Difference between revisions of "Team:UNITN-Trento/Test"

 
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<header>
 
<header>
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<h2><strong>Interlab Study</strong></h2>
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<h2><strong>InterLab Measurement Study</strong></h2>
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<p>I have no idea what I'm doing!</p>
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<p>What happens when GFP meets different promoters? Are they all the same? Here is what we found out!</p>
 
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<ul class="buttons">
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<li><p id="interlab_intro_btn" class="button small bstyle1" style="line-height:2em;" onclick="javascript:scrollToID('interlab_intro')"><span class="primary">Introduction</span> <span class="primary">&amp; Achievements</span></p></li> 
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<li><p id="interlab_design_btn" class="button small bstyle3"  onclick="javascript:scrollToID('interlab_design')"><span class="primary">Experimental</span> <span class="primary">Design</span></p></li>
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<li><p id="interlab_protocols_btn" class="button small bstyle5" onclick="javascript:scrollToID('interlab_protocols')"><span class="primary">Experiments</span><span class="primary">&amp; Protocols</span></p></li>
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<li><p id="interlab_final_btn" class="button small bstyle7" style="line-height:2em;" onclick="javascript:scrollToID('interlab_final')"><span class="primary">Final Discussion</span> <span class="primary">&amp; Results</span></p></li>
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<a class="anchor-off" name="interlab_intro" id="interlab_intro"></a>
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<p class="wow fadeInDown">The characterization of standard parts has always been one of the main concerns in Synthetic Biology. For this very same reason, iGEM teams from all around the World were suggested to take part in the biggest Measurement Interlab Study ever conducted and the 2015 UNITN iGEM Team answered the call. The goal of this Second International Measurement Interlab Study is to assemble <b>three different devices</b>, each one containing a promoter with a screening plasmid intermediate and collect as many fluorescence data as possible. The three different promoters will differently affect the GFP production and thating plasmid intermediate and collect as iGEM teams are free to use any technique to measure their devices as long the obtained data are solid and reproducible.</p> 
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<span class="rotate-box-icon tals"><i class="faa flaticon-up151"></i></span>
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<h4>Clear Data and Protocols</h4>
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<p>We have listed in the &rdquo;InterLab Study&rdquo; page:
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<ul>
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<li>All devices measured for this study</li>
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<li>All protocols developed and adopted</li>
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<li>Sequencing data for all measurements devices</li>
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<h4><span>3</span> BioBrick devices</h4>
 
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<p>We used the three BioBrick devices listed in the &rdquo;Required Devices&rdquo; section.</p>
 
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<h4><span> 5</span> techniques </h4>
 
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<p>We used <span class="i_enph italic">in-vivo</span> and <span class="i_enph italic">in-vitro</span> techniques for measuring RNA and proteins levels:
 
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<ul>
 
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<li>Plate Reader</li>
 
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<li>Spectrofluorimeter</li>
 
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<li>FACS</li>
 
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<li>RT-qPCR</li>
 
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<li>Cell-Free Extract</li>
 
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</ul>
 
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</p>
 
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<h4> Statistical Reliability </h4>
 
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<p>We have three biological replicates for each measurement, with positive and negative controls</p>
 
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<span class="rotate-box-icon"><i class="faa flaticon-lists10"></i></span>
 
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<h4>Worksheet and Protocol</h4>
 
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<p>We have completed the InterLab Worksheet and the InterLab Protocol</p>
 
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<span class="rotate-box-icon"><i class="faa flaticon-lab2"></i></span>
 
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<div class="rotate-box-info">
 
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<h4>Technical replicates </h4>
 
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<p>We have technical replicates for each sample</p>
 
