Difference between revisions of "Team:Aix-Marseille/Notebook2"

 
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color: #FFFFFF !important;
 
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} .bg-1 {
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background: url(http://i.imgur.com/FrsJhjG.jpg);background-repeat:no-repeat;
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background: url(https://static.igem.org/mediawiki/2015/f/f3/FrsJhjG.jpg);background-repeat:no-repeat;
 
         background-size:cover;
 
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min-height: 600px;
 
min-height: 600px;
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               new WOW().init();
 
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               </script>
 
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<meta name="viewport" content="width=device-width, initial-scale=1">
 
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</head>
 
</head>
 
<body>
 
<body>
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                         </button>
 
                         </button>
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                         <a href="/Team:Aix Marseille" class="navbar-brand hidden-lg hidden-md hidden-sm" ><http://i.imgur.com/oHSATfM.png" class="img-responsive" width="80" height="80"></a>
+
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                             <div class="collapse navbar-collapse  " id="bs-example-navbar-collapse-1">
 
                             <div class="collapse navbar-collapse  " id="bs-example-navbar-collapse-1">
 
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−
                                 <li><a href="/Team:Aix-Marseille"  class="logo"><img src="http://i.imgur.com/oHSATfM.png" class="img-responsive" width="85" height="85"></a></li>
+
                                 <li><a href="/Team:Aix-Marseille"  class="logo"><img src="https://static.igem.org/mediawiki/2015/6/6e/OHSATfM.png" class="img-responsive" width="85" height="85"></a></li>
 
                                     <li  ><a href="/Team:Aix-Marseille">Home</a></li>
 
                                     <li  ><a href="/Team:Aix-Marseille">Home</a></li>
 
                                       <li class="dropdown " id="dropdown"><a href="#" class="dropdown-toggle"  data-toggle="dropdown">Project</a>
 
                                       <li class="dropdown " id="dropdown"><a href="#" class="dropdown-toggle"  data-toggle="dropdown">Project</a>
 
                                       <ul class="dropdown-menu">
 
                                       <ul class="dropdown-menu">
−
                                         <li ><a href="/Team:Aix-Marseille/Description">Overview</a></li>
+
                                         <li ><a href="/Team:Aix-Marseille/Project/Description">Overview</a></li>
 
                                         <li ><a href="/Team:Aix-Marseille/Design">Project design</a></li>
 
                                         <li ><a href="/Team:Aix-Marseille/Design">Project design</a></li>
 
                                         <li ><a href="/Team:Aix-Marseille/Parts">Parts</a></li>                                   
 
                                         <li ><a href="/Team:Aix-Marseille/Parts">Parts</a></li>                                   
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                                         <li ><a href="/Team:Aix-Marseille/Notebook2">Calendar</a></li>
 
                                         <li ><a href="/Team:Aix-Marseille/Notebook2">Calendar</a></li>
 
                                         <li ><a href="/Team:Aix-Marseille/Measurement">Results</a></li>
 
                                         <li ><a href="/Team:Aix-Marseille/Measurement">Results</a></li>
−
                                         <li ><a href="/Team:Aix-Marseille/Protocols2">Protocols2</a></li>
+
                                         <li ><a href="/Team:Aix-Marseille/Protocols2">Protocols</a></li>
 
                                         <li ><a href="/Team:Aix-Marseille/Sequencing/data">Sequencing data</a></li>                                   
 
                                         <li ><a href="/Team:Aix-Marseille/Sequencing/data">Sequencing data</a></li>                                   
 
                                       </ul>
 
                                       </ul>
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                                       </ul>     
 
                                       </ul>     
 
                                     </li>
 
                                     </li>
−
                        
+
                       <li class="dropdown " id="dropdown"><a href="/Team:Aix-Marseille/Achievement" class="dropdown-toggle"  data-toggle="dropdown">Achievement</a></li>
 
                                   </li>
 
                                   </li>
  
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     </header>
 
     </header>
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<li>BBa_J23103 + I13504 + pSB1K3</li>
 
<li>BBa_J23103 + I13504 + pSB1K3</li>
 
<li>BBa_K525998 + I13401 + pSB1K3</li>
 
<li>BBa_K525998 + I13401 + pSB1K3</li>
 +
</ul>
 
<p>Transformation of ligation products</p>
 
<p>Transformation of ligation products</p>
 
<p>PCR on some M1 clones.</p>
 
<p>PCR on some M1 clones.</p>
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<li>BBa_K525998 + I13401 + pSB1K3</li>
 
<li>BBa_K525998 + I13401 + pSB1K3</li>
 
<li>And PCR  M1 relaunch </li>
 
<li>And PCR  M1 relaunch </li>
 +
</ul>
  
 
<p>Starters launched of positive clones</p>
 
<p>Starters launched of positive clones</p>
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   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo11">July 15</button>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo11">July 15</button>
 
   <div id="demo11" class="collapse">
 
   <div id="demo11" class="collapse">
−
Transformation of J23103 + I13504 + pSB1K3 into E.coli (DH5-Alpha)
+
Transformation of J23103 + I13504 + pSB1K3 into <i>E.coli</i> (DH5-Alpha)
 
</div>
 
</div>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo12">July 16</button>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo12">July 16</button>
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<p>Ligation in pSB1C3 </p>
 
<p>Ligation in pSB1C3 </p>
  
−
<p>Transformation into E.coli (DH5-Alpha) of:</p>
+
<p>Transformation into <i>E.coli</i> (DH5-Alpha) of:</p>
 
<ul>
 
<ul>
 
<li>J23100 + I13504 + pSB1C3</li>
 
<li>J23100 + I13504 + pSB1C3</li>
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<div id="main" style="position:relative;top:0px;left:172px;width:1000px;" class="menu-container">
 
<div id="main" style="position:relative;top:0px;left:172px;width:1000px;" class="menu-container">
−
<button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo14">June 1</button>
+
<button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo14">July 20</button>
 
   <div id="demo14" class="collapse">
 
   <div id="demo14" class="collapse">
−
A new supervisor named Valerie Prima joined our team.
+
<p>PCR on many transformants:</p>
−
The team presented our projet “Chew fight” to the laboratory, they liked it and will support us during the summer.
+
<ul>
 +
<li>J23100 + I13504 + pSB1C3</li>
 +
<li>J23101 + I13504 + pSB1C3</li>
 +
<li>J23118 + I13504 + pSB1C3</li>
 +
<li>J23106 + I13504 + pSB1C3</li>
 +
<li>J23117 + I13504 + pSB1C3</li>
 +
<li>J23103 + I13504 + pSB1C3</li>
 +
<li>K525998 + I13401 + pSB1C3</li>
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo15">June 10</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo15">July 21</button>
 
   <div id="demo15" class="collapse">
 
   <div id="demo15" class="collapse">
−
Our project was published in the local newspaper “Grand Luminy”
+
<p>Electrophoresis of the PCR products from July 20th</p>
 +
<p>Starters on positive clones</p>
 +
 
 +
<p>Transformation of J23117 + I13504 + pSB1C3 because it was negative</p>
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo16">June 12</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo16">July 22</button>
 
