Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Sep

From 2014.igem.org

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<ul>
<ul>
<li>Bands as expected (~1200 bp)</li>
<li>Bands as expected (~1200 bp)</li>
-
                 <div class="element" style="height:350px; width:150px; text-align:center">
+
                 <div class="element" style="height:350px; width:200px; text-align:center">
-
                               <a href="https://static.igem.org/mediawiki/2014/9/9a/Bielefeld_CeBiTec_2014-10-14_adhA_Kontrollverdau_09_01.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/9/9a/Bielefeld_CeBiTec_2014-10-14_adhA_Kontrollverdau_09_01.png" height="230px"></a><br><font size="1">Agarose gel from the restriction digestion with <i>EcoR</i>I and <i>Pst</i>I. As a Ladder we used <a href="http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/generuler-1-kb-dna-ladder-ready-to-use-250-to-10000-bp">GeneRuler™ 1 kb DNA Ladder from Thermo Scientific</a>. </font>
+
                               <a href="https://static.igem.org/mediawiki/2014/9/9a/Bielefeld_CeBiTec_2014-10-14_adhA_Kontrollverdau_09_01.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/9/9a/Bielefeld_CeBiTec_2014-10-14_adhA_Kontrollverdau_09_01.png" height="230px"></a><br><font size="2">Agarose gel from the restriction digestion of pSB1K3_<i>adhA</i> with <i>EcoR</i>I and <i>Pst</i>I. As a Ladder we used the <a href="http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/generuler-1-kb-dna-ladder-ready-to-use-250-to-10000-bp">GeneRuler™ 1 kb DNA Ladder. The fragments in both lines have the right sizes.</a>. </font>
                         </div>
                         </div>
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> of pSB1A2_T7_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> </li>
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> of pSB1A2_T7_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> </li>
<ul>
<ul>
-
<li>Bands not as expected (~7000bp)</li>
+
<li>Bands not as expected (Backbone: ~ 2200 and insert: ~ 7500bp)</li>
  </ul> </ul></ul></ul></ul>
  </ul> </ul></ul></ul></ul>
<ul>
<ul>
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<ul>
<ul>
<li>Annealing temperature: 65 °C</li>
<li>Annealing temperature: 65 °C</li>
-
<li>Bands not as expected (~1800 bp)</li>
+
<li>Bands not as expected (~ 1800 bp)</li>
</ul>
</ul>
</ul>
</ul>
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<ul>
<ul>
<li>Annealing temperature: 65°C</li>
<li>Annealing temperature: 65°C</li>
-
<li>Bands not as expected (~3000 bp)</li>
+
<li>Bands not as expected (~ 3000 bp)</li>
</ul>
</ul>
</ul>
</ul>
</ul>
</ul>
-
</ul>  
+
</ul></ul>  
-
<br>
+
-
 
+
-
</ul>  
+
<br>
<br>
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             </div>
             </div>
             <div class="content" style="margin-right:10%; margin-left:10%">
             <div class="content" style="margin-right:10%; margin-left:10%">
 +
<ul>
 +
<li><b><i>pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></i></b></li>
 +
<ul>
 +
<li>This week we tried to reclone <i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> into pSB1C3 </li>
 +
                <ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> of pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> and pSB1C3_<i>RFP</i> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> </li>
 +
<ul>
 +
<li>Bands as expected <br>(~ 7500 bp for <i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> and ~ 2200 bp for pSB1C3)</li>
 +
</ul><li>Clean up and Ligation as explained in the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a></li>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li> 
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvD_kivD" target="_blank">fw_ilvD_kivD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_kivD" target="_blank">rv_pSB1C3_kivD</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 65 °C</li>
 +
<li>Bands not as expected (~ 1800 bp)</li>
 +
</ul>
 +
</ul></u></ul></ul>
 +
<ul>
 +
<li><b><i>pSB1C3_<i>adhA</i></b></li>
 +
<ul>
 +
<li>This week we tried to reclone <i>adhA</i> into pSB1C3 </li>
 +
                <ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> of pSB1K3_<i>adhA</i></i> and pSB1C3_<i>RFP</i> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> </li>
 +
<ul>
 +
<li>Bands as expected <br>(~ 1200 bp for <i>adhA</i> and ~ 2200 bp for pSB1C3)</li>
 +
</ul><li>Clean up and Ligation as explained in the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a></li>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li> 
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_pSB1C3_adhA" target="_blank">fw_pSB1C3_adhA</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_adhA" target="_blank">rev_pSB1C3_adhA</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 65 °C</li>
 +
<li>Bands not as expected (~ 1200 bp)</li>
 +
</ul></ul>
 +
</ul>
 +
 +
<ul>
 +
<li><b><i>pSB1A2_T7_<i>adhA</i></i></b></li>
 +
<ul>
 +
<li>This week we tried to contruct the pSB1A2_T7_<i>adhA</i>-Plasmid </li>
 +
                     
