Team:Fudan Lux/Notebook

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   <div class="wrapper"> <a href="https://2012.igem.org/Team:Fudan_Lux><img id="logo" src="https://static.igem.org/mediawiki/2012/5/5d/Logo_64px.png" alt="Nova"></a>  
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         <li><a href="https://2012.igem.org/wiki/index.php?title=Team:Fudan_Lux/project_introduction">Project<span class="subheader">Cool</span></a>
         <li><a href="https://2012.igem.org/wiki/index.php?title=Team:Fudan_Lux/project_introduction">Project<span class="subheader">Cool</span></a>
           <ul style="display: none; visibility: hidden; ">
           <ul style="display: none; visibility: hidden; ">
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             <li><a href="https://2012.igem.org/wiki/index.php?title=Team:Fudan_Lux/project_introduction">Introduction</a></li>
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             <li><a href="https://2012.igem.org/wiki/index.php?title=Team:Fudan_Lux/project_introduction">Overview</a></li>
             <li><a href="https://2012.igem.org/wiki/index.php?title=Team:Fudan_Lux/biowave">Project Biowave</a></li>
             <li><a href="https://2012.igem.org/wiki/index.php?title=Team:Fudan_Lux/biowave">Project Biowave</a></li>
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             <li><a href="https://2012.igem.org/wiki/index.php?title=Team:Fudan_Lux/nanotube">nanotubling</a></li>
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             <li><a href="https://2012.igem.org/wiki/index.php?title=Team:Fudan_Lux/nanotube">Project Bacto-Trafficking</a></li>
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             <li><a href="https://2012.igem.org/wiki/index.php?title=Team:Fudan_Lux/result">Result</a></li>
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             <li><a href="https://2012.igem.org/wiki/index.php?title=Team:Fudan_Lux/result">Results</a></li>
           </ul>
           </ul>
         </li>
         </li>
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             <li><a href="href="https://2012.igem.org/wiki/index.php?title=Team:Fudan_Lux/labcloud">Project Labcloud</a></li>
             <li><a href="href="https://2012.igem.org/wiki/index.php?title=Team:Fudan_Lux/labcloud">Project Labcloud</a></li>
             <li><a href="href="https://2012.igem.org/wiki/index.php?title=Team:Fudan_Lux/Associations">Associations</a></li>
             <li><a href="href="https://2012.igem.org/wiki/index.php?title=Team:Fudan_Lux/Associations">Associations</a></li>
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            <li><a href="href="https://2012.igem.org/wiki/index.php?title=Team:Fudan_Lux/tianmu">Tianmu mountain</a></li>
 