   <div id="demo16" class="collapse">
 
   <div id="demo16" class="collapse">
−
Welcome to Ella De Gaullejaque who comes from Paris and joined the team! The game Marseille VS Paris will begin !
+
<p>PCR of J23117 + I13504 + pSB1C3</p>
 +
<p>Starters on positive clones</p>
 +
 
 +
<p>The sequences are bad we have to do it again except for M3 which was the only good one</p>
 +
 
 +
<p>Starter of I13504 to make a stock of positive control</p>
 +
 
 +
<p>Plasmid purification of </p>
 +
<ul>
 +
<li>J23100 + I13504 + pSB1C3</li>
 +
<li>J23101 + I13504 + pSB1C3</li>
 +
<li>J23118 + I13504 + pSB1C3</li>
 +
<li>J23106 + I13504 + pSB1C3</li>
 +
<li>J23103 + I13504 + pSB1C3</li>
 +
<li>K525998 + I13401 + pSB1C3</li>
 +
</ul>
 +
 
 +
<p>Digestion E/P and electrophoresis to see if the insertion is good</p>
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo17">June 15</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo17">July 23</button>
 
   <div id="demo17" class="collapse">
 
   <div id="demo17" class="collapse">
−
Meeting with the whole team (members, advisors, instructors) to finalize the timeline of the experiments.
+
<p>We took the wrong BioBrick!!!</p>
−
We created a money pot on Leetchi.com to collect money online
+
<p>Transformation of the right I13504 into <i>E.coli</i> (TG1), two starter cultures launched </p>
 
</div>
 
</div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo18">June 16</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo18">July 24</button>
 
   <div id="demo18" class="collapse">
 
   <div id="demo18" class="collapse">
−
The linker between the laccase and the cytochrome has been designed by Wilson.
+
<p>DNA purification of I13504. Digestion X/P of I13504</p>
−
https://www.facebook.com/photo.php?fbid=10207511567166932&set=gm.830198770403621&type=1
+
<p>Digestion E/S of J23100, K823005, J23118, K823008, K823013</p>
−
The sequences needed were ordered (IDT).
+
<p>Ligation:</p>
 +
<ul>
 +
<li>BBa_J23100 + I13504 + pSB1C3</li>
 +
<li>BBa_K823005 + I13504 + pSB1C3</li>
 +
<li>BBa_J23118 + I13504 + pSB1C3</li>
 +
<li>BBa_K823013 + I13504 + pSB1C3</li>
 +
<li>BBa_J23103 + I13504 + pSB1C3</li>
 +
<li>BBa_K525998 + I13401 + pSB1C3</li>
 +
</ul>
 +
<p>But we did not get enough pSB1C3 only BBa_J23100 + I13504 + pSB1C3</p>
 +
<p>BBa_K823005 + I13504 + pSB1C3 could be launched</p>
 +
<p>Transformation of BBa_J23100 + I13504 + pSB1C3</p>
 +
<p>BBa_K823005 + I13504 + pSB1C3 into <i>E.coli</i> (TG1)</p>
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo19">June 17</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo19">July 27</button>
 
   <div id="demo19" class="collapse">
 
   <div id="demo19" class="collapse">
−
Writing of a newsletter about our project and the synthetic biology.
+
<p>PCR of BBa_J23100 + I13504 + pSB1C3 and
−
Successful application for being sponsored by the french embassy in USA.
+
BBa_K823005 + I13504 + pSB1C3</p>
 +
 
 +
<p>Preparation of pSB1C3 Backbone with a digestion E/P of BBa_K525998 electrophoresis and gel purification of the highest band</p>
 +
 
 +
<p>Ligation:</p>
 +
<ul>
 +
<li>BBa_J23118 + I13504 + pSB1C3</li>
 +
<li>BBa_K823013 + I13504 + pSB1C3</li>
 +
<li>BBa_J23103 + I13504 + pSB1C3</li>
 +
<li>BBa_K525998 + I13401 + pSB1C3</li>
 +
</ul>
 +
<p>Transformation into <i>E.coli</i> (TG1)</p>
 +
<ul>
 +
<li>BBa_J23118 + I13504 + pSB1C3</li>
 +
<li>BBa_K823013 + I13504 + pSB1C3</li>
 +
<li>BBa_J23103 + I13504 + pSB1C3</li>
 +
<li>BBa_K525998 + I13401 + pSB1C3</li>
 +
<li>M1/M2</li>
 +
</ul>
 +
<p>PCR on a green colony seen in the “magic box” => no positive results</p>
 +
<p>Thermal shock forgot for the transformations, we have to do it again!! </p>
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo20">June 18</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo20">July 28</button>
 
   <div id="demo20" class="collapse">
 
   <div id="demo20" class="collapse">
−
First day at the laboratory, meeting of everyone.
+
<p>PCR of J23100 + I13504 + pSB1C3 and
−
Cleaning and room arrangement.
+
BBa_K823005 + I13504 + pSB1C3</p>
−
https://www.facebook.com/photo.php?fbid=102sa06853955166402&set=gm.834872003269631&type=1
+
 
 +
<p>Starters launched on positive results</p>
 +
 
 +
<p>Transformation into <i>E.coli</i> (TG1)</p>
 +
<ul>
 +
<li>BBa_J23118 + I13504 + pSB1C3</li>
 +
<li>BBa_K823013 + I13504 + pSB1C3</li>
 +
<li>BBa_J23103 + I13504 + pSB1C3</li>
 +
<li>BBa_K525998 + I13401 + pSB1C3</li>
 +
<li>M1/M2</li>
 +
</ul>
 +
<p>Plasmid purification of J23100 + I13504 + pSB1C3</p>
 +
<p>E/P digestion of J23100 + I13504 + pSB1C3 and electrophoresis to see the insertion of the BioBrick</p>
 +
 
 +
<p>Preparation of starters to use the TECAN, a plate reader we use to mesure GFP</p>
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo21">June 19</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo21">July 29</button>
 
   <div id="demo21" class="collapse">
 
   <div id="demo21" class="collapse">
−
Familiarization and training sessions with technics, supervised by the instructors. We learned basics, as running a PCR, and where we can find everything needed to work in the lab.
+
<p>We learned how to use the TECAN</p>
 +
 
 +
<p>PCR on M1, M2, BBa_K823013 + I13504 + pSB1C3, BBa_K823013 + I13504 + pSB1C3</p>
 +
 
 +
<p>Transformation of BBa_K823005 + I13504 + pSB1C3 into <i>E.coli</i> (TG1)</p>
 +
 
 +
<p>Ligation overnight:</p>
 +
<ul>
 +
<li>BBa_J23118 + I13504 + pSB1C3</li>
 +
<li>BBa_K823013 + I13504 + pSB1C3</li>
 +
<li>BBa_J23103 + I13504 + pSB1C3</li>
 +
<li>BBa_K525998 + I13401 + pSB1C3</li>
 +
</ul>
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo22">June 22</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo22">July 30</button>
 