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<section  id="interlab_design"  class="wrapper style4 container" style="margin-top:1em;">
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<div class="content">
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<header>
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<h3 class="wow fadeInDown">Experimental Design</h3>
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</header>
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<p class="wow fadeInDown">We used the three mandatory devices for the measurement study:
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<ul class="arrowed">
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<li>Device 1: <a class="i_linker" target="_blank" href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> + <a class="i_linker" href="http://parts.igem.org/Part:BBa_I13504">BBa_I13504</a> in pSB1C3</li>
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<li>Device 2: <a class="i_linker" target="_blank" href="http://parts.igem.org/Part:BBa_J23106">BBa_J23106</a> + <a class="i_linker" href="http://parts.igem.org/Part:BBa_I13504">BBa_I13504</a> in pSB1C3</li>
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<li>Device 3: <a class="i_linker" target="_blank" href="http://parts.igem.org/Part:BBa_J23117">BBa_J23117</a> + <a class="i_linker" href="http://parts.igem.org/Part:BBa_I13504">BBa_I13504</a> in pSB1C3</li>
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<li>Negative control: <a class="i_linker" target="_blank" href="http://parts.igem.org/Part:BBa_R0040">BBa_R0040</a> in psB1C3</li>
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<li>Positive control: <a class="i_linker" target="_blank" href="http://parts.igem.org/Part:BBa_I20270">BBa_I20270</a> in pSB1C3.</li>
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</ul> </p>
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<div class="row">
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<div class="5u 12u(narrower)">
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<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/f/f5/Unitn_pics_interlab_GFP.png"><img src="https://static.igem.org/mediawiki/2015/b/bd/Unitn_pics_interlab_GFP_thumb.jpg" alt="" style="width:100%;"/></a>
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</div>
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<div class="7u 12u(narrower)">
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<p>The measurement devices were prepared by amplifying the reporter (BBa_I20270) by PCR. The amplified insert was then cut with <span class="i_enph">XbaI</span> and <span class="i_enph">PstI</span> and ligated into the plasmid containing the promoter previously cut with <span class="i_enph">SpEI</span> and <span class="i_enph">PstI</span>. All the devices were confirmed by restriction digestion as well as DNA sequencing.</p>
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<div class="6u 12u(narrower) rotate-box-superhover"> 
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<h4 class="header4"><div href="#" class="rotate-box square-icon" style="text-align:center;">
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<span class="rotate-box-icon"><i class="faa flaticon-bacteria"></i></span>
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</div><br />In-vivo Measurements </h4> 
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<p>The confirmed devices were then transformed in different bacterial strains of E. Coli, NEB10&beta;, NEB Express, and JM109. Each measurement was taken at the same optical density to allow a more precise comparison of the data. For each device we have 3 biological and 3 technical measurements for each used technique. We measured in vivo fluorescence emission in different ways using Tecan Infinite 200 PRO plate reader, Varian Cary Eclipse spectrofluorimeter, and BD FACSCanto FACS.</p>
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<div class="6u 12u(narrower) rotate-box-superhover"> 
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<h4 class="header4"><div href="#" class="rotate-box square-icon" style="text-align:center;">
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<span class="rotate-box-icon"><i class="faa flaticon-bacteria"></i></span>
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</div><br />In-vitro Measurements </h4>
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<p>We also focused of transcription since the characterization is about promoters. To do so we we performed RT-qPCR using a BioRad CFX96 Touch&trade; Real-Time PCR Detection System. Additionally, we performed an in vitro characterization study, by measuring the fluorescence intensities of each device with a Cell Free <span class="i_enph italic">E. coli</span> <span class="i_enph">S30</span> Extract System with a Circular DNA Real Time PCR.</p>
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</div> 
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</div> 
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</section>
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</section>
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<section  id="interlab_protocols"  class="wrapper style4 container" style="margin-top:1em;">
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<div class="content">
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<section>
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<header>
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<h3 class="wow fadeInDown">Experiments &amp; Protocols</h3>
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</header>
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<h4 class="header4 displayControl" style="position:relative">Extraction from the Registry</h4>
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<div style="display:none;">
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<p>All the parts needed for the InterLab Study were extracted from the 2015 iGEM Registry Distribution Kit.</p>
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<table class="standard_table">
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<thead>
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<tr>
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<td>Part ID</td>
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<td>Description</td>
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<td>Plasmid Backbone</td>
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<td>2015 Registry Location</td>
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</tr>
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</thead>
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<tbody>
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<tr>
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<td>BBa_K823005</td>
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<td>Anderson promoter J23101</td>
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<td>pSB1C3</td>
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<td>Plate 1; 20K</td>
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</tr>
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<tr>
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<td>BBa_K823008</td>
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<td>Anderson promoter J23106</td>
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<td>pSB1C3</td>
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<td>Plate 1; 22A</td>
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</tr>
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<tr>
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<td>BBa_K823013</td>
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<td>Anderson promoter J23117</td>
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<td>pSB1C3</td>
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<td>Plate 1; 22K</td>
