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<p>For the assemblies constructuion we addopted the following method:</p>

<br>

<p>We used the pSB1C3 vector from the Biobrick BBa_J04450. Using this biobrick we added a red/white selection, where the red colonies are those which do not have the assembly, and the white ones are the correct ones.</p>

<p>See our <a href="https://2014.igem.org/Team:Brasil-SP/Notebook/Protocols">Protocol</a>

<h1>Main Assembly Map</h1>

<!--estou colocando os fichamentos dos assemblies aqui, ainda preciso acabar os que faltam mas ja da pra experimentar com a organização destes-->

<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>

       <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>BBa_143012</li>

         <li>BBa_K081001</li>

         <li>BBa_E0840</li>

         <li>BBa_K316037</li>

         <li>BBa_K316018</li>

         <li>BBa_K316015</li>

         <li>BBa_K316016</li>

         <li>BBa_K143031</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>BBa_143012</li>

         <li>BBa_K081001</li>

         <li>BBa_E0840</li>

         <li>BBa_B0015</li>

         <li>BBa_B0015</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>RBS + lasR (BBa_C0079)</li>

         <li>BBa_J04450</li>

         <li>BBa_B0015</li>

       <li>BBa_K823003</li>

       <li>BBa_B0015</li>

         <li>BBa_J04450</li>

         <li>BBa_B0015</li>

         <li>BBa_J04450 (EP)</li>

         <li>BBa_J04450 (XS)</li>

         <li>BBa_K823003</li>

         <li>BBa_K143055</li>

         <li>BBa_J04450</li>

         <li>BBa_B0015</li>

         <li>BBa_J04450 (EP)</li>

         <li>BBa_J04450 (XS)</li>

         <li>BBa_K823003</li>

         <li>BBa_316016</li>

         <li>BBa_K143055</li>

         <li>BBa_K316016</li>

         <li>BBa_K316016</li>

         <li>BBa_K316016</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><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 <a href="https://static.igem.org/mediawiki/2014/0/03/PCR_Product_Clonig.pdf" style="color:#f05151">Ligation</a> in the PUC19 vector</li>

<thead><h2>August</h2>

         <li>BBa_K143055</li>

         <li>Plac BBa_K143055</li>

         <li>BBa_P0312</li>

         <li>Plac BBa_K143055</li>

         <li>BBa_P0312</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>BBa_P0312</li>

       <li>Prepare Plac BBa_K143055 for sequencing</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>

         <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><a href="https://static.igem.org/mediawiki/2014/6/63/QuickChange_PCR.pdf" style="color:#f05151">QuickChange PCR</a> to mutate the cleavage site of the BBa_316037 to a Cathepsin S recognition site</li>

       <li><a href="https://static.igem.org/mediawiki/2014/6/63/QuickChange_PCR.pdf" style="color:#f05151">QuickChange PCR</a> to mutate the cleavage site of the BBa_316037 to a Cathepsin S recognition site</li>

       <li>Prepare inoculum of the mutated BBa_K316037</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><a href="https://static.igem.org/mediawiki/2014/3/35/PCR_for_synthesis.pdf" style="color:#f05151">PCR for synthesis</a> of the BBa_143015 (BBa_143055 without RBS)</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><a href="https://static.igem.org/mediawiki/2014/0/03/PCR_Product_Clonig.pdf" style="color:#f05151">Ligation</a> of the synthesized BBa_K143015 in TOPO vector followed by <a href="https://static.igem.org/mediawiki/2014/a/a5/Transformation_in_Escherichia_coli_DH5.pdf" style="color:#f05151"> Transformation</a></li>

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

       <li>Inoculum of the BBa_K143015</li>

       <li><a href="https://static.igem.org/mediawiki/2014/6/6f/Purelink_quick_plasmid_qrc.pdf">Miniprep</a> 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> of the BBa_K143015</li>

<thead><h2>October</h2>

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

     <td>07/10</td>

     <td>08/10</td>

     <td>09/10</td>

     <td>10/10</td>