   <div id="demo22" class="collapse">
 
   <div id="demo22" class="collapse">
−
Training sessions for PCR
+
<p>Transformation of ligation products:</p>
 +
<ul>
 +
<li>BBa_J23118 + I13504 + pSB1C3</li>
 +
<li>BBa_K823013 + I13504 + pSB1C3</li>
 +
<li>BBa_J23103 + I13504 + pSB1C3</li>
 +
<li>BBa_K525998 + I13401 + pSB1C3</li>
 +
</ul>
 +
<p>PCR on the only clone of BBa_K823005 + I13504 + pSB1C3 we got => positive result=> starter launched</p>
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo23">June 23</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo23">July 31</button>
 
   <div id="demo23" class="collapse">
 
   <div id="demo23" class="collapse">
−
<p>Training sessions for bacteria transformation</p>
+
<p>Plasmid purification of BBa_K823005 + I13504 + pSB1C3 and E/P digestion to see the right insertion of the insert</p>
−
<p>Registration for measurement </p>
+
 
 +
<p>PCR on green colonies on “the magic box”</p>
 
</div>
 
</div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo24">June 24</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo24">August 3</button>
 
   <div id="demo24" class="collapse">
 
   <div id="demo24" class="collapse">
−
<p>Training session for Gel purification, PCR clean up, plasmid purification (miniprep)</p>
+
<p>PCR of BBa_K823005 + I13504 + pSB1C3 done again because of bad results</p>
−
<p>First purchases of the lab material.</p>
+
 
 +
<p>Starter launched on positive results</p>
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo25">June 25</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo25">August 4</button>
 
   <div id="demo25" class="collapse">
 
   <div id="demo25" class="collapse">
−
<p>Resuspension of biobricks K863011, K863006, K525998, K844000, E0240, K823005, K823012, I20260 and transformation into E.coli (DH5-alpha). </p>
+
<p>Starter of BBa_K823013 + I13504 + pSB1C3 and on BBa_K823008 + I13504 + pSB1C3 launched</p>
−
<p>Negative results (no colony on the plates).</p>
+
 
 +
<p>J23100 + I13504 + pSB1C3 good sequencing!!!</p>
 +
 
 +
<p>Ligation overnight BBa_J23101 + I13504 + pSB1C3</p>
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo26">June 26</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo26">August 5</button>
 
   <div id="demo26" class="collapse">
 
   <div id="demo26" class="collapse">
−
<p>New try for Resuspension of biobricks K863011, K863006, K525998, K844000, E0240, K823005, K823012, I20260 and transformation into E.coli (DH5-alpha). </p>
+
<p>Transformation of J23101 + I13504 + pSB1C3 into <i>E.coli</i> (TG1)</p>
−
<p>Positive results (several colonies on the plate).</p>
+
 
 +
<p>BBa_K823008 + I13504 + pSB1C3 prepared to send for sequencing </p>
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo27">June 29</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo27">August 6</button>
 
   <div id="demo27" class="collapse">
 
   <div id="demo27" class="collapse">
−
<p>First meeting with the director of innovations and communications from the cleaning company named ONET. </p>
+
<p>PCR on J23101 + I13504 + pSB1C3 </p>
−
<p>We talked about our idea and our Chew fight project in order to be sponsored.</p>
+
<p>Starter of positive clones</p>
 +
 
 +
<p>Ligation J23103 + I13504 + pSB1C3</p>
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo28">June 30</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo28">August 7</button>
 
   <div id="demo28" class="collapse">
 
   <div id="demo28" class="collapse">
−
Plasmid purification (miniprep) on K863011, K863006, K525998, K844000, E0240, K823005, K823012, I20260 - digestion J23101 et J23115(E/S) et E0240 (X/P) + plasmide (?) I20260 (E/P Dpn1)
+
<p>Transformation of J23101 + I13504 + pSB1C3 into <i>E.coli</i> (TG1)</p>
 +
 
 +
<p>Plasmid purification of J23101 + I13504 + pSB1C3 starters</p>
 
   </div>
 
   </div>
 
</div>
 
</div>
Line 416: Line 510:
  
 
<div id="main" style="position:relative;top:0px;left:172px;width:1000px;" class="menu-container">
 
<div id="main" style="position:relative;top:0px;left:172px;width:1000px;" class="menu-container">
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo29">July 6</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo29">August 10</button>
 