             <li><a href="href="https://2012.igem.org/wiki/index.php?title=Team:Fudan_Lux/other practise">Other Practise</a></li>
             <li><a href="href="https://2012.igem.org/wiki/index.php?title=Team:Fudan_Lux/other practise">Other Practise</a></li>
           </ul>
           </ul>
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<div id="page-title">
<div id="page-title">
<span class="title">Note Book</span>
<span class="title">Note Book</span>
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<span class="subtitle">Tabs</span>
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<span class="subtitle">Record iGEMers in summer.</span>
</div>
</div>
<!-- ENDS title -->
<!-- ENDS title -->
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<!-- the tabs -->
<!-- the tabs -->
<ul class="tabs">
<ul class="tabs">
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<li><a href="#"><span>March</span></a></li>
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<li><a href="#"><span>week 1</span></a></li>
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<li><a href="#"><span>April</span></a></li>
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<li><a href="#"><span>week 2</span></a></li>
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<li><a href="#"><span>May</span></a></li>
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<li><a href="#"><span>week 3~week 4</span></a></li>
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<li><a href="#"><span>June</Span></a></li>
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<li><a href="#"><span>week 5</Span></a></li>
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<li><a href="#"><span>July~August</span></a></li>
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<li><a href="#"><span>week 6~week 10</span></a></li>
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<li><a href="#"><span>September</Span></a></li>
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<li><a href="#"><span>week 11</Span></a></li>
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<li><a href="#"><span>October</span></a></li>
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<li><a href="#"><span>week 12</span></a></li>
</ul>
</ul>
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<div>
<div>
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<p>We summarized our recent brain storm result. At last we chose to make a light-signal oscillator <img  src="https://static.igem.org/mediawiki/2012/1/1d/Discussion.jpg" style="float:right;width:300px;"alt="test">in E.coli and possibilities to use nanotubes.
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<p>We summarized our recent brain storm result. At last we chose to make a light-signal oscillator <img  src="https://static.igem.org/mediawiki/2012/1/1d/Discussion.jpg" style="float:right;width:300px;"alt="test" class="myimg">in E.coli and possibilities to use nanotubes.
The first thing for the next week is to find an efficient light sensor and a stable lighting part.
The first thing for the next week is to find an efficient light sensor and a stable lighting part.
</p>
</p>
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<div>
<div>
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<h1>HTML Ipsum Presents</h1>
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<p>Discuss the establishment of the gene circuits.<img src="https://static.igem.org/mediawiki/2012/6/64/Gene_circuits1.jpg" style="float:right;width:300px;"alt="test"></p>
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<p>Discuss the establishment of the gene circuits.</p>
 +
<img src="https://static.igem.org/mediawiki/2012/6/64/Gene_circuits1.jpg" style="width:400px;"alt="test" class="myimg">
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<img src="https://static.igem.org/mediawiki/igem.org/d/da/Gene_circuits2.jpg" style="width:400px;"alt="test" class="myimg"><br><br>
 +
<p>Get lux without operon and promoter from k325909 by PCR.
 +
We used longPCR Enzyme and Overlapping PCR.
 +
</p><br>
 +
<p>Measure the growth curve of dH5a transformed by lux with araBAD (k325909)</p>
 +
<img src="http://partsregistry.org/wiki/images/4/4c/Growth_curve_of_K325909.jpg" style="width:500px;"alt="test" class="myimg"><br><br>
 +
<p>Transform tetR (<a href="http://partsregistry.org/Part:BBa_C0040">C0040</a>) from the kit. Transfer araBAD (<a href=" http://partsregistry.org/Part:BBa_I0500">I0500</a>) and tetR (<a href="http://partsregistry.org/Part:BBa_C0040">C0040</a>) with RBS into pSB1C3 by 3A ligation. Transform it into dH5a.</p><br><p>Transfer tetR with promoter and RFP with ptetO as promoter (<a href="http://partsregistry.org/Part:BBa_J61002">J61002</a>) into pSB1C3 by 3A ligation. Transform it into dH5a.</p><br>
 +
<p>Sorry to find the ligation is failed. But fortunately, we decide to change the reporter into GFP (<a href="http://partsregistry.org/Part:BBa_I13522">I13522</a>). Transform the plasmid from the kit into dH5a.</p><br>
 +
<p>Design protein composed by lov domain and modified helix turn helix (abbr. <a href="http://partsregistry.org/Part:BBa_K785004">lov-HTH</a>).</p>
 +
<img src="https://static.igem.org/mediawiki/2012/f/fd/Lov_demestration.jpg" style="width:500px;"alt="test" class="myimg"><br>
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<h2>Header Level 2</h2>
 
