |
| | + | </tr> |
| | + | <tr> |
| | + | <td class='row'>17.31</td> |
| | + | <td class='row'>739.4</td> |
| | + | <td class='row'>1.39</td> |
| | + | <td class='row'>0.38</td> |
| | + | <td class='row'>5.07</td> |
| | + | </tr> |
| | + | </table> |
| | + | </center> |
| | + | <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. |
| | + | <div align="right"><small><a href="#indice" title="">^top</a></small></div> |
| | + | <br> |
| | + | <br> |
| | + | <br> |
| | | | |
| | + | </html> |
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| | {{end}} | | {{end}} |
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