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<a href="https://2014.igem.org/Team:Brasil-SP/Parts"style="color:#000000"> Parts</a></td>

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<a href="https://2014.igem.org/Team:Brasil-SP/Attributions"style="color:#000000"> Attributions </a></td>

<td align ="center"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="55px"></a> </td>

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<h1>Main Assembly Map</h1>

 

<img src="https://static.igem.org/mediawiki/2014/8/87/Mapa.png" style="width:100%;height:900px"/>

 

<h1>Assemblies forms</h1>

 

 

 

 

 

 

 

 

 

 

 

<h1>Characterization Assemblies</h1>

 

 

 

 

 

<img src="https://static.igem.org/mediawiki/2014/d/d5/KI_w.png" width="300" height="170px"/>

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

<h1><strong>Life Inside the LAB</stron></h1>

<thead><h2>July</h2>

     <th>Monday</th>  

     <th>Tuesday</th>

     <th>Wednesday</th>

     <th>Thursday</th>

     <th>Friday</th>

     <th>Saturday</th>

     <th>Sunday</th>

       <li><a href="https://static.igem.org/mediawiki/2014/7/74/PCR.pdf" style="color:#f05151">PCR</a> lasR (BBa_C0079). The purpose of this PCR was to add the RBS (BBa_K143021), through addition of the sequence in the primer foward, and also to remove the LVA tag. In our primers we only added the restriction sites X and S, so we still need to put it in the pSB1C3 for the Biobrick standard completion. When standardized this part will be called BBa_K1521001.</li>

       <li>Purification of lasR <a href="https://static.igem.org/mediawiki/2014/7/74/PCR.pdf" style="color:#f05151">PCR</a> product </li>

       <li>Transformation of the Biobricks:</li>

         <li>BBa_143012</li>

         <li>BBa_K081001</li>

         <li>BBa_E0840</li>

         <li>BBa_K316037</li>

         <li>BBa_K316018</li>

         <li>BBa_K316015</li>

       <li>Transformation of the Biobricks sent by the Imperial College Team:</li>

         <li>BBa_K316016</li>

         <li>BBa_K143031</li>

       <li>Miniprep, Quantification of the plasmidial DNA samples and <a href="https://static.igem.org/mediawiki/2014/4/42/Restriction_Anaysis_and_Digestion_in_large_scale.pdf" style="color:#f05151">Restriction Analysis</a>:</li>

         <li>BBa_143012</li>

         <li>BBa_K081001</li>

         <li>BBa_E0840</li>

         <li>BBa_143012</li>

         <li>BBa_K081001</li>

         <li>BBa_E0840</li>

       <li>Miniprep, Quantification and <a href="https://static.igem.org/mediawiki/2014/4/42/Restriction_Anaysis_and_Digestion_in_large_scale.pdf" style="color:#f05151">Restriction Analysis</a>:</li>

         <li>BBa_143012</li>

         <li>BBa_K081001</li>

         <li>BBa_E0840</li>

       <li>Transformation:</li>

         <li>BBa_B0015</li>

         <li>BBa_B0015</li>

       <li>Miniprep, Quantification and <a href="https://static.igem.org/mediawiki/2014/4/42/Restriction_Anaysis_and_Digestion_in_large_scale.pdf" style="color:#f05151">Restriction Analysis</a>:</li>

         <li>BBa_B0015 (restriction analysis fail, repeat)</li>

       <li><a href="https://static.igem.org/mediawiki/2014/7/74/PCR.pdf" style="color:#f05151">PCR</a> the qteE gene for amplification. We also added the RBS (BBa_K143021) using the foward primer. In our primers we only added the restriction sites X and S, so we still need to put it in the pSB1C3 for the Biobrick standard completion. When standardized this biobrick will be called BBa_K1521000.</li>

       <li>Transformation:</li>

         <li>RBS + lasR (BBa_C0079)</li>

       <li>Purification:</li>

       <li>Ligation of <a href="https://static.igem.org/mediawiki/2014/7/74/PCR.pdf" style="color:#f05151">PCR</a> qteE in pGEM T-easy vector</li>

       <li>Transformation:</li>

         <li>BBa_J04450</li>

         <li>BBa_B0015</li>

     <li>Tranformation:</li>

       <li>BBa_K823003</li>

       <li>BBa_B0015</li>

       <li>Glycerol Stock and Miniprep:<li>

         <li>BBa_J04450</li>

         <li>BBa_B0015</li>

         <li>BBa_J04450 (EP)</li>

         <li>BBa_J04450 (XS)</li>

         <li>BBa_K823003</li>

       <li>Transformation</li>

         <li>BBa_K143055</li>

       <li>Glycerol Stock and Miniprep:</li>

         <li>BBa_J04450</li>

         <li>BBa_B0015</li>

         <li>BBa_J04450 (EP)</li>

         <li>BBa_J04450 (XS)</li>

         <li>BBa_K823003</li>

       <li>Transformation:</li>

         <li>BBa_316016</li>

         <li>BBa_K143055</li>

         <li>BBa_K316016</li>

       <li>Miniprep, <a href="https://static.igem.org/mediawiki/2014/4/42/Restriction_Anaysis_and_Digestion_in_large_scale.pdf" style="color:#f05151">Restriction analysis</a> and <a href="https://static.igem.org/mediawiki/2014/4/42/Restriction_Anaysis_and_Digestion_in_large_scale.pdf" style="color:#f05151">Digestion</a>:</li>