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<blockquote><p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Vivamus magna. Cras in mi at felis aliquet congue. Ut a est eget ligula molestie gravida. Curabitur massa. Donec eleifend, libero at sagittis mollis, tellus est malesuada tellus, at luctus turpis elit sit amet quam. Vivamus pretium ornare est.</p></blockquote>
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<h3>Header Level 3</h3>
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<ul>
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  <li>Lorem ipsum dolor sit amet, consectetuer adipiscing elit.</li>
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  <li>Aliquam tincidunt mauris eu risus.</li>
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</ul>
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<pre><code>
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#header h1 a {
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display: block;
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width: 300px;
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height: 80px;
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</code></pre>
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</div>
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<div>
<div>
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<dl>
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<p>Design protein composed by lov domain and tetR (abbr. lov-tetR).</p><br>
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  <dt>Definition list</dt>
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<p>Send the synthetic orders to the gene synthesis company.</p><br>
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  <dd>Consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna
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<p>Half of our members went to Tianmu Mount, Zhejiang Province. They spent a week there to participate a fieldwork organized by the Life Science Institute. During the fieldwork, they paid a lot of attention to a China’s specific firefly, Qiongyuying. They observed the behavior of Qiongyuying, especially the synchrony.</p>
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aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea
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<img src="https://static.igem.org/mediawiki/2012/c/cf/Firefly1.jpg" style="width:300px;"alt="test" class="myimg"><br><br>
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commodo consequat.</dd>
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<p>Our instructor and four of our members went to meet Richard J Roberts after the reward ceremony of COMBREX, a project to accelerate the functional annotation of prokaryotic genomes. They talked for about an hour and we presented our project to him. Richard advised that we should make our project more practical and functional.</p>
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  <dt>Lorem ipsum dolor sit amet</dt>
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<img src="https://static.igem.org/mediawiki/2012/c/c6/Richard.jpg" style="width:400px;"alt="test"class=“myimg"><br><br>
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  <dd>Consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna
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<p>After negotiating with the principal of volunteer teaching program in our institute, one of our members, with all members of Fudan_Lux’s hope and responsibility on his shoulders, went to Shangrao, Jiangxi Province to do the volunteer teaching.</p>
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aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea
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<div><img src="https://static.igem.org/mediawiki/2012/8/87/VT1.jpg" style="width:250px;"alt="test class="myimg"">
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commodo consequat.</dd>
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<img src="https://static.igem.org/mediawiki/2012/3/3b/VT2.jpg" style="width:250px;"alt="test" class="myimg"></div><br>
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</dl>
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<p>Make an appointment with Li Jin, Hongyan Wang etc. to show our projects.</p><br>
 +
<p>Receive lov-HTH in nonstandard vector.</p><br>
 +
<p>Design primers to make clones of lov-HTH, and transfer lov-HTH with double terminators into pSB1C3.</p><br>
</div>
</div>
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<div>
<div>
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<p>Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo. Quisque sit amet est et sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis. Praesent dapibus, neque id cursus faucibus, tortor neque egestas augue, eu vulputate magna eros eu erat. Aliquam erat volutpat. Nam dui mi, tincidunt quis, accumsan porttitor, facilisis luctus, metus</p>
