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| - | Results
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| - | <table id="toc" class="toc"><tr><td><div id="toctitle"><h2>Contents</h2></div>
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| - | <ul>
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| - | <li class="toclevel-1"><a href="#assembly"><span class="tocnumber">1</span> <span class="toctext">Part assembly</span></a>
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| - | <li class="toclevel-1"><a href="#characterization"><span class="tocnumber">1</span> <span class="toctext">Characterization of basic modules</span></a>
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| - | <li class="toclevel-2"><a href="#promoters"><span class="tocnumber">2.1</span> <span class="toctext">Promoter characterization</span></a></li>
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| - | <li class="toclevel-2"><a href="#enzymes"><span class="tocnumber">2.2</span> <span class="toctext">Characterization of the activity of the enzymes AiiA and LuxI</span></a></li>
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| - | <li class="toclevel-2"><a href="#rbs"><span class="tocnumber">2.3</span> <span class="toctext">Characterization of RBS efficiency</span></a></li>
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| - | <li class="toclevel-1"><a href="#growth"><span class="tocnumber">3</span> <span class="toctext">Identification of bacterial growth parameters</span></a></li>
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| - | <li class="toclevel-1"><a href="#HSL"><span class="tocnumber">4</span> <span class="toctext">Estimation of the spontaneous degradation of HSL in M9 medium and in cultures at different pH values</span></a></li>
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| - | <li class="toclevel-1"><a href="#t9002"><span class="tocnumber">5</span> <span class="toctext">Characterization of BBa_T9002 biosensor</span></a></li>
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| - | NB: unless differently specified, all tests were performed in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#MG1655Z1'><em>E. coli</em> MGZ1</a> in M9 supplemented medium at 37°C. For the cloning of the parts, <a href='https://2011.igem.org/Team:UNIPV-Pavia/Protocols#TOP10'><em>E. coli</em> TOP10</a> was used.
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| - | <a name='assembly'></a><h1>Parts assembly</h1>
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| - | All the parts have been cloned with success. The part name, plasmids and quality controls are reported in the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Freezer'>Freezer section</a>.
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| - | <div align="right"><small><a href="#indice" title="">^top</a></small></div>
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| - | <a name='characterization'></a><h1>Characterization of basic modules</h1>
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| - | <!--------pTet and pLux----------->
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| - | <a name='promoters'></a><h2>Characterization of promoters pTet and pLux</h2>
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| - | <p>Inducible and constitutive promoters were assembled upstream of different coding sequences containing an RBS from the Community collection.</p>
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| - | <p>The assembled RBSs are:</p>
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| - | <tr><td class="row"><b>BioBrick code</b></td><td><b> Declared efficiency</b></td></tr>
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| - | <tr><td class="row">BBa_B0030 </td><td class="row"> 0,6</td></tr>
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| - | <tr><td class="row">BBa_B0031 </td><td class="row"> 0,07</td></tr>
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| - | <tr><td class="row">BBa_B0032 </td><td class="row"> 0,3</td></tr>
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| - | <tr><td class="row">BBa_B0034 </td><td class="row"> 1</td></tr>
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| - | </table></div>
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| - | <div align="justify"><p>For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range.</p>
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| - | <p>The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein.
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| - | RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP).</p>
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| - | <p>For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (<b>S<sub>cell</sub></b>) and <b>R.P.U.s</b> (Relative Promoter Units) as explained in <a href='https://2011.igem.org/Team:UNIPV-Pavia/Measurements'>measurements</a> section.</p>
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| - | <p>Operative parameters of the promoter are derived from the estimated Hill equations obtained by <em>nonlinear least squares</em> fitting (<em>lsqnonlin</em> Matlab routine) of the <a href='https://2011.igem.org/Team:UNIPV-Pavia/Project/Modelling#Equations_for_gene_networks'>Hill function</a> expressed in RPUs:</p><p></p>
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| - | <p><ol><ul><li><b>
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| - | RPU<sub>max</sub></b> is equal to the α and represents the maximum promoter activity</p>
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| - | </li><p><li><b>
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| - | RPU<sub>min</sub></b> is equal to the α * δ represents the minimum promoter activity</p>
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| - | </li><p><li>
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| - | <b>Switch point</b> is computed as the abscissa of the inflection point of the Hill curve and it is representative of the position of linear region</p>
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| - | <b>Linearity boundaries</b> are determined as the intersection between the tangent line to the inflection point and the upper and lower horizontal boundaries of the Hill curve.</div></li></p>
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