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
 +
<ul>
 +
<li>Backbone pSB1A2_T7(digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
 +
<ul>
 +
<li>pSB1A2_T7</li>
 +
</ul>
 +
<li>Insert pSB1K3_<i>adhA</i>(digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
 +
<ul>
 +
<li><i>adhA</i></li>
 +
</ul>
 +
</ul>
 +
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_pSB1C3_adhA" target="_blank">fw_pSB1C3_adhA</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_adhA" target="_blank">rev_pSB1C3_adhA</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 65 °C</li>
 +
<li>Bands not as expected (~ 1200 bp)</li>
 +
</ul></ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><b><i>pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i>_<i>adhA</i></i></b></li>
 +
<ul>
 +
<li>This week we tried to contruct the pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i>_<i>adhA</i>-Plasmid by <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#NEBBioBrick" target="_blank">NEB BioBrick Assembly</a></li>
 +
                      <ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#NEBBioBrick" target="_blank">NEB BioBrick Assembly</a></li>
 +
<ul>
 +
<li>Upstream part pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>)</li>
 +
<ul>
 +
<li><i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></li>
 +
</ul>
 +
<li>Downstream part pSB1K3_<i>adhA</i>(digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
 +
<ul>
 +
<li><i>adhA</i></li>
 +
</ul>
 +
                                  <li>Destination part pSB1C3_RFP (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
 +
<ul>
 +
<li><i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></li>
 +
</ul>
 +
</ul>
 +
</ul><ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvD_kivD" target="_blank">fw_ilvD_kivD </a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_adhA" target="_blank">rev_pSB1C3_adhA</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 65°C</li>
 +
<li>Bands not as expected (~ 3000 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul><li>Until now we didn't purified Inserts out of the gel if the backbone had another antibiotic resistence. We were using the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">PCR purification</a>. But because it didn't worked out, we analyzed all our cut samples by gelelectrophorese. Thereby we discovered that the pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> has an illigal restriction side. <br> Because of the integration of RBS's between all genes in the <i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i>-Plasmid, a restriction side between <i>alsS</i> and a RBS occurs.<br>New primers were ordered: <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvC_alsS-new" target="_blank">rv_ilvC_alsS-new</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC-new" target="_blank">fw_alsS_ilvC-new</a></li></ul>
 +
</ul>
 +
</ul>
 +
         </div>
         </div>
       </div>
       </div>
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             </div>
             </div>
             <div class="content" style="margin-right:10%; margin-left:10%">
             <div class="content" style="margin-right:10%; margin-left:10%">
 +
 +
<ul>
 +
<li><b><i>pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></i></b></li>
 +
<ul>
 +
<li>This week the new primers <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvC_alsS-new" target="_blank">rv_ilvC_alsS-new</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC-new" target="_blank">fw_alsS_ilvC-new</a> arrived. We tried to remove the illegale restriction side.</li>
 +
                    <ul>
 +
<li>We tried different <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a>s to increase the chance to get a correct BioBrick. Therefore we amplified five parts for two combination possibilities in the Gibson Assembly:
 +
<ol><li>  <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_kivD_pSB1C3" target="_blank">fw_kivD_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_alsS_pSB1C3" target="_blank">rv_alsS_pSB1C3</a> on pSB1C3_RFP <br>&#8594; pSB1C3 with ~ 2200 bp</li>
 +
 +
<li>  <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primerfw_pSB1C3_alsS" target="_blank">fw_pSB1C3_alsS</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvC_alsS-new" target="_blank">rv_ilvC_alsS-new</a> on pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> <br>&#8594; <i>alsS</i> with ~ 1800 bp</li>