         <li>Ligation</li>

         <li>Transformation</li>

       <li>Prepare more competent cells</li>

         <li>BBa_K316016</li>

       <li>Miniprep:</li>

         <li>BBa_K316016</li>

         <li>Miniprep</li>

         <li>OBS: This analysis was reapeted because we had problems confirming the assembly. The issue was that bot the construction and the pSB1C3 vector had similar size. Check out the Lab Journal report of the day for more information.</li>

         <li>Ligation</li>

         <li>Transformation</li>

       <li><a href="https://static.igem.org/mediawiki/2014/7/74/PCR.pdf" style="color:#f05151">PCR</a> BBa_K143055 for RBS removal</li>

       <li>PCR of Plac BBa_K143055 was followed by a <a href="https://static.igem.org/mediawiki/2014/a/af/Agarose_Gel_Electrophoresis_and_DNA_Gel_Purification.pdf"  style="color:#f05151">Gel Purification</a> and Ligation in the PUC19 vector</li>

<thead><h2>August</h2>

     <th>Monday</th>

     <th>Tuesday</th>

     <th>Wednesday</th>

     <th>Thursday</th>

     <th>Friday</th>

     <th>Saturday</th>

     <th>Sunday</th>

       <li>Transformation:</li>

         <li>BBa_K143055</li>

         <li>Miniprep</li>

         <li>Plac BBa_K143055</li>

       <li>Transformation</li>

         <li>BBa_P0312</li>

       <li>Miniprep:</li>

         <li>Plac BBa_K143055</li>

         <li>BBa_P0312</li>

         <li>Miniprep</li>

         <li>And once again! <a href="https://static.igem.org/mediawiki/2014/4/42/Restriction_Anaysis_and_Digestion_in_large_scale.pdf" style="color:#f05151">Restriction analysis</a> (this BBa_K316016 is tough)</li>

       <li>Miniprep and <a href="https://static.igem.org/mediawiki/2014/4/42/Restriction_Anaysis_and_Digestion_in_large_scale.pdf" style="color:#f05151">Restriction analysis</a>:</li>

         <li>BBa_P0312</li>

       <li>Prepare Plac BBa_K143055 for sequencing</li>

       <li><a href="https://static.igem.org/mediawiki/2014/a/af/Agarose_Gel_Electrophoresis_and_DNA_Gel_Purification.pdf"  style="color:#f05151">Gel Purification</a> and Ligation in pSB1C3 for BBiobrick standard:</li>

         <li>Ligation</li>

         <li>Transformation</li>

         <li>Miniprep</li>

         <li>Repeat <a href="https://static.igem.org/mediawiki/2014/4/42/Restriction_Anaysis_and_Digestion_in_large_scale.pdf" style="color:#f05151">restriction analysis</a> adding the enzyme NdeI (for more info check out the Lab Journal)</li>

         <li>BBa_J04450</li>

         <li>BBa_K143055</li>

       <li><a href="https://static.igem.org/mediawiki/2014/7/74/PCR.pdf" style="color:#f05151">PCR</a> BBa_K143055 with higher melting temperature (check out our Lab Journal for more info)</li>

<thead><h2>September</h2>

     <td>Monday</td>

     <td>Tuesday</td>

     <td>Wednesday</td>

     <td>Thursday</td>

     <td>Friday</td>

     <td>Saturday</td>

     <td>Sunday</td>

         <li><a href="https://static.igem.org/mediawiki/2014/4/42/Restriction_Anaysis_and_Digestion_in_large_scale.pdf" style="color:#f05151">Digestion</a> of the part BII with EcoRV (for more info check out our Lab Journal)

         <li>Ligation</li>

         <li>Transformation</li>

         <li>Miniprep</li>

       <li>QuickChange PCR to mutate the cleavage site of the BBa_316037 to a Cathepsin S recognition site</li>

       <li>Transformation of the QuickCHange PCR product</li>

       <li>QuickChange PCR to mutate the cleavage site of the BBa_316037 to a Cathepsin S recognition site</li>

       <li>Transformation of the QuickCHange PCR product</li>

       <li>Prepare inoculum of the mutated BBa_K316037</li>

         <li>Ligation</li>

         <li>Tranformation</li>

       <li>We had far to many difficulties trying to remove the RBS from the BBa_K143055, so we decided to synthesize it using PCR.</li>

       <li>PCR for BBa_143015 (BBa_143055 without RBS)</li>

         <li>Repeat Ligation</li>

         <li>Miniprep</li>

         <li>Transformation</li>

       <li><a href="https://static.igem.org/mediawiki/2014/a/af/Agarose_Gel_Electrophoresis_and_DNA_Gel_Purification.pdf"  style="color:#f05151">Gel Purification</a> of the BBa_K143015</li>

         <li>Miniprep</li>

       <li>LIgation of the synthesized BBa_K143015 in TOPO vector followed by Transformation</li>

       <li>Replate BBa_K143015. We forgot the X-gal :(</li>

       <li>Inoculum of the BBa_K143015</li>

         <li>Digestão EXSP</li>

         <li>Digestão EXSP</li>

       <li>Miniprep and Rstriction Analysis of the BBa_K143015</li>

         <li>Ligation</li>

         <li>Transformation</li>

         <li>Miniprep</li>

         <li>Ligation</li>

         <li>Transformation</li>

<thead><h2>October</h2>

     <td>Monday</td>

     <td>Tuesday</td>

     <td>Wednesday</td>

     <td>Thursday</td>

     <td>Friday</td>

     <td>Saturday</td>

     <td>Sunday</td>

         <li>Miniprep</li>

       <li>Assemblies KIX, CIII and KXVI</li>

         <li>Digestão EXSP</li>