   <div id="demo29" class="collapse">
 
   <div id="demo29" class="collapse">
−
Reception of pipettes borrowed from Polytech school. All the pipettes were calibrated
+
<p>J23101 + I13504 + pSB1C3 prepared to send for sequencing </p>
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo30">July 7</button>
+
−
  <div id="demo30" class="collapse">
+
−
Reception of the microcentrifuge, gel electrophoresis, western blot and SDS-page materials, borrowed from Polytech school.
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo31">July 8</button>
+
−
  <div id="demo31" class="collapse">
+
−
ONET company presented our project to the Executive office.
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo32">July 9</button>
+
−
  <div id="demo32" class="collapse">
+
−
Positive answer from ONET: it sponsored our project with 7000 euros !
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo33">July 10</button>
+
−
  <div id="demo33" class="collapse">
+
−
Resuspension of the biobrick “12” (cyt C Shewanella) received from IDT, ligation into pSB1C3, transformation into DH5-alpha.
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo34">July 15</button>
+
−
  <div id="demo34" class="collapse">
+
−
<p>Colony PCR on BL21 strain to amplify the 3 parts of ccm operon. Oligos were too concentrated and inhibited the reaction.</p>
+
−
<p>Colony PCR and electrophoresis to check the size of the biobrick “12” (cytochrome c Shewanella). Positive result.</p>
+
−
<p>Resuspension of biobricks “05”, “08”, “30”, “11” received from IDT. E/P digestion, ligation into pSB1C3, transformation into E.coli (TG1). Negative result (no colony on the plate).</p>
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width:110px;" data-target="#demo35">July 16</button>
+
−
  <div id="demo35" class="collapse">
+
−
<p>Second try to resuspend the biobricks “05”, “08”, “30”, “11” received from IDT and resuspension of the biobrick “13” received from IDT. E/P digestion, ligation into pSB1C3, transformation into E.Coli (TG1). Positive result (several colonies on the plate).</p>
+
−
<p>Second try for Colony PCR on DH5-alpha strain to amplify the 3 parts of ccm operon with diluted oligos at 1/10. Positive result except for part 1: there are some contaminants due to nonspecific hybridization.</p>
+
−
<p>Reception of oligos from SIGMA to amplify the laccase of E.coli and T.thermophilus.</p>
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo36">July 17</button>
+
−
  <div id="demo36" class="collapse">
+
−
<p>Third try for colony PCR on DH5-alpha to amplify part 1 of ccm operon. The Tm changed from 55 to 60°C. Positive result.</p>
+
−
<p>Colony PCR and electrophoresis to check the size of the biobricks “05”, “08”, “30”, “11” and “13”. Positive results except for “08” et “11”.</p>
+
−
<p>Starters of “05”, “13”, “30”.</p>
+
−
<p>Plasmid purification (miniprep) of the biobrick “12”, E/P digestion to check the size of the insert. Positive result.</p>
+
−
<p>High fidelity DNA amplification of biobricks “09” and “10” by PCR with the Q5 polymerase in order to remove the codon-STOP. PCR clean up. Negative result (nonspecific amplification).</p>
+
−
<p>Reception of the biobrick “15” from IDT.</p>
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo37">July 20</button>
+
−
  <div id="demo37" class="collapse">
+
−
<p>Colony PCR and electrophoresis to check the size of the biobricks “08” and “11”. Positive result for “08” but not for “11”. Starter of “08”.</p>
+
−
<p>New DH5-alpha competent cells done.</p>
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo38">July 21</button>
+
−
  <div id="demo38" class="collapse">
+
−
<p>Second try for high fidelity DNA amplification of biobricks “09” and “10” by PCR with the Q5 polymerase in order to remove the codon-STOP (the new synthesized biobricks are called respectively “35” and “36”). PCR clean up. Negative result (non-specific amplification). An electrophoresis was done on the total PCR product and was extracted and purified by PCR clean up.</p>
+
−
<p>Miniprep of “08”. E/P digestion to check the size of the insert. Negative result (unexpected size).</p>
+
−
<p>Resuspension of biobrick “15” received from IDT. E/P digestion, ligation into pSB1C3, transformation into E.coli (DH5-alpha). Negative result (no colony on the plate).
+
−
<p>New TG1 competent cells done.</p>
+
−
<p>Transformation efficiency test with the transformation efficiency kit of iGEM on the new DH5-alpha competent cells. Positive result (the DH5-alpha cells are competent enough to be transformed with our constructions).</p>
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo39">July 22</button>
+
−
  <div id="demo39" class="collapse">
+
−
<p>Colony PCR and electrophoresis to check the size of the biobricks “05” and “13”. Positive result. Starters of « 05 » and « 13 ».</p>
+
−
<p>SLIC reaction to ligate the 3 parts of ccm operon into the pSB1C3 plasmid. Transformation into E.coli (TG1). Negative result (no colony on the plate). </p>
+
−
<p>SLIC reaction to ligate the new biobricks “35” and “36” into pSB1C3. Transformation into E.coli (TG1). Negative result (no colony on the plate).</p>
+
−
<p>Transformation efficiency test with the transformation efficiency kit of iGEM on the new TG1 competent cells. Positive result (the TG1 cells are competent enough to be transformed with our constructions)</p>
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo40">July 23</button>
+
−
  <div id="demo40" class="collapse">
+
−
<p>Miniprep of “05” and “13” and E/P digestion to check the size of the insert. </p>
+
−
<p>New try to transform “08” into pSB1C3 into DH5-alpha.</p>
+
−
<p>Resuspension of biobricks “09” and “10” from the registry, transformation into TG1.</p>
+
−
<p>Construction of “30-02”, “12-02” : E/X digestion of “30” and “12”, S/P digestion of “02”, ligation into pSB1A3, transformation into TG1. Negative result (no colony on the plate).</p>
+
−
<p>New try for the SLIC reaction (ccm operon), “35” and “36”. Negative result (no colony on the plate).</p>
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo41">July 24</button>
+
−
  <div id="demo41" class="collapse">
+
−
<p>New try with different volumes for resuspension of biobricks “15” and “08” received from IDT. E/P digestion, ligation into pSB1C3, transformation into E.coli (TG1). Positive result (several colonies on the plate).</p>
+
−
<p>Colony PCR and electrophoresis to check the size of the biobricks”08”, “09”, “10”,”12-02”, “30-02”, “32” and “34”. Positive result for “09” and “10” (expected size). Starters of « 09 » and « 10 ».</p>
+
−
<p>Negative result for “32”, “34”, “12-02”, “30-02” (No colony on the plate) and for “08” (unexpected size).<
+
−
<p>New try to construct “30-02”, “12-02”: E/X digestion of “30” and “12”, S/P digestion of “02”, ligation into pSB1A3, transformation into TG1. Positive result (several colonies on the plate).
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo42">July 27</button>
+
−
  <div id="demo43" class="collapse">
+
−
<p>Colony PCR and electrophoresis of “08”, “15”, “12-02” and “30-02” to check the size of the insert. Positive results for “15”, “12-02” and “13-02” (expected size) but negative result for “08” (several sizes observed). Starters of “08”, “15”, “12-02” and “30-02”.</p>
+
−
<p>Plasmid purification (miniprep) of “05”, “09” and “10”. Problem encountered: genomic DNA contamination.</p>
+
−
<p>E/P digestion and electrophoresis to check the size of the insert of “05”, “09”, and “10”. </p>
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo44">July 28</button>
+
−
  <div id="demo44" class="collapse">
+
−
<p>Miniprep of “08”, “15”, 12-02”, “30-02”. E/P digestion and electrophoresis to check the size of the insert of “08”, “15”, 12-02”, “30-02”. Positive results for “15”, “12-02”, “30-02” (expected size) and negative result for “08” (unexpected size). </p>
+
−
<p>Comment: constructs “12-02” and “30-02” have to be compared with the single “12” and “30” to check if “02” was added.  </p>
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo45">July 29</button>
+
−
  <div id="demo46" class="collapse">
+
−
<p>Colony PCR and electrophoresis to check the size of the insert of “08”. Negative result</p>
+
−
<p>Electrophoresis of “12”, “12-02”, “30” and “30-02”. No result (no DNA observed on the agarose gel)</p>
+
−
<p>Construction of “09-02”, “10-02” and “13-02”: E/S digestion of “09”, “10” and “13”. X/P digestion of “02”. Ligation into pSB1A3, transformation into TG1. Positive results for “09-02” and “13-02” (1 colony on the plate). Negative result for “10-02” (no colony on the plate).</p>
+
−
<p>Resuspension of biobrick “11” and new try for the “05” and the “08” received from IDT. E/P digestion and ligation into pSB1C3. Transformation into TG1. Positive result for “11” (several colonies on the plate) but not for “05” and “08” (no colony on the plate) </p>
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo46">July 30</button>
+
−
  <div id="demo46" class="collapse">
+
−
<p>Colony PCR and electrophoresis to check the size of the insert of « 11 », “09-02” and “13-02”. Uncertain results (problems: DNA is not observed on the agarose gel). “13-02” seems good: starter of “13-02”.</p>
+
−
<p>New try with new oligos for the colony PCR and electrophoresis of « 08 ». No result (no DNA observed on the agarose gel).</p>
+
−
<p>New try with new volumes for resuspension of the biobrick “05” received from IDT. E/P digestion and ligation into pSB1C3. Transformation into TG1. Negative result (no colony on the plate).</p>
+
−
<p>New try to construct “09-02”, “10-02” and “13-02”. E/S digestion of “09”, “10” and “13”. X/P digestion of “02”. Ligation overnight into pSB1A3, transformation into TG1 (07/31). Positive result for “13-02” (several colonies on the plate. Negative result for “09-02” and “10-02” (no colony on the plate).</p>
+
−
<p>E/P digestion to check the size of the insert of “30”, “30-02”, “12”, “12-02”. Results seems good for “12” and “12-02”: send for sequencing. Negative results for “30”, “30-02” (unexpected sizes).</p>
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo47">July 31</button>
+
−
  <div id="demo47" class="collapse">
+
−
<p>Miniprep of « 13-02 ». E/P digestion and electrophoresis to check the size of the insert. Positive result (expected size).</p>
+
−
<p>Colony PCR and electrophoresis to check the size of the insert of “13-02”. Negative result (unexpected size).</p>
+
−
<p>Transformation of “09-02”, “10-02” and “13-02” into TG1.</p>
+
−
<p>pSB1C3 backbone was running out. New backbone done: E/P digestion on measurement biobricks, electrophoresis and purification of the digested plasmid by gel extraction and PCR clean up. </p><p>Problem encountered: no DNA observed on the agarose gel. </p>
+
−
  </div>
+
−
</div>
+
  