 +
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_kivD" target="_blank">rv_pSB1C3_kivD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC-new" target="_blank">fw_alsS_ilvC-new</a> on pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> <br>&#8594; <i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> with ~ 5200 bp </li>
 +
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC-new" target="_blank">fw_alsS_ilvC-new</a> on pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> <br>&#8594; <i>ilvC</i>_<i>ilvD</i> with ~ 3600 bp</li>
 +
 +
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvD_kivD" target="_blank">fw_ilvD_kivD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_kivD" target="_blank">rv_pSB1C3_kivD</a>  on pSB1K3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> <br>&#8594; <i>kivD</i> with ~ 1800 bp</li></ol>
 +
<ul>
 +
<li>Annealing temperature: 65 &deg;C</li>
 +
<li>Bands as expected (see above)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li>PCR products were <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a> out of the gel</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with the previous PCR products:  <ul>
 +
<li>1., 2. and 3.</li><li>1., 2., 4. and 5. </li>
 +
</ul></li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
 +
</ul>
 +
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primerfw_pSB1C3_alsS" target="_blank">fw_pSB1C3_alsS</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvC_alsS-new" target="_blank">rv_ilvC_alsS-new</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 65 °C</li>
 +
<li>Bands as expected (~ 1800 bp)</li>
 +
</ul>
 +
</ul>
 +
 +
<li>Liquid cultures for a restriction digest were prepared.</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3-<i>alsS</i>-<i>ilvC</i>-<i>ilvD</i>-<i>kivD</i> without illigale restriction side</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a></li>
 +
              <ul>
 +
                  <li>Bands as expected (backbone: 2,2 kb and insert: 7 kb)</li>
 +
              </ul>
 +
<li>Successful sequencing</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><b><i>pSB1C3_adhA</i></b></li>
 +
<ul>
 +
<li>This week we tried to construct the pSB1C3_<i>adhA</i> using Gibson Assembly.</li>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of the pSB1C3_RFP for pSB1C3 as backbone (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_adhA_pSB1C3" target="_blank">fw_adhA_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_adhA_pSB1C3" target="_blank">rev_adhA_pSB1C3</a>) and pSB1K3_<i>adhA</i> for <i>adhA</i> as insert (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_adhA" target="_blank">rev_pSB1C3_adhA</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_pSB1C3_adhA" target="_blank">fw_pSB1C3_adhA</a>)  </li>
 +
<ul>
 +
<li>Annealing temperature: 65 &deg;C</li>
 +
<li>Bands as expected (~ 1200 bp for the <i>adhA</i> and ~ 2200 bp for the backbone)</li>
 +
</ul>
 +
 +
<li>PCR products were <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a> out of the gel</li>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>adhA</i> and pSB1C3</li>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_adhA" target="_blank">rev_pSB1C3_adhA</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_pSB1C3_adhA" target="_blank">fw_pSB1C3_adhA</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 65 °C</li>
 +
<li>Bands as expected (~ 1200 bp)</li>
 +
</ul>
 +
 +
<li>Liquid cultures for a restriction digest were prepared.</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3-<i>adhA</i></li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a></li>
 +
              <ul>
 +
                  <li>Bands as expected (backbone: 2,2 kb and insert: 1,2 kb)</li>
 +
              </ul>
 +
<li>Successful sequencing</li>
 +
</ul>
 +
</ul>
 +
         </div>
         </div>
       </div>
       </div>
Line 229: Line 408:
             </div>
             </div>
             <div class="content" style="margin-right:10%; margin-left:10%">
             <div class="content" style="margin-right:10%; margin-left:10%">
 +
<ul>
 +
<li><b><i>pSB1C3_ptac_alsS_ilvC_ilcD_kivD</i></b></li>
 +
<ul>
 +
<li>This week we tried to combine the pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> contruct (without illegale restriction side) with the <i>ptac</i> promotor.</li>
 +
            <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix and Prefix)</li>
