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

<p>We used the pSB1C3 vector from the Biobrick <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a>. 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>

 

<div align=center><img src="https://static.igem.org/mediawiki/2014/2/25/Red_and_WHite_plate_.jpg" width="700" height="265"/></div>

 

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

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       <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><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>

         <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>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.</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><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>

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

     <td>07/10</td>

     <td>08/10</td>

     <td>09/10</td>

     <td>10/10</td>