 +
<p>Starter of BBa_K823013 + I13504 + pSB1C3 launched => preparation for sequencing</p>
  
−
<div id="main" style="position:relative;top:0px;left:172px;width:1000px;" class="menu-container">
+
<p>Isolation of BBa_K823008 + I13504 + pSB1C3</p>
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo48">August 3</button>
+
−
  <div id="demo48" class="collapse">
+
−
<p>Colony PCR and electrophoresis to check the size of the insert of “09-02”, “10-02” and “13-02”. Positive results (expected sizes). Starters of “09-02”, “10-02” and “13-02.</p>
+
−
<p>New try for making pSB1C3 backbone: E/P digestion of measurement biobricks, electrophoresis and purification of the digested plasmid by gel extraction and PCR clean up. </p>
+
−
<p>Resuspension of the biobrick “07” (cotA) received from IDT. E/P digestion and ligation into pSB1A3, transformation into TG1</p>
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo49">August 4</button>
+
−
  <div id="demo49" class="collapse">
+
−
<p>Colony PCR and electrophoresis to check the size of the insert on “07” => we got a positive clone, starter of this clone launched</p>
+
−
<p>Plasmid purification of “13-02” “09-02” “10-02”</p>
+
−
<p>E/P digestion of “05” and “11” and ligation “05” + pSB1K3 and “11” + pSB1K3 </p>
+
−
<p>Transformation of ligation products into E.coli (TG1)</p>
+
−
<p>PCR on “07” and “08” => seems to be unclean with the Q5 high fidelity DNA amplification</p>
+
−
<p>X/P digestion of “15” </p>
+
−
<p>Overnight ligation “12”+”02”+pSB1A3 and “01”+”15”+pSB1T3</p>
+
−
<p>“30-02” and “08” got bad results in sequencing</p>
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo50">August 5</button>
+
−
  <div id="demo50" class="collapse">
+
−
<p>Transformation of ”01-15” and “12-02” into E.coli (TG1)</p>
+
−
<p>Plasmid purification of “09-02” “07” “13-02” “09-02” “10-02” => low concentration of “09-02” but we got good result for “13-02”</p>
+
−
<p>Transformation of “12-02” and “01-15” into TG1</p>
+
−
<p>PCR clean up “07” “08” “24” ==> low plasmid concentration of “07” and “08” to do again. </p>
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo51">August 6</button>
+
−
  <div id="demo51" class="collapse">
+
−
<p>Colony PCR of “05” “11” “12-02” “01-15” => good but only for “12-02”</p>
+
−
 
+
−
<p>PCR clean up of “08” and “07” → PCR products contaminated → PCR clean up</p>
+
  
−
<p>Transformation of “10-02” and “09-02”→ Wrong size starter relaunched</p>
+
<p>X/P digestion of I13504</p>
  
−
<p>Ligation “09-02” and “10-02” into  pSB1A3 and “01-15” into pSB1T3</p>
+
<p>Ligation BBa_K823008 + I13504 + pSB1C3</p>
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo52">August 7</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo30">August 11</button>
−
   <div id="demo52" class="collapse">
+
   <div id="demo30" class="collapse">
−
<p>Colony PCR of “05” and “01-15” --> no results on this PCR </p>
+
Transformation BBa_K823008 + I13504 + pSB1C3 into <i>E.coli</i> (TG1)
−
 
+
−
<p>Transformation of “09-02” “10-02” “01-15” into E.coli (TG1)</p>
+
−
 
+
−
<p>Ligation “05” + pSB1K3 and “11” + pSB1K3</p>
+
−
 
+
−
<p>Plasmid purification of “12-02” --> Bad results with electrophoresis</p>
+
−
 
+
−
<p>For “05” “07” “08” “11” → PCR with Q5 high fidelity polymerase and “PCR clean up”</p>
+
−
 
+
−
<p>Preparation of BL21 competent cells </p>
+
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo53">August 10</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo31">August 12</button>
−
   <div id="demo53" class="collapse">
+
   <div id="demo31" class="collapse">
−
<p>Colony PCR of “01-15” → No results => Ligation with the digestion product “01-15” relaunched</p>
+
<p>BBa_K823005 + I13504 + pSB1C3 has the right sequence!</p>
  
−
<p>For “05” “07” “08” “11” : Digestion E/S + Ligation pSB1C3 + Transformation into TG1</p>
+
<p>Ligation J23103 + I13504 + pSB1C3</p>
 +
<p>Transformation of J23103 + I13504 + pSB1C3 into <i>E.coli</i> (TG1)</p>
  
−
<p>For “30” : Digestion E/P + Ligation into pSB1K3</p>
+
<p>Send for sequencing BBa_K823008 + I13504 + pSB1C3 and BBa_K823013 + I13504 + pSB1C3</p>
  
−
<p>For “01-15” : Ligation into pSB1T3</p>
+
<p>Plasmid purification of BBa_K823013 + I13504 + pSB1C3</p>
−
 
+
−
<p>PCR clean up of “28” </p>
+
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo54">August 11</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo32">August 16</button>
−
   <div id="demo54" class="collapse">
+
   <div id="demo32" class="collapse">
−
<p>Colony PCR of “09-02” “10-02” “05” “07” “08” “11”</p>
+
PCR screening colonies of J23119 + I13504 + pSB1C3
−
 
+
−
<p>Preparation of TG1 competent cells </p>
+
−
 
+
−
<p>We used the wrong “02” Biobrick we have to do everything again with the good one →  Colony PCR of the “new” “02” →  size = ok </p>
+
−
 
+
−
<p>Transformation of “30” “01-01” “02” into TG1 </p>
+
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo55">August 12</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo33">August 17</button>
−
   <div id="demo55" class="collapse">
+
   <div id="demo33" class="collapse">
−
<p>Preparation of the backbone pSB1A3</p>
+
<p>BBa_K823013 + I13504 + pSB1C3 send for sequencing</p>
  
−
<p>Ligation of “05” “08” “07” “11” into pSB1A3</p>
+
<p>Starter launched on positive clones from the PCR done on August 16</p>
−
 
+
−
<p>Colony PCR of “30” “21-17” “23-17” “01-15”</p>
+
−
<p>For “35” et “36” : Digestion E/S + ligation into pSB1A3 + transformation into TG1</p>
+
−
<p>For “37” “38” : Digestion E/P + Ligation into pSB1K3 + transformation into “supercompetent” cells</p>
+
−
 