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<p>Receive lov-tetR in nonstandard vector.</p><br>
 +
<p>Do double digestion by E.coliI HF and SpeI, and transfer lov-tetR into pSB1C3.</p><br>
 +
<p>Make an appointment with prof. Guoping Zhao, who establishes China’s first synthesis biology laboratory in Shanghai. He talked directly about the impropriation of our tumor project and the lack of application about our biowave project.</p><br>
</div>
</div>
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<div>
<div>
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<p>Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo. Quisque sit amet est et sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis. Praesent dapibus, neque id cursus faucibus, tortor neque egestas augue, eu vulputate magna eros eu erat. Aliquam erat volutpat. Nam dui mi, tincidunt quis, accumsan porttitor, facilisis luctus, metus</p>
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<p>The double digestion and the ligation held us for more than 4 weeks!</p>
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<img src="https://static.igem.org/mediawiki/2012/8/86/YuAn.jpg" style="width:400px;"alt="test" class="myimg"><br><br>
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<p>In order to do the ligation more efficiently, we decide to do the ligation according to this table.</p>
 +
<table style="border-collapse:collapse;" width="96%" align="center" border="1" cellspace="0" cellpadding="3"><tbody>
 +
<tr><td valign="top">NO.&nbsp;<br>
 +
</td><td valign="top">Composition
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</td><td valign="top">Backbone
 +
</td><td valign="top">Code
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</td></tr>
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<tr><td valign="top">P1 14N
 +
</td><td valign="top">araBAD
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</td><td valign="top">pSB2K3
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</td><td valign="top">I0050
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</td></tr>
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<tr><td valign="top">P1 6G
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</td><td valign="top">pLac
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</td><td valign="top">pSB1A2
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</td><td valign="top">R0011
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</td></tr>
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<tr><td valign="top">P1 6N
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</td><td valign="top">T7 promoter
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</td><td valign="top">pSB1AK8
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</td><td valign="top">I712074
 +
</td></tr>
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<tr><td valign="top">P1 2M
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</td><td valign="top">Rbs(1.0)
 +
</td><td valign="top">pSB1A2
 +
</td><td valign="top">B0034
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</td></tr>
 +
<tr><td style="">P1 2I</td><td style="">Rbs(0.3)</td><td style="">pSB1A2&nbsp;&nbsp;</td><td style="">B0032&nbsp;</td></tr>
 +
<tr><td style="">P1 2G&nbsp;</td><td style="">Rbs(0.07)</td><td style="">pSB1A2&nbsp;&nbsp;</td><td style="">B0031&nbsp;</td></tr></tbody></table>
 +
<p>1.araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-GFP</p>
 +
<p>2.araBAD-Rbs(0.3)-lov-HTH-Terminator-ptetO-GFP</p>
 +
<p>3.araBAD-Rbs(0.07)-lov-HTH-Terminator-ptetO-GFP</p>
 +
<p>4.araBAD-Rbs(1.0)-lov-tetR-Terminator-ptetO-GFP</p>
 +
<p>5.araBAD-Rbs(0.3)-lov- tetR -Terminator-ptetO-GFP</p>
 +
<p>6.araBAD-Rbs(0.07)-lov- tetR -Terminator-ptetO-GFP</p>
 +
<p>7.araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-CFP</p>
 +
<p>8.araBAD-Rbs(0.3)-lov-HTH-Terminator-ptetO-CFP</p>
 +
<p>9.araBAD-Rbs(0.07)-lov-HTH-Terminator-ptetO-CFP</p>
 +
<p>10.araBAD-Rbs(1.0)-lov-tetR-Terminator-ptetO-CFP</p>
 +
<p>11.araBAD-Rbs(0.3)-lov- tetR -Terminator-ptetO-CFP</p>
 +
<p>12.araBAD-Rbs(0.07)-lov- tetR -Terminator-ptetO-CFP</p>
 +
<p>13.araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-lux</p>
 +
<p>14.araBAD-Rbs(0.3)-lov-HTH-Terminator-ptetO- lux</p>
 +
<p>15.araBAD-Rbs(0.07)-lov-HTH-Terminator-ptetO- lux</p>
 +
<p>16.araBAD-Rbs(1.0)-lov-tetR-Terminator-ptetO- lux</p>
 +
<p>17.araBAD-Rbs(0.3)-lov- tetR -Terminator-ptetO- lux&nbsp;</p>
 +
<p>18.araBAD-Rbs(0.07)-lov- tetR -Terminator-ptetO- lux</p>
 +
<p>19.pLac-Rbs(1.0)-lov-HTH-Terminator-ptetO-GFP</p>
 +
<p>20.pLac -Rbs(0.3)-lov-HTH-Terminator-ptetO-GFP</p>
 +
<p>21.pLac -Rbs(0.07)-lov-HTH-Terminator-ptetO-GFP</p>
 +