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</tr>
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<tr>
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<td>BBa_I12504</td>
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<td>RBS + GFP + 2 terminators</td>
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<td>pSB1A2</td>
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<td>Plate 4; 21J</td>
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</tr>
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<tr>
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<td>BBa_R0040</td>
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<td>TetR sequence</td>
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<td>pSB1C3</td>
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<td>Plate 2; 6F</td>
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</tr>
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<tr>
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<td>BBa_I20270</td>
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<td>Promoter MeasKit</td>
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<td>pSB1C3</td>
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<td>Plate 3; 8P</td>
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</tr>
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</tbody>
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</table>
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</div>
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<h4 class="header4 displayControl" style="position:relative">Polymerase Chain Reaction (PCR)</h4>
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<div class="row" style="display:none;">
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<div class="3u 12u(narrower)">
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<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/a/ae/Unitn_pics_interlab_gel_GFP1.jpg"><img src="https://static.igem.org/mediawiki/2015/a/ae/Unitn_pics_interlab_gel_GFP1.jpg" alt="" style="width:100%; max-width:163px;"/></a>
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</div>
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<div class="9u 12u(narrower)">
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<p>The reporter gene (i.e. <span class="i_enph">GFPmut3b</span> from part <a class="i_linker" href="http://parts.igem.org/Part:BBa_I13504">BBa_I13504</a>) was amplified using the Phusion polymerase from New England Biolabs and primers matching the prefix and suffix.</p>
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<p>We used <span class="i_enph quantity">50 ng</span> of template with an annealing temperature of <span class="i_enph quantity">59 &deg;C</span> and an extension time of <span class="i_enph quantity">90 seconds</span>. The PCR was confirmed by electrophoresis and subsequently purified with NucleoSpin Gel and PCR Clean-Up Kit from Macherey-Nigel. <span class="i_enph quantity">125 ng</span> of purified PCR were digested with <span class="i_enph quantity">1 μl</span> of <span class="i_enph">XbaI</span> and <span class="i_enph quantity">1 μl</span> of <span class="i_enph">PstI</span> overnight at <span class="i_enph quantity">37&deg;C</span>. The following morning the sample was treated with 1 ul of <span class="i_enph">DpnI</span> for 2 hour at <span class="i_enph quantity">37&deg;C</span> and the enzymes were deactivated for <span class="i_enph quantity">20 min</span>  at <span class="i_enph quantity">80 &deg;C</span>.</p>
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</div>
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</div>
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<h4 class="header4 displayControl" style="position:relative">Restriction Digestion of plasmids containing the promoter</h4>
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<div style="display:none;">
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<p>Each promoter containing plasmid was digested with <span class="i_enph quantity">1 μl</span> of <span class="i_enph">SpeI</span> and <span class="i_enph quantity">1 μl</span> of <span class="i_enph">PstI</span> at <span class="i_enph quantity">37&deg;C</span> overnight. The day after add <span class="i_enph quantity">1 μl</span> of phosphatase (CIP from New England Biolabs) for <span class="i_enph quantity">2 hours</span> at <span class="i_enph quantity">37&deg;C</span>. The enzymes were then heat deactivated.</p>
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<p>The digestion reactions were assembled in this way:</p>
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<table style="width:40%;" class="standard_table">
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<thead>
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<tr>
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<td class="empty"></td>
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<td>PCR Products</td>
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<td>Plasmids</td>
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</tr>
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</thead>
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<tbody>
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<tr>
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<td>Template</td>
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<td>3000 ng</td>
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<td>2000 ng</td>
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</tr>
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<tr>
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<td>Enzyme 1</td>
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<td>2 µl</td>
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<td>1.5 µl</td>
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</tr>
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<tr>
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<td>Enzyme 2</td>
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<td>2 µl</td>
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<td>1.5 µl</td>
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</tr>
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<tr>
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<td>Buffer (Stock 10X)</td>
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<td>5 µl</td>
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<td>5 µl</td>
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</tr>
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<tr>
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<td>BSA (Stock 10X)</td>
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<td>5 µl</td>
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<td>5 µl</td>
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</tr>
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<tr>
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<td>Water</td>
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<td>Up to 50 µl</td>
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<td>Up to 50 µl</td>
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</tr>
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</tbody>
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</table>
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</div>
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<h4 class="header4 displayControl" style="position:relative" >Ligation</h4>
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<div style="display:none;">
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<p>The ligation reactions were assembled and incubated at room temperature for <span class="i_enph quantity">1 hour</span> according to the table below:</p>
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<table class="standard_table">
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<thead>