+
−
<p>Plasmid purification of “02” --> Low concentration of plasmid, starter relaunched</p>
+
−
 
+
−
<p>For “02” : Digestion X/P </p>
+
−
<p>For “13” : Digestion E/S</p>
+
−
 
+
−
<p>Ligation of “35-02” “36-02” “12-02” “13-02”  into pSB1A3</p>
+
−
 
+
−
<p>PCR  on “09” et “10” to remove the stop codon</p>
+
−
 
+
−
<p>Transformation of “05” “07” “08” “11” into  “supercompetent” cells </p>
+
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo56">August 13</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo34">August 18</button>
−
   <div id="demo56" class="collapse">
+
   <div id="demo34" class="collapse">
−
<p>Colony PCR on “05” “07” “08” “21-17” “11” “35” “36” “37” “38”</p>
+
<p>Plasmid purification and digestion E/P to check the insertion => wrong results </p>
 +
<p>Try again on the same clone</p>
  
−
<p>Transformation of “12-02” “13-02” “35-02” “36-02” “36-02” into TG1</p>
+
<p>Ligations: BBa_K823008 + I13504 + pSB1C3 and BBa_K823013 + I13504 + pSB1C3</p>
−
 
+
−
<p>PCR clean up of “35” “36” </p>
+
−
 
+
−
<p>Digestion E/S of “12” “35” “36” “16” “13”</p>
+
−
 
+
−
<p>For “12-02” “13-02” “35-02” “36-02” : Ligation into pSB1A3 + transformation in TG1</p>
+
−
<p>For “01-15” : Ligation into pSB1T3 + transformation into TG1</p>
+
−
<p>For “16-17” : Ligation into pSB1C3 + transformation into TG1</p>
+
−
 
+
−
<p>Colony PCR on “38” “05” “07” “36” ”08” “11”</p>
+
−
 
+
−
<p>For “39” “30” : Digestion E/P + Ligation into pSB1K3</p>
+
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo57">August 14</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width:110px;" data-target="#demo35">August 19</button>
−
   <div id="demo57" class="collapse">
+
   <div id="demo35" class="collapse">
−
<p>Colony PCR on “12-02” “13-02” “39” “36-02” “23-17” “35-02”</p>
+
<p>BBa_K823008 + I13504 + pSB1C3 and BBa_K823013 + I13504 + pSB1C3 and J23100 + I13504 + pSB1C3 send for sequencing</p>
  
−
<p>Plasmid purification of “36” “38” “07” “05” “35” “11” “37” --> Verification ok for “36” “38” “35” “37” </p>
+
<p>J23103 + I13504 + pSB1C3 has a good sequencing result</p>
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo58">August 15</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo36">August 20</button>
−
   <div id="demo58" class="collapse">
+
   <div id="demo36" class="collapse">
−
<p>Colony PCR on “05” “07” “08” “11” “16-17” “21-17” “05” “07” “08” “11” “16-17” “21-17” --> Bad results for all</p>
+
J23119 + I13504 + pSB1C3 send for sequencing
−
 
+
−
<p>Plasmid purification of  “38” “05” “11” “08” “39” “12-02” “13-02” “35-02” “23-17” “35” “01-15” “16-17” “21-17” --> Problem with the ladder, we cannot read the gel</p>
+
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo59">August 17</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo37">August 23</button>
−
   <div id="demo59" class="collapse">
+
   <div id="demo37" class="collapse">
−
<p>For “38” “39” “08” “05” : Digestion E/S</p>
+
<p>BBa_K823008 + I13504 + pSB1C3 and BBa_K823013 + I13504 + pSB1C3 => good sequencing result</p>
−
<p>For “12” “13” “39” “35” “35-02” “13-02” “12-02” : Digestion X/P</p>
+
−
 
+
−
<p>For “38-12” “38-13” “39-02” “38-39”  “08-02” “36” “05-02”: Ligation into pSB1A3</p>
+
−
<p>For “01-3502” “01-1302” “01-1202” “01-35” : Ligation into pSB1T3</p>
+
−
<p>For “21-17” “22-17” : Ligation into pSB1C3</p>
+
−
        ==> Transformation for all ligation products into TG1
+
  
−
<p>Colony PCR of “07” “05” “08” “11” with “pool” technic</p>
+
<p>Transformation of ligation products of J23119 + I13504 + pSB1C3 and BBa_K823013 + I13504 + pSB1C3</p>
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo60">August 18</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo38">August 22</button>
−
   <div id="demo60" class="collapse">
+
   <div id="demo38" class="collapse">
−
<p>Colony PCR of “11” “7” => starters launched on positive clones</p>
+
PCR on J23119 + I13504 + pSB1C3 and BBa_K823013 + I13504 + pSB1C3 
−
 
+
−
<p>Plasmid verification of “05” “07” “08” “11” and “16-17”</p>
+
−
<p>“07” and “08” have to be relaunched </p>
+
−
 
+
−
<p>Colony PCR on transformants from August 17 => Starters on positive clones launched and the constructions “01-3502” and “38-39” have to be rebuilt</p>
+
−
 
+
−
<p>Preparation of the backbone pSB1A3</p>
+
−
 
+
−
<p>Ligation of “01-1302” “01-1202” “01-35” into pSB1T3</p>
+
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo61">August 19</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo39">August 24</button>
−
   <div id="demo61" class="collapse">
+
   <div id="demo39" class="collapse">
−
<p>Starters launched of “05” and “11”</p>
+
<p>Transformation of J23119 + I13504 + pSB1C3 and K823013 + I13504 + pSB1C3  into <i>E.coli</i> (DH5-Alpha)</p>
−
<p>Preparation of the backbone pSB1A3</p>
+
−
<p>Sequencing results “12-02” “13-02” “35-02” “01-15” are ok</p>
+
−
<p>“08” is bad and “05” couldn’t be sequenced</p>
+
−
<p>Plasmid purification of “30” “38-13” “39-02” “08-02” “05-02” “38-12” “11” “07” “16-17”</p>
+
−
<p>“38-13” “39-02” “16-17” “38-12” send for sequencing</p>
+
−
<p>“30” “08-02” “05-02” “11” “07” are bad we cannot use its </p>
+
−
  </div>
+
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo62">August 20</button>
+
−
  <div id="demo62" class="collapse">
+
−
<p>Colony PCR of “05-02” “01-1302”</p>
+
  
−
<p>Plasmid purification of “05” and “11” and of 2 starters to make some backbones (pSB1A3 and pSB1T3)</p>
+
<p>First try of construction of a box to observe GFP called “magic-box”. First try with coomasie blue as light filter, public lab spectrometer, yellow filter and a webcam</p>
−
<p>The digestion to prepare pSB1T3 is bad, starter relaunched</p>
+
−
 