 +
<ul>
 +
<li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
 +
<ul>
 +
<li>pSB1C3_<i>ptac</i></li>
 +
</ul>
 +
<li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
 +
<ul>
 +
<li><i><i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></i></li>
 +
</ul>
 +
              AND:
 +
              <li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>)</li>
 +
<ul>
 +
<li><i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i>_pSB1C3</li>
 +
</ul>
 +
<li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>)</li>
 +
<ul>
 +
<li><i><i>ptac</i></i></li>
 +
</ul>
 +
</ul>
 +
          <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvC_alsS-new" target="_blank">rv_ilvC_alsS-new</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 55°C</li>
 +
<li>Bands as expected (~ 3000 bp)</li>
 +
</ul>
 +
<li>Liquid culture for a restriction digest was prepared.</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3-<i>ptac</i>_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a></li>
 +
              <ul>
 +
                  <li>Bands as expected (backbone: ~ 2200 bp and insert: ~ 9500 bp)</li>
 +
              </ul>
 +
<li>Successful sequencing</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><b><i>pSB1C3_ptac_adhA</i></b></li>
 +
<ul>
 +
<li>This week we tried to combine the pSB1C3_<i>adhA</i> contruct with the <i>ptac</i> promotor.</li>
 +
            <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix and Prefix)</li>
 +
<ul>
 +
<li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
 +
<ul>
 +
<li>pSB1C3_<i>ptac</i></li>
 +
</ul>
 +
<li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
 +
<ul>
 +
<li><i>adhA</i></li>
 +
</ul>
 +
              AND:
 +
              <li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>)</li>
 +
<ul>
 +
<li><i>adhA</i>_pSB1C3</li>
 +
</ul>
 +
<li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>)</li>
 +
<ul>
 +
<li><i>ptac</i></li>
 +
</ul>
 +
</ul>
 +
          <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_adhA" target="_blank">rev_pSB1C3_adhA</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 55°C</li>
 +
<li>Bands as expected (~ 2800 bp)</li>
 +
</ul>
 +
<li>Liquid cultures for a restriction digest were prepared.</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3-<i>adhA</i></li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a></li>
 +
              <ul>
 +
                  <li>Bands as expected (backbone: ~ 2200 kb and insert: ~ 2400 bp)</li>
 +
              </ul>
 +
<li>Successful sequencing</li>
 +
</ul>
 +
</ul>
 +
 +
 +
<ul>
 +
<li><b><i>pSB1C3_alsS_ilvC_ilcD_kivD_adhA</i></b></li>
 +
<ul>
 +
<li>This week we tried to combine the pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i> contruct (without illegale restriction side) with the <i>adhA</i> from <i>L. lactis</i>.</li>
 +
            <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix and Prefix)</li>
 +
<ul>
 +
<li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>)</li>
 +
<ul>
 +
<li>pSB1C3_<i>adhA</i></li>
 +
</ul>
 +
<li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
 +
<ul>
 +
<li><i><i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></i></li>
 +
</ul>
 +
              AND:
 +
              <li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
 +
<ul>
 +
<li>pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i></li>
 +
</ul>
 +
<li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
 +
<ul>
 +
<li><i><i>ptac</i></i></li>
 +
</ul>
 +
</ul>
 +
          <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvD_kivD" target="_blank">fw_ilvD_kivD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_adhA" target="_blank">rev_pSB1C3_adhA</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 55°C</li>
 +
<li>Bands as expected (~ 3000 bp)</li>
 +
</ul>
 +
<li>Liquid culture for a restriction digest was prepared.</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_<i>alsS</i>_<i>ilvC</i>_<i>ilvD</i>_<i>kivD</i>_<i>adhA</i></li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a></li>
 +
              <ul>
 +
                  <li>Bands as expected (backbone: ~ 2200 bp and insert: ~ 9500 bp)</li>
 +
              </ul>
 +
<li>Successful sequencing</li>
 +
</ul>
 +
</ul>
 +
 +
<ul>
 +
<li><b><i>pSB1A2_T7_<i>adhA</i></i></b></li>
 +
<ul>
 +
<li>This week we tried to contruct the pSB1A2_T7_<i>adhA</i>-Plasmid </li>
 +
                     