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<tr>
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<td></td>
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<td>Plasmid: Insert = 1 : 3</td>
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<td>Control</td>
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</tr>
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</thead>
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<tbody>
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<tr>
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<td>Vector</td>
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<td>100 ng</td>
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<td>100 ng</td>
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</tr>
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<tr>
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<td>Insert</td>
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<td>125 ng</td>
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<td>-</td>
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</tr>
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<tr>
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<td>10X Buffer</td>
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<td>2 µl</td>
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<td>2 µl</td>
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</tr>
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<tr>
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<td>Buffer (Stock 10X)</td>
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<td>5 µl</td>
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<td>5 µl</td>
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</tr>
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<tr>
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<td>T4 - DNA Ligase</td>
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<td>2 µl</td>
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<td>2 µl</td>
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</tr>
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<tr>
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<td>Water</td>
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<td>Up to 20 µl</td>
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<td>Up to 20 µl</td>
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</tr>
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</tbody>
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</table>
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<div class="row">
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<div class="7u 12u(narrower)">
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<p>Subsequently <span class="i_enph quantity">10 μl</span> of the ligation mixture were transformed and plated into LB-agar plates with the proper antibiotic resistance.</p>
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<p style="margin-top:1em;" >Correct clones were screened by restriction digestion and confirmed by sequencing:</p>
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<ul class="arrowed" style="margin-bottom:1em;">
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<li>BBa_J23101 + BBa_I13504 <a class="i_linker" target="_blank" style="font-size:0.8em;" href="https://static.igem.org/mediawiki/2015/b/b2/Unitn_fasta_interlab_GFPK05.fasta.txt">[Sequencing Data]</a></li>
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<li>BBa_J23106 + BBa_I13504 <a class="i_linker" target="_blank" style="font-size:0.8em;" href="https://static.igem.org/mediawiki/2015/6/62/Unitn_fasta_interlab_GFPK08.fasta.txt">[Sequencing Data]</a></li>
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<li>BBa_J23117 + BBa_I13504 <a class="i_linker" target="_blank" style="font-size:0.8em;" href="https://static.igem.org/mediawiki/2015/8/83/Unitn_fasta_interlab_GFPK13.fasta.txt">[Sequencing Data]</a></li>
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</ul>
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<p>The confirmed devices were transfected in NEB10&beta;, JM109, NEB Express.</p>
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</div>
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<div class="5u 12u(narrower)">
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<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/f/f0/Unitn_pics_interlab_GFP2.png"><img src="https://static.igem.org/mediawiki/2015/8/8a/Unitn_pics_interlab_GFP2_thumb.jpg" title="Correct clones were screened by restriction digestion" alt="" style="width:100%; max-width:400px;"/></a>
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</div>
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<h4 class="header4 displayControl" style="position:relative">Glycerol stocks preparation and Sample Growth  </h4>
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<div style="display:none;">
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<p>A single colony was inoculated with a sterile pipette tip in a test tube with <span class="i_enph quantity">10 ml</span> of LB and antibiotic (1000:1 LB to antibiotic ratio) and placed in the thermoshaker (<span class="i_enph quantity">190 rpm</span>, <span class="i_enph quantity">37°C</span>. When the culture got cloudy</span>, <span class="i_enph quantity">40 ml</span> of LB+antibiotic were added to reach a final volume of <span class="i_enph quantity">50 ml</span>. The cells were grown until an OD600 of <span class="i_enph quantity">0.5</span> and then centrifuged at <span class="i_enph quantity">4100 rpm</span> for <span class="i_enph quantity">10 minutes</span> at <span class="i_enph quantity">4 °C</span>. The supernatant was discarded and the cells were resuspend in <span class="i_enph quantity">5 ml</span> of LB + antibiotic + glycerol (<span class="i_enph quantity">20% v/v</span>). The cells were kept on ice and were promptly aliquoted into <span class="i_enph quantity">200 μl</span> tubes and frozen at <span class="i_enph quantity">-80°C</span> immediately. From this protocol we obtained a <span class="i_enph quantity">10X</span> concentrated glycerol stock for each sample.</p>
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<p>The glycerol stock was thaw and added into <span class="i_enph quantity">10 ml</span> of LB with antibiotic, giving a  starting culture with an OD600 of <span class="i_enph quantity">0.1</span>. The sample were grown in a <span class="i_enph quantity">50 mL</span> conical  plastic tube in the termoshaker at <span class="i_enph quantity">37°C</span> and were grown until an OD600 <span class="i_enph quantity">0.7</span>. At this  point 3 mL of the culture were transferred in a new tube, centrifuged it, and stored at <span class="i_enph quantity">-20°C</span>.</p>
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</div>
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<h4 class="header4 displayControl" style="position:relative">Fluorescence readings:<br/>Tecan INFINITE &reg; 200 PRO Plate Reader  </h4>
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<h4 class="header4 displayControl" style="position:relative">Fluorescence readings:<br/>Cary Eclipse Fluorescence Spectrophotometer  </h4>
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<h4 class="header4 displayControl">Fluorescence readings:<br/>BioRad CFX96 TouchTM Real-Time PCR Detection System  </h4>
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<h4 class="header4 displayControl" style="position:relative">Fluorescence readings:<br/>E. coli S30 Extract System for DNA Circular  </h4>
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<h3 class="wow fadeInDown">Final Discussion</h3>
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Latest revision as of 14:24, 17 September 2015

InterLab Measurement Study

What happens when GFP meets different promoters? Are they all the same? Here is what we found out!

The Interlab Measurement Study