+
−
<p>Ligation of “38-39” “11-02” into pSB1A3</p>
+
−
<p>“38-11” into pSB1C3</p>
+
−
<p>“01-1202” “05-02” “01-36” “01-1302” “01-3502” “01-35” “21-17” “22-17” into pSB1K3</p>
+
−
<p>Transformation of previous ligation products into DH5-Alpha for “21-17” and “22-17”, into TG1 for “11-02” “38-11” “05-02” and “38-39” and into BL21 for “01-1202” “01-36” “01-1302” “01-3502” and “01-35”</p>
+
−
 
+
−
<p>“38-13” “39-02” “11” and “38-12” sent for sequencing</p>
+
−
<p>Sequencing result of “05” => bad </p>
+
−
 
+
−
<p>Transformation of “18-17” “19-17” “20-17” and “23-17” into DH5-Alpha</p>
+
−
 
+
−
<p>Colony PCR of “07”</p>
+
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo63">August 21</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo40">August 25</button>
−
   <div id="demo63" class="collapse">
+
   <div id="demo40" class="collapse">
−
<p>Digestion E/S fof “16” “38” “11” “22”, X/P for “17” “39” “02” “11” “35”</p>
+
<p>J23119 + I13504 + pSB1C3 (obtained the August 24th)  send for sequencing </p>
  
−
<p>Ligation of “16-17” “22-17” + pSB1C3 “38-39” “11-02” “38-11” + pSB1A3</p>
+
<p>Construction of the magic-box without the public lab spectrometer</p>
−
<p>“01-35” “05” “07” “08” “30” + pSB1K3</p>
+
<p>Coomasie blue concentration determination and first assay with the magic box</p>
−
 
+
−
<p>Transformation of “30” “08” “07” “05” into C2987 (“supercompetent” cells)</p>
+
−
 
+
−
<p>Transformation of “38-39” “11-02” “38-11” and “01-35” into TG1</p>
+
−
 
+
−
<p>Plasmid purification of “01-1202” “01-3502” “05” “01-1302” “07” and of pSBT3 to get some backbone.</p>
+
−
 
+
−
<p>Colony PCR of “08” “01-3502” “01-1202” “01-1302” “01-36”</p>
+
−
 
+
−
<p>We got many bad results today so bad luck, but we got some good results also.</p>
+
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo64">August 22</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo41">August 26</button>
−
   <div id="demo64" class="collapse">
+
   <div id="demo41" class="collapse">
−
<p>Colony PCR of “38-39” “38-11” “11-02” “01-35” </p>
+
<p>Sequencing results of J23119 + I13504 + pSB1C3 => bad</p>
−
<p>Starters launched of positive clones</p>
+
<p>K823013 + I13504 + pSB1C3 clone 5 => good </p>
 +
<p>K823013 + I13504 + pSB1C3 clone 2 => bad</p>
  
−
<p>Transformation of overnight ligation produtcs (“16-17” and “22-17” from August 21)</p>
+
<p>Isolation of K525998 + I13401 + pSB1C3, K823008 + I13504 + pSB1C3, J23119 + I13504 + pSB1K3 and K823013 + I13504 + pSB1K3</p>
  
−
<p>Plasmid purification to make some pSB1A3 and pSB1T3</p>
+
<p>E/P digestion of J23119 + I13504 + pSB1K3</p>
  
−
<p>First protein expression test, we maybe succeed to express our first laccase </p>
+
<p>Ligation overnight:</p>
−
  </div>
+
<p>J23119 + I13504 + pSB1C3 </p>
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo65">August 23</button>
+
−
  <div id="demo65" class="collapse">
+
−
<p>Colony PCR of “16-17” and “22-17” launched overnight => green colonies!!</p>
+
  
−
<p>Starters launched of positive results</p>
+
<p>tarter launched on another  J23119 + I13504 + pSB1C3 clone</p>
  
−
<p>Plasmid purification of “22”-17” “16-17” and ‘01-35” => all results are good!!!!</p>
+
<p>Measure of fluorescent intensity with different bacterial OD</p>
−
 
+
<p>Determination of the medium that will be use => LB or PBS1X</p>
−
<p>Digestion E/S for “38” and X/P for “39”, ligation overnight into pSB1A3</p>
+
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo66">August 24</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo43">August 27</button>
−
   <div id="demo66" class="collapse">
+
   <div id="demo43" class="collapse">
−
<p>Transformation of “30” “08” “07” “11” “05” into TG1</p>
+
<p>Ligation K823013 + I13504 + pSB1C3</p>
  
−
<p>Plasmid purification of “16-17” “01-1302” “01-1202” “22-17” “01-3502”</p>
+
<p>Transformation of K823013 + I13504 +pSB1K3 into <i>E.coli</i> (DH5-Alpha)</p>
  
−
<p>Colony PCR of “05” “07” “08” “11” => no positive results!</p>
+
<p>Test on many machines to observe GFP => confocal microscope, magic-box, TECAN, and fluorimeter</p>
−
 
+
−
<p>Q5 PCR of “05” “07” “08” “11” “30” => positive results </p>
+
−
 
+
−
<p>“05” and “01-35” sent for sequencing </p>
+
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo67">August 25</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo44">August 28</button>
−
   <div id="demo67" class="collapse">
+
   <div id="demo44" class="collapse">
−
<p>Transformation of “05” “07” “08” “11” “30” into TG1 </p>
+
<p>Transformation of K823013 + I13504 +pSB1C3 gives no transformants</p>
−
 
+
−
<p>Plasmid purification of “01-36” “19-17” “18-17” “23-17” “20-17” => good results</p>
+
−
 
+
−
<p>“01-1202” “01-1302” “01-3502” sent for sequencing</p>
+
−
 
+
−
<p>Sequencing results of “01-35” “38-12” “38-13” “39-02” => good results “05” “11” => bad results</p>
+
  
−
<p>Starters launched of “39” “01-1302” “01-1202” “01-3502” “01-15” “13-02” “12-02” “38”</p>
+
<p>Measure of GFP intensity with TECAN and confocal microscope on “18-17”, “19-17”, “20-17”, “21-17”, “22-17”, “23-17”, “01-24”, “33”  and “32”</p>
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo68">August 26</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo45">August 31</button>
−
   <div id="demo68" class="collapse">
+
   <div id="demo45" class="collapse">
−
Protein expression => the cytochrome c and the laccase are expressed.
+
Digestion E/P of K823013 + I13504 + pSB1C3
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo69">August 27</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo46">September 3</button>
−
   <div id="demo69" class="collapse">
+
   <div id="demo46" class="collapse">
−
 
+
<p>Measure of GFP intensity with fluorimeter and magic box on “18-17”, “19-17”, “20-17”, “21-17”, “22-17”, “23-17”, “01-24”, “33”  and “32”</p>
−
<p>Isolation and starter launched of “32”</p>
+
−
 
+
−
<p>Plasmid purification of “39” “01-1302” “01-1202” “01-3502” “01-15” “13-02” “12-02” “38” “16-17”=> no results!</p>
+
−
 
+
−
<p>Ligation of “40” “41” “42” “43” “44” “45” “46” “47” “48” “22-17” + pSB1C3</p>
+
−
<p>“30” + pSB1K3</p>
+
−
 