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
 +
<ul>
 +
<li>Backbone pSB1A2_T7(digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Spe</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
 +
<ul>
 +
<li>pSB1A2_T7</li>
 +
</ul>
 +
<li>Insert pSB1C3_<i>adhA</i>(digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li>
 +
<ul>
 +
<li><i>adhA</i></li>
 +
</ul>
 +
</ul>
 +
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_pSB1C3_adhA" target="_blank">fw_pSB1C3_adhA</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev_pSB1C3_adhA" target="_blank">rev_pSB1C3_adhA</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 65 °C</li>
 +
<li>Bands as expected (~ 1200 bp)</li>
 +
</ul>
 +
<li>Liquid culture for a restriction digest was prepared.</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1A2-<i>T7</i>_<i>adhA</i></li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>EcoR</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a></li>
 +
              <ul>
 +
                  <li>Bands as expected (backbone: ~ 2200 bp and insert: ~ 1300 bp)</li>
 +
              </ul>
 +
<li>Successful sequencing</li>
 +
<li>For the protein expression analysis of AdhA we made a <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Cultivation for Expression of recombinant proteins" target="_blank">cultivation</a>. Samples were taken like explained in the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#FastCellLysisforSDS-PAGE" target="_blank">cell lysis for a SDS-PAGE Protocol</a>. Protein expression was induced when the culture reached a OD<sub>600</sub> 0,8 with rhamnose. The first sample was taken before the induction. Additionalle we took samples one, two and three and 20 hours later. Of these samples, we made a <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis%20%28SDS-PAGE%29" target="_blank">SDS Page</a>
 +
 +
<div class="element" style="height:300px; width:450px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/5/53/Bielefeld-CeBiTec_14-10-16_SDS_T7_adhA.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/5/53/Bielefeld-CeBiTec_14-10-16_SDS_T7_adhA.jpg" height="230px"></a><br><font size="1">SDS page from pSB1A2_<i>T7</i>_<i>adhA</i>
 +
<br>The sizes of the AdhA is ~ 35 Da</font>
 +
                    </div>
 +
 +
 +
</li>
 +
</ul>
 +
</ul>
 +
 +
 +
<ul>
 +
<li><b><i>GC-MS analysis of the isobutanol production</i></b></li>
 +
<ul>
 +
<li>For the analysis of the isobutanol production we decided to use a GC-MS analysis. Because we will cultivate in LB medium, we made a calibration line with LB and the following concentrations of isobutanol for further analysis and quantification of our samples.</li>
 +
              <ul>
 +
                  <li>0,5 % isobutanol</li>
 +
                  <li>0,1 % isobutanol</li>
 +
                  <li>0,05 % isobutanol</li>
 +
                  <li>0,01 % isobutanol</li>
 +
                  <li>0,001 % isobutanol</li>
 +
              </ul>
 +
Additionally we prepared GC-grade aceton with 1% 2-Butanol as an internal standard. >br>The samples were treated as described below:
 +
<ul><li>Solvent extraction with 100&#181;l LB-sample and 900&#181;l aceton</li>
 +
<li>5 min centrifugation</li>
 +
<li>150 &#181;l supernatant in the GC-MS vials for the analysis </li></ul> </li>
 +
        <li>The detection of isobutanol works for all tested concentrations but the 2-butanol peak was to high, so we need to do this again with 0.1% 2-Butanol.</li>
 +
</ul>
 +
</ul>
 +
         </div>
         </div>
       </div>
       </div>