<p>22.pLac -Rbs(1.0)-lov-tetR-Terminator-ptetO-GFP</p>
 +
<p>23.pLac -Rbs(0.3)-lov- tetR -Terminator-ptetO-GFP</p>
 +
<p>24.pLac -Rbs(0.07)-lov- tetR -Terminator-ptetO-GFP</p>
 +
<p>25.pLac-Rbs(1.0)-lov-HTH-Terminator-ptetO-CFP</p>
 +
<p>26.pLac -Rbs(0.3)-lov-HTH-Terminator-ptetO-CFP</p>
 +
<p>27.pLac -Rbs(0.07)-lov-HTH-Terminator-ptetO-CFP</p>
 +
<p>28.pLac -Rbs(1.0)-lov-tetR-Terminator-ptetO-CFP</p>
 +
<p>29.pLac -Rbs(0.3)-lov- tetR -Terminator-ptetO-CFP</p>
 +
<p>30.pLac -Rbs(0.07)-lov- tetR -Terminator-ptetO-CFP</p>
 +
<p>31.pLac-Rbs(1.0)-lov-HTH-Terminator-ptetO- lux</p>
 +
<p>32.pLac -Rbs(0.3)-lov-HTH-Terminator-ptetO- lux</p>
 +
<p>33.pLac -Rbs(0.07)-lov-HTH-Terminator-ptetO- lux</p>
 +
<p>34.pLac -Rbs(1.0)-lov-tetR-Terminator-ptetO- lux</p>
 +
<p>35.pLac -Rbs(0.3)-lov- tetR -Terminator-ptetO- lux</p>
 +
<p>36.pLac -Rbs(0.07)-lov- tetR -Terminator-ptetO- lux</p>
 +
<p>37.T7 promoter-Rbs(1.0)-lov-HTH-Terminator-ptetO-GFP</p>
 +
<p>38.T7 promoter -Rbs(0.3)-lov-HTH-Terminator-ptetO-GFP</p>
 +
<p>39.T7 promoter -Rbs(0.07)-lov-HTH-Terminator-ptetO-GFP</p>
 +
<p>40.T7 promoter -Rbs(1.0)-lov-tetR-Terminator-ptetO-GFP</p>
 +
<p>41.T7 promoter -Rbs(0.3)-lov- tetR -Terminator-ptetO-GFP</p>
 +
<p>42.T7 promoter -Rbs(0.07)-lov- tetR -Terminator-ptetO-GFP</p>
 +
<p>43.T7 promoter-Rbs(1.0)-lov-HTH-Terminator-ptetO-CFP</p>
 +
<p>44.T7 promoter -Rbs(0.3)-lov-HTH-Terminator-ptetO-CFP</p>
 +
<p>45.T7 promoter -Rbs(0.07)-lov-HTH-Terminator-ptetO-CFP</p>
 +
<p>46.T7 promoter -Rbs(1.0)-lov-tetR-Terminator-ptetO-CFP</p>
 +
<p>47.T7 promoter -Rbs(0.3)-lov- tetR -Terminator-ptetO-CFP</p>
 +
<p>48.T7 promoter -Rbs(0.07)-lov- tetR -Terminator-ptetO-CFP</p>
 +
<p>49.T7 promoter-Rbs(1.0)-lov-HTH-Terminator-ptetO-lux</p>
 +
<p>50.T7 promoter -Rbs(0.3)-lov-HTH-Terminator-ptetO- lux</p>
 +
<p>51.T7 promoter -Rbs(0.07)-lov-HTH-Terminator-ptetO- lux</p>
 +
<p>52.T7 promoter -Rbs(1.0)-lov-tetR-Terminator-ptetO- lux</p>
 +
<p>53.T7 promoter -Rbs(0.3)-lov- tetR -Terminator-ptetO- lux</p>
 +
<p>54.T7 promoter -Rbs(0.07)-lov- tetR -Terminator-ptetO- lux</p>
 +
<p>Transform the parts mentioned in the box into dH5a.</p><br>
 +
<p>Pick colonies to cultivate. And extract plasmids.</p><br>
 +
<p>Do all the double digestions and ligations together for the first time. The order is:</p>
 +
<p>1.Promoter + Rbs & Terminator + Reporter</p>
 +
<p>2.Promoter-Rbs + modified protein</p>
 +
<p>3.Promoter-Rbs-modified protein + Terminator-Reporter</p>
 +
<br>
 +
<p>Transform fails for several ligation products.</p><br>
 +
<p>At last, we choose 3 gene circuits for further investigation:</p>
 +
<p>1.araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-GFP</p>
 +
<p>2.T7 promoter -Rbs(0.3)-lov-HTH-Terminator-ptetO-GFP</p>
 +
<p>3.araBAD-Rbs(1.0)-lov-tetR-Terminator-ptetO-GFP</p>
 +
<p>4.araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-lux</p>
 +
<p>5.T7 promoter -Rbs(0.3)-lov-HTH-Terminator-ptetO-lux<p>
 +
<p>6.araBAD-Rbs(1.0)-lov-tetR-Terminator-ptetO-lux</p>
 +
<br>
 +
<p>We run a SDS-PAGE gel to confirm the expression of the modified protein. The result is positive. </p><br>
 +
<p>In order to verify the modified protein’s characteristic, Juntao Mai does the western-blot. The result is partly positive. Under equal quantity induction of arabinose (0.3%), samples under 450nm wavelength light shows a 30% GFP decay compared with samples incubated in the dark.</p><br>
 +
<p>We detected the lov-HTH, whose HTH part is modified, by reversed transcription PCR. We‘ve done a series of RT PCR with samples induced by 0.3% arabinose during their incubation and samples incubated without arabinose at the first place. In order to get rid of the effect of light, we incubated all the cells in the dark. We can easily find lov-HTH (modified) mRNA in samples induced by arabinose.</p><br>
 +
<p>We investigated the time course of the transcription of the arabinose-induced GFP using quantitative real-time PCR. We combined different conditions together and did a series of QRT PCR. We can get the detail in this table.</p>
 +
<img src="https://static.igem.org/mediawiki/2012/9/9b/QRTPCR.JPG" style="width:500px;"alt="test" class="myimg"><br><br>
 +
<p>We decide to use EMSA (Electrophoretic Mobility Shift Assay) to verify if the modified protein can be efficient binding the specific part in ptetO.</p><br>
 +
<p>Considering the time, cost and the possibility, we give up EMSA.</p><br>
 +
<p>With propose of testing the protein’s repression ability, we do a fluorescence intensity test. Under an arabinose induction gradient between 0 to 0.3%, we investigate the time course of the two groups of cells’ GFP fluorescence intensity and the absorbance of OD600 at the same time. One group of them is incubated in the 470nm wavelength light and the other one was incubated in the dark. The two curves of RFU per OD600 present 30% differences which is positive.</p><br>
 +
 