+
−
<p>Transformation of “30” “40” “45” “48” into TG1</p>
+
−
<p>Transformations didn’t work! => “40” “45” “48” relaunched </p>
+
−
 
+
−
<p>Sequencing results “01-1302” => without promoter</p>
+
−
<p>“01-1202” and “01-3502” are good</p>
+
  
 +
<p>Sequencing result: “23-17” is bad </p>
 
   </div>
 
   </div>
−
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo70">August 31</button>
+
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo47">September 4</button>
−
   <div id="demo70" class="collapse">
+
   <div id="demo47" class="collapse">
−
<p>Colony PCR of “40” “45” “48” </p>
+
Preparation of standard curve of Atton 488 for magic-box, fluorimeter and TECAN
−
 
+
−
<p>Starters launched of “18-17” “19-17” “20-17” “21-17” “22-17” “23-17” “32” “33” “01-24” each in triplicate</p>
+
−
 
+
−
<p>Stock of backbone pSB1C3 done</p>
+
−
 
+
−
<p>Preparation of SDS-PAGE, of solutions and of the column to make the protein purification</p>
+
−
 
+
−
<p>Digestion E/P of “22-17” “43” “44” “47” and digestion X/P of “1302” “3902”</p>
+
−
 
+
−
<p>Ligation “43” “44” “47” into pSB1C3</p>
+
−
<p>“013002” into pSB1K3</p>
+
−
<p>“01-3902” “01-1302” into pSB1C3 </p>
+
 
   </div>
 
   </div>
 
</div>
 
</div>
  
−
<div id="main" style="position:relative;top:0px;left:172px;width:1000px;" class="menu-container">
+
<!-- start social section -->
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo71">September 1</button>
+
    <section style="padding:30px 0px;" class="arrow_box" id="ethics">
−
  <div id="demo71" class="collapse">
+
   
−
<p>Colony PCR of “40” “45” “48” all clones are positive, starters launched</p>
+
    <div class="container">
 +
    <div class="row">
 +
    <div class="col-md-12 text-center">
 +
<h1 class="blue"><div align ="center">MORE INFORMATION ON FACEBOOK !</div></h1>
 +
                    <ul class="list-inline space80 icon">
 +
                        <li><div align ="center"><a href="https://www.facebook.com/iGEM.AMU?fref=ts"><img src="https://static.igem.org/mediawiki/2015/e/ed/FacebookAMU.png" width="200" height="200" class="img-grey mautomargin"></a></li></div>
 +
                    </ul>
  
−
<p>Protein purification of “01-3502” “01-1202” </p>
+
                </div>
−
<p>SDS-PAGE of “01-3502” “01-1202”</p>
+
    </div>
 +
    </div>
  
−
<p>01-3502 purified and pure!!!! We got our first protein which is a laccase from E.coli</p>
+
    </section>
  
−
<p>Starters launched of “38” “13-02” “01-36” “01-15” “01-3502” “01-1202” “37” “35-02” “12-02” “35” “36” “01-35” “39” “40” “45” “48” </p>
 
−
  </div>
 
−
 
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo72">September 2</button>
 
−
  <div id="demo72" class="collapse">
 
−
<p>Plasmid purification of “01-36” “40” “39” “48” “45” “37” “36” “01-1202” “01-36” “01-15” “35-02” “35” “38” “13-02” “12-02” “01-3502” “01-35”</p>
 
−
 
−
<p>Protein expression of 01-3502 and 01-1202</p>
 
−
 
−
<p>Colony PCR of “01-15” beause we have doubt concerning colonies => all colonies were positive</p>
 
−
 
−
<p>Ligation of “43” “44” “47” “03002” “01-1302” “013902” into pSB1C3</p>
 
−
<p>Transformation of “43” “44” “47” “013002” into C2987 and “01-1302” “01-3902” “3813-02” “3812-02” into TG1</p>
 
−
  </div>
 
−
  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo73">September 3</button>
 
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<p>Verification digestion on “40” “38” “01-15” “48” “45” “01-36” “01-1202” “01-36” “37” </p>
 
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<p>Plasmid purification of “01-1202” </p>
 
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<p>Colony PCR of “3812-02” “3813-02” “01-3902” “01-1302” starters launched on positive results</p>
 
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<p>No colony for “43” “44” “47” “013002”</p>
 
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<p>Preparation of the backbone pSB1C3</p>
 
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<p>Transformation of “013002” “41” “44” “47” into C2987</p>
 
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  <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo74">September 4</button>
 
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<p>Plasmid purification of “45” “01-36” “381302” “38” “01-1202” “3812-02” “3813-02”</p>
 
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<p>Colony PCR of “47” “01-3002” “43” “44” => almost everything is positive!!! Great day!</p>
 
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<button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo75">September 7</button>
 
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<p>Plasmid purification (miniprep) of “44” “47” “43” “38” “01-1302” “01-1202” “01-3002” “45” </p>
 
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<p>“01-1302” “01-36” “3813-02” seems good so they are send for sequencing.</p>
 
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<p>Construction : Digestion E/S of “38” “01-36” “01-35” “01” and Digestion X/P of “44” “47” “43” “48” “44” “45” “1302” “3813-02” “39-02” “40” “3812-02”</p>
 
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<p>Starter launched on “01-1202” “01-3002” for production and on “0136” for miniprep</p>
 
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  <div id="demo76" class="collapse">
 
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<p>Ligation in pSB1C3 of “0136-02” “01-381302” “01-381202” “01-3902” “01-1302” “38-3902” “0136-40” “0136-45” “0136-48” “0135-43” “0135-44” “0135-47”</>
 
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<p>Transformation of “01-3002” in BL21, of pSB4K5 and pSB3T5 in TG1 and transformation of ligation product in TG1</p>
 
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<p>Purification of “01-1202” (1) and (2):</p>
 
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    <p>Induction by IPTG </p>
 
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    <p>Storing the cells at -80°C </p>
 
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<button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo77">September 9</button>
 
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  <div id="demo77" class="collapse">
 
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<p>Colony PCR of “013002” “013002” => all results are positive</p>
 
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<p>Production of “01-1202” : Sonication of cells, centrifugation 10 min at 4500 rpm, ultracentrifugation 45 min at 4500 rpm, solubilizing membrane in Triton 1% during 2h and incubation overnight.</p>
 
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  </div>
 
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<button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo78">September 10</button>
 
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  <div id="demo78" class="collapse">
 
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<p>Preparation of SDS Page 15% and a western blot with products of yesterday => results are not good so we have to resart the production</p>
 
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  </div>
 
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<button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo79">September 14</button>
 
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<p>Culture of “01-1202” and “01-3002” in anaerobic condition, cells were centrifuged 10 min at 4500 rpm and pellet were stored at -20°C</p>
 
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<p>A western blot was done → Production of Cytochrome, monomer in the membrane and dimer in the cytoplasm</p>
 
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<p>The next step is to purify “01-1202” and “01-3002” and start a culture on 01-3502 in anaerobic condition</p>
 
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Latest revision as of 00:07, 19 September 2015

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Chew figth project, for the iGEM competition. See you soon in Boston !