Latest revision as of 11:49, 17 October 2014


September

Week 1     09/01 - 09/07
Week 1     09/01 - 09/07
  • T7_alsS_ilvC_ilvD_kivD
    • This week we tried to verify the positive cutures that were identified last week by a restriction digest
  • pSB1K3_alsS_ilvC_ilcD_kivD_adhA

Week 2     09/08 - 09/14
Week 2     09/08 - 09/14

Week 3     09/15 - 09/21
Week 3     09/15 - 09/21
  • pSB1C3_alsS_ilvC_ilvD_kivD_adhA
    • This week we tried to contruct the pSB1C3_alsS_ilvC_ilvD_kivD_adhA-Plasmid by NEB BioBrick Assembly
      • NEB BioBrick Assembly
        • Upstream part pSB1K3_alsS_ilvC_ilvD_kivD (digested with EcoRI, SpeI)
          • alsS_ilvC_ilvD_kivD
        • Downstream part pSB1K3_adhA(digested with XbaI, PstI)
          • adhA
        • Destination part pSB1C3_RFP (digested with EcoRI, PstI)
          • alsS_ilvC_ilvD_kivD
      • Until now we didn't purified Inserts out of the gel if the backbone had another antibiotic resistence. We were using the PCR purification. But because it didn't worked out, we analyzed all our cut samples by gelelectrophorese. Thereby we discovered that the pSB1K3_alsS_ilvC_ilvD_kivD has an illigal restriction side.
        Because of the integration of RBS's between all genes in the alsS_ilvC_ilvD_kivD-Plasmid, a restriction side between alsS and a RBS occurs.
        New primers were ordered: rv_ilvC_alsS-new and fw_alsS_ilvC-new
Week 4     09/22 - 09/28
Week 4     09/22 - 09/28
Week 5     09/29 - 10/05
Week 5 09/29 - 10/05
  • pSB1C3_ptac_alsS_ilvC_ilcD_kivD
    • This week we tried to combine the pSB1C3_alsS_ilvC_ilvD_kivD contruct (without illegale restriction side) with the ptac promotor.
    • BioBrick Assembly (Suffix and Prefix)
      • Backbone (digested with SpeI, PstI)
        • pSB1C3_ptac
      • Insert (digested with XbaI, PstI)
        • alsS_ilvC_ilvD_kivD
        AND:
      • Backbone (digested with EcoRI, XbaI)
        • alsS_ilvC_ilvD_kivD_pSB1C3
      • Insert (digested with EcoRI, SpeI)
        • ptac
    • Transformation with electrocompotetent cells
    • Colony PCR (VF-Primer, rv_ilvC_alsS-new)
      • Annealing temperature: 55°C
      • Bands as expected (~ 3000 bp)
    • Liquid culture for a restriction digest was prepared.
    • Plasmid isolation of pSB1C3-ptac_alsS_ilvC_ilvD_kivD
    • Restriction digestion with XbaI and PstI
      • Bands as expected (backbone: ~ 2200 bp and insert: ~ 9500 bp)
    • Successful sequencing
  • pSB1C3_alsS_ilvC_ilcD_kivD_adhA
    • This week we tried to combine the pSB1C3_alsS_ilvC_ilvD_kivD contruct (without illegale restriction side) with the adhA from L. lactis.
    • BioBrick Assembly (Suffix and Prefix)
      • Backbone (digested with SpeI, SpeI)
        • pSB1C3_adhA
      • Insert (digested with EcoRI, PstI)
        • alsS_ilvC_ilvD_kivD
        AND:
      • Backbone (digested with SpeI, PstI)
        • pSB1C3_alsS_ilvC_ilvD_kivD
      • Insert (digested with XbaI, PstI)
        • ptac
    • Transformation with electrocompotetent cells
    • Colony PCR (fw_ilvD_kivD, rev_pSB1C3_adhA)
      • Annealing temperature: 55°C
      • Bands as expected (~ 3000 bp)
    • Liquid culture for a restriction digest was prepared.
    • Plasmid isolation of pSB1C3_alsS_ilvC_ilvD_kivD_adhA
    • Restriction digestion with EcoRI and PstI
      • Bands as expected (backbone: ~ 2200 bp and insert: ~ 9500 bp)
    • Successful sequencing
  • pSB1A2_T7_adhA
    • This week we tried to contruct the pSB1A2_T7_adhA-Plasmid
      • Colony PCR (fw_pSB1C3_adhA, rev_pSB1C3_adhA)
        • Annealing temperature: 65 °C
        • Bands as expected (~ 1200 bp)
      • Liquid culture for a restriction digest was prepared.
      • Plasmid isolation of pSB1A2-T7_adhA
      • Restriction digestion with EcoRI and PstI
        • Bands as expected (backbone: ~ 2200 bp and insert: ~ 1300 bp)
      • Successful sequencing
      • For the protein expression analysis of AdhA we made a cultivation. Samples were taken like explained in the cell lysis for a SDS-PAGE Protocol. Protein expression was induced when the culture reached a OD600 0,8 with rhamnose. The first sample was taken before the induction. Additionalle we took samples one, two and three and 20 hours later. Of these samples, we made a SDS Page

        SDS page from pSB1A2_T7_adhA
        The sizes of the AdhA is ~ 35 Da
    • GC-MS analysis of the isobutanol production
      • For the analysis of the isobutanol production we decided to use a GC-MS analysis. Because we will cultivate in LB medium, we made a calibration line with LB and the following concentrations of isobutanol for further analysis and quantification of our samples.
        • 0,5 % isobutanol
        • 0,1 % isobutanol
        • 0,05 % isobutanol
        • 0,01 % isobutanol
        • 0,001 % isobutanol
        Additionally we prepared GC-grade aceton with 1% 2-Butanol as an internal standard. >br>The samples were treated as described below:
        • Solvent extraction with 100µl LB-sample and 900µl aceton
        • 5 min centrifugation
        • 150 µl supernatant in the GC-MS vials for the analysis
      • The detection of isobutanol works for all tested concentrations but the 2-butanol peak was to high, so we need to do this again with 0.1% 2-Butanol.

Retrieved from "http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Sep"