 +
 
</div>
</div>
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<div>
<div>
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<p>Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo. Quisque sit amet est et sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis. Praesent dapibus, neque id cursus faucibus, tortor neque egestas augue, eu vulputate magna eros eu erat. Aliquam erat volutpat. Nam dui mi, tincidunt quis, accumsan porttitor, facilisis luctus, metus</p>
+
<p>With one of our classmates’ help, Zining Hou make a wood box for shooting pictures in the dark.</p>
 +
<img src="https://static.igem.org/mediawiki/igem.org/0/03/Wood_box.jpg" style="width:300px;"alt="test" class="myimg"><br><br>
 +
<p>We make our first double layer disk to shoot.</p><br>
 +
<p>Our instructor helps us make a single colony mathematic model.</p>
 +
<img src="https://static.igem.org/mediawiki/igem.org/a/a5/Stimulation.jpg" style="width:500px;"alt="test" class="myimg"><br><br>
 +
<p>We get a series of photos which show a positive result.
 +
(araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-lux)
 +
</p><br>
 +
<p>We compare araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-lux, araBAD-Rbs(1.0)-lov-tetR-Terminator-ptetO-lux and T7 promoter-Rbs(0.3)-lov-HTH-Terminator-ptetO-lux by putting four double layer disks together (we use ptetO-lux as control).
 +
Unfortunately, the light emitted from different disks interfere each other. So we couldn’t get a usable result. But good news is that we assure the light emitted by lux is strong enough to be a signal.
 +
</p><br>
 +
<p>Organize our first association recruiting with <a href="https://2012.igem.org/Team:Fudan_D">Fudan_D</a>, our cooperating team.</p>
 +
<img src="https://static.igem.org/mediawiki/2012/c/cf/Association.jpg" style="width:500px;"alt="test" class="myimg"><br><br>
 +
<p>Build the wiki</p><br>
</div>
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<p>Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo. Quisque sit amet est et sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt cond
+
 
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<!-- Twitter -->
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<li class="col">
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<h6>About the Team</h6>
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He he he he!
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</li>
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</ul>
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<div id="bottom-text">All rights reserved. Tempalate Edit by Fudan-lux team <a href="http://www.luiszuno.com">Powered By Luiszuno.com</a> </div>
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Latest revision as of 22:52, 26 September 2012

NOVA

Note Book Record iGEMers in summer.

We summarized our recent brain storm result. At last we chose to make a light-signal oscillator testin E.coli and possibilities to use nanotubes. The first thing for the next week is to find an efficient light sensor and a stable lighting part.

Discuss the establishment of the gene circuits.

test test

Get lux without operon and promoter from k325909 by PCR. We used longPCR Enzyme and Overlapping PCR.


Measure the growth curve of dH5a transformed by lux with araBAD (k325909)

test

Transform tetR (C0040) from the kit. Transfer araBAD (I0500) and tetR (C0040) with RBS into pSB1C3 by 3A ligation. Transform it into dH5a.


Transfer tetR with promoter and RFP with ptetO as promoter (J61002) into pSB1C3 by 3A ligation. Transform it into dH5a.


Sorry to find the ligation is failed. But fortunately, we decide to change the reporter into GFP (I13522). Transform the plasmid from the kit into dH5a.


Design protein composed by lov domain and modified helix turn helix (abbr. lov-HTH).

test

Design protein composed by lov domain and tetR (abbr. lov-tetR).


Send the synthetic orders to the gene synthesis company.


Half of our members went to Tianmu Mount, Zhejiang Province. They spent a week there to participate a fieldwork organized by the Life Science Institute. During the fieldwork, they paid a lot of attention to a China’s specific firefly, Qiongyuying. They observed the behavior of Qiongyuying, especially the synchrony.

test

Our instructor and four of our members went to meet Richard J Roberts after the reward ceremony of COMBREX, a project to accelerate the functional annotation of prokaryotic genomes. They talked for about an hour and we presented our project to him. Richard advised that we should make our project more practical and functional.

test

After negotiating with the principal of volunteer teaching program in our institute, one of our members, with all members of Fudan_Lux’s hope and responsibility on his shoulders, went to Shangrao, Jiangxi Province to do the volunteer teaching.

test class= test

Make an appointment with Li Jin, Hongyan Wang etc. to show our projects.


Receive lov-HTH in nonstandard vector.


Design primers to make clones of lov-HTH, and transfer lov-HTH with double terminators into pSB1C3.


Receive lov-tetR in nonstandard vector.


Do double digestion by E.coliI HF and SpeI, and transfer lov-tetR into pSB1C3.


Make an appointment with prof. Guoping Zhao, who establishes China’s first synthesis biology laboratory in Shanghai. He talked directly about the impropriation of our tumor project and the lack of application about our biowave project.


The double digestion and the ligation held us for more than 4 weeks!

test

In order to do the ligation more efficiently, we decide to do the ligation according to this table.

NO. 
Composition Backbone Code
P1 14N araBAD pSB2K3 I0050
P1 6G pLac pSB1A2 R0011
P1 6N T7 promoter pSB1AK8 I712074
P1 2M Rbs(1.0) pSB1A2 B0034
P1 2IRbs(0.3)pSB1A2  B0032 
P1 2G Rbs(0.07)pSB1A2  B0031 

1.araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-GFP

2.araBAD-Rbs(0.3)-lov-HTH-Terminator-ptetO-GFP

3.araBAD-Rbs(0.07)-lov-HTH-Terminator-ptetO-GFP

4.araBAD-Rbs(1.0)-lov-tetR-Terminator-ptetO-GFP

5.araBAD-Rbs(0.3)-lov- tetR -Terminator-ptetO-GFP

6.araBAD-Rbs(0.07)-lov- tetR -Terminator-ptetO-GFP

7.araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-CFP

8.araBAD-Rbs(0.3)-lov-HTH-Terminator-ptetO-CFP

9.araBAD-Rbs(0.07)-lov-HTH-Terminator-ptetO-CFP

10.araBAD-Rbs(1.0)-lov-tetR-Terminator-ptetO-CFP

11.araBAD-Rbs(0.3)-lov- tetR -Terminator-ptetO-CFP

12.araBAD-Rbs(0.07)-lov- tetR -Terminator-ptetO-CFP

13.araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-lux

14.araBAD-Rbs(0.3)-lov-HTH-Terminator-ptetO- lux

15.araBAD-Rbs(0.07)-lov-HTH-Terminator-ptetO- lux

16.araBAD-Rbs(1.0)-lov-tetR-Terminator-ptetO- lux

17.araBAD-Rbs(0.3)-lov- tetR -Terminator-ptetO- lux 

18.araBAD-Rbs(0.07)-lov- tetR -Terminator-ptetO- lux

19.pLac-Rbs(1.0)-lov-HTH-Terminator-ptetO-GFP

20.pLac -Rbs(0.3)-lov-HTH-Terminator-ptetO-GFP

21.pLac -Rbs(0.07)-lov-HTH-Terminator-ptetO-GFP

22.pLac -Rbs(1.0)-lov-tetR-Terminator-ptetO-GFP

23.pLac -Rbs(0.3)-lov- tetR -Terminator-ptetO-GFP

24.pLac -Rbs(0.07)-lov- tetR -Terminator-ptetO-GFP

25.pLac-Rbs(1.0)-lov-HTH-Terminator-ptetO-CFP

26.pLac -Rbs(0.3)-lov-HTH-Terminator-ptetO-CFP

27.pLac -Rbs(0.07)-lov-HTH-Terminator-ptetO-CFP

28.pLac -Rbs(1.0)-lov-tetR-Terminator-ptetO-CFP

29.pLac -Rbs(0.3)-lov- tetR -Terminator-ptetO-CFP

30.pLac -Rbs(0.07)-lov- tetR -Terminator-ptetO-CFP

31.pLac-Rbs(1.0)-lov-HTH-Terminator-ptetO- lux

32.pLac -Rbs(0.3)-lov-HTH-Terminator-ptetO- lux

33.pLac -Rbs(0.07)-lov-HTH-Terminator-ptetO- lux

34.pLac -Rbs(1.0)-lov-tetR-Terminator-ptetO- lux

35.pLac -Rbs(0.3)-lov- tetR -Terminator-ptetO- lux

36.pLac -Rbs(0.07)-lov- tetR -Terminator-ptetO- lux

37.T7 promoter-Rbs(1.0)-lov-HTH-Terminator-ptetO-GFP

38.T7 promoter -Rbs(0.3)-lov-HTH-Terminator-ptetO-GFP

39.T7 promoter -Rbs(0.07)-lov-HTH-Terminator-ptetO-GFP

40.T7 promoter -Rbs(1.0)-lov-tetR-Terminator-ptetO-GFP

41.T7 promoter -Rbs(0.3)-lov- tetR -Terminator-ptetO-GFP

42.T7 promoter -Rbs(0.07)-lov- tetR -Terminator-ptetO-GFP

43.T7 promoter-Rbs(1.0)-lov-HTH-Terminator-ptetO-CFP

44.T7 promoter -Rbs(0.3)-lov-HTH-Terminator-ptetO-CFP

45.T7 promoter -Rbs(0.07)-lov-HTH-Terminator-ptetO-CFP

46.T7 promoter -Rbs(1.0)-lov-tetR-Terminator-ptetO-CFP

47.T7 promoter -Rbs(0.3)-lov- tetR -Terminator-ptetO-CFP

48.T7 promoter -Rbs(0.07)-lov- tetR -Terminator-ptetO-CFP

49.T7 promoter-Rbs(1.0)-lov-HTH-Terminator-ptetO-lux

50.T7 promoter -Rbs(0.3)-lov-HTH-Terminator-ptetO- lux

51.T7 promoter -Rbs(0.07)-lov-HTH-Terminator-ptetO- lux

52.T7 promoter -Rbs(1.0)-lov-tetR-Terminator-ptetO- lux

53.T7 promoter -Rbs(0.3)-lov- tetR -Terminator-ptetO- lux

54.T7 promoter -Rbs(0.07)-lov- tetR -Terminator-ptetO- lux

Transform the parts mentioned in the box into dH5a.


Pick colonies to cultivate. And extract plasmids.


Do all the double digestions and ligations together for the first time. The order is:

1.Promoter + Rbs & Terminator + Reporter

2.Promoter-Rbs + modified protein

3.Promoter-Rbs-modified protein + Terminator-Reporter


Transform fails for several ligation products.


At last, we choose 3 gene circuits for further investigation:

1.araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-GFP

2.T7 promoter -Rbs(0.3)-lov-HTH-Terminator-ptetO-GFP

3.araBAD-Rbs(1.0)-lov-tetR-Terminator-ptetO-GFP

4.araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-lux

5.T7 promoter -Rbs(0.3)-lov-HTH-Terminator-ptetO-lux

6.araBAD-Rbs(1.0)-lov-tetR-Terminator-ptetO-lux


We run a SDS-PAGE gel to confirm the expression of the modified protein. The result is positive.


In order to verify the modified protein’s characteristic, Juntao Mai does the western-blot. The result is partly positive. Under equal quantity induction of arabinose (0.3%), samples under 450nm wavelength light shows a 30% GFP decay compared with samples incubated in the dark.


We detected the lov-HTH, whose HTH part is modified, by reversed transcription PCR. We‘ve done a series of RT PCR with samples induced by 0.3% arabinose during their incubation and samples incubated without arabinose at the first place. In order to get rid of the effect of light, we incubated all the cells in the dark. We can easily find lov-HTH (modified) mRNA in samples induced by arabinose.


We investigated the time course of the transcription of the arabinose-induced GFP using quantitative real-time PCR. We combined different conditions together and did a series of QRT PCR. We can get the detail in this table.

test

We decide to use EMSA (Electrophoretic Mobility Shift Assay) to verify if the modified protein can be efficient binding the specific part in ptetO.


Considering the time, cost and the possibility, we give up EMSA.


With propose of testing the protein’s repression ability, we do a fluorescence intensity test. Under an arabinose induction gradient between 0 to 0.3%, we investigate the time course of the two groups of cells’ GFP fluorescence intensity and the absorbance of OD600 at the same time. One group of them is incubated in the 470nm wavelength light and the other one was incubated in the dark. The two curves of RFU per OD600 present 30% differences which is positive.


With one of our classmates’ help, Zining Hou make a wood box for shooting pictures in the dark.

test

We make our first double layer disk to shoot.


Our instructor helps us make a single colony mathematic model.

test

We get a series of photos which show a positive result. (araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-lux)


We compare araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-lux, araBAD-Rbs(1.0)-lov-tetR-Terminator-ptetO-lux and T7 promoter-Rbs(0.3)-lov-HTH-Terminator-ptetO-lux by putting four double layer disks together (we use ptetO-lux as control). Unfortunately, the light emitted from different disks interfere each other. So we couldn’t get a usable result. But good news is that we assure the light emitted by lux is strong enough to be a signal.


Organize our first association recruiting with Fudan_D, our cooperating team.

test

Build the wiki


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