Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Sep

From 2014.igem.org

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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> <!-- glpX -->
 +
                <li><i><b>glpX</b></i></li>
 +
                <ul>
 +
                  <li>We tried to find and isolate pSB1K3_glpX.</li>
 +
                  <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#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR-Primer</a>)
 +
                    </li>
 +
            <ul>
 +
      <li>Annealing temperature: 55 °C</li>
 +
      <li>Bands as expected (~1300 bp)</li>
 +
            </ul>
 +
                  </ul>
 +
                  <ul>
 +
            <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1K3_glpX</li>
 +
                  </ul>
 +
                </ul>
 +
              </ul> <!-- /csoS1-4 -->
 +
 +
              <br>
 +
               <ul> <!-- csoS1-4 für GFP Fusion -->
               <ul> <!-- csoS1-4 für GFP Fusion -->
                 <li><i><b>csoS1-4</b></i></li>
                 <li><i><b>csoS1-4</b></i></li>
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                   </ul>
                   </ul>
                 </ul>
                 </ul>
-
               </ul>
+
               </ul> <!-- /csoS1-4 -->
               <br>
               <br>
               <ul> <!-- csoS1-4 Fusion mit GFP -->
               <ul> <!-- csoS1-4 Fusion mit GFP -->
-
                 <li><b><i>csoS1-4</i> and GFP</b></li>
+
                 <li><b><i>csoS1-4</i> and GFP <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1465222" target="_blank">(BBa_K1465222)</a></b></li>
                 <ul>
                 <ul>
                   <li>We tried to assemble the shell proteins of the carboxysome and GFP.</li>
                   <li>We tried to assemble the shell proteins of the carboxysome and GFP.</li>
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                   </ul>
                   </ul>
                   <ul>
                   <ul>
-
                     <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>Dpn</i>I</a></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>Dpn</i>I</a></li>
                   </ul>
                   </ul>
                   <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#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
                   </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-csoS1A-GFP" target="_blank">fw-csoS1A-GFP</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv-csoS1B-GF" target="_blank">rv-csoS1B-GF</a>)
 +
                    </li>
 +
            <ul>
 +
      <li>Annealing temperature: 68 °C</li>
 +
      <li>Bands as expected (~1000 bp)</li>
 +
            </ul>
 +
                  </ul>
 +
                  <ul>
 +
            <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_csoS1-4_GFP</li>
 +
                  </ul>
 +
                  <ul>
 +
            <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>Not</i>I</a></li>
 +
            <ul>
 +
      <li>Bands as expected (~2000 bp and ~2400 bp)</li>
 +
                      <div class="element" style="height:350px; width:120px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/b/ba/Bielefeld_CeBiTec_2014-10-14_csoS1-4_gfp_Kontrollverdau_09_04.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/b/ba/Bielefeld_CeBiTec_2014-10-14_csoS1-4_gfp_Kontrollverdau_09_04.png" height="230px"></a><br><font size="1">Agarose gel from restriction digestion. 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>
 +
                    </div>
 +
            </ul>
 +
                  </ul>
                 </ul>
                 </ul>
-
               </ul>
+
               </ul> <!-- /csoS1-4 and GFP -->
        
        
 +
              <br>
 +
 +
              <ul> <!-- csoS1-4_GFP Fusion mit T7 -->
 +
<li><b><i>csoS1-4_GFP</i> and T7</b></li>
 +
                <ul>
 +
                  <li>We tried to assemble our pSB1C3_csoS1-4_GFP construct with the T7 promoter.</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 (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 (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>csoS1-4_GFP</i></li>
 +
      </ul>
 +
            </ul>
 +
                  </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/Primer#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_csoS4A_PstI" target="_blank">rv_csoS4A_PstI</a>)
 +
                    </li>
 +
            <ul>
 +
      <li>Annealing temperature: 55 °C</li>
 +
      <li>Bands as expected (~320 bp)</li>
 +
            </ul>
 +
                  </ul>
 +
 +
 +
                 
 +
                </ul>
 +
              </ul> <!-- /csoS1-4_GFP Fusion mit T7-->
 +
               <br>
               <br>
               <ul> <!-- csoS1-4 Fusion mit can -->
               <ul> <!-- csoS1-4 Fusion mit can -->
-
        <li><b><i>can</i> and <i>csoS1-4</i></b></li>
+
<li><b><i>can</i> and <i>csoS1-4</i> <a href="parts.igem.org/wiki/index.php?title=Part:BBa_K1465208" target="_blank">(BBa_K1465208)</a></b></li>
        <ul>
        <ul>
  <li>This week we tried to find positive clones of our transformation.</li>
  <li>This week we tried to find positive clones of our transformation.</li>
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            <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_can_csoS1-4</li>
            <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_can_csoS1-4</li>
                   </ul>
                   </ul>
 +
                  <ul>
 +
            <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 (~2100 bp and ~3300 bp)</li>
 +
                      <div class="element" style="height:350px; width:120px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/c/ce/Bielefeld_CeBiTec_2014-10-14_can_csoS1-4_Kontrollverdau_09_02.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/c/ce/Bielefeld_CeBiTec_2014-10-14_can_csoS1-4_Kontrollverdau_09_02.png" height="230px"></a><br><font size="1">Agarose gel from restriction digestion. 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>
 +
                    </div>
 +
 +
            </ul>
 +
                  </ul>
 +
 +
                </ul>
 +
              </ul> <!-- /csoS1-4 Fusion mit can -->
                   <br>
                   <br>
-
                   <li><b><i>Hneap</i> and <i>T7</i></b></li>
+
              <ul> <!-- can_csoS1-4_csoS1D Fusion -->
 +
<li><b><i>can_csoS1-4</i> and <i>csoS1D</i> <a href="parts.igem.org/wiki/index.php?title=Part:BBa_K1465209" target="_blank">(BBa_K1465209)</a></b></li>
 +
                <ul>
 +
                   <li>We tried to assemble our pSB1C3_can_csoS1-4 construct with <i>csoS1D</i>.</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 (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_can_csoS1-4</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>csoS1D</i></li>
 +
        </ul>
 +
              </ul>
 +
                    </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/Primer#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR-Primer</a>)
 +
                      </li>
 +
              <ul>
 +
        <li>Annealing temperature: 55 °C</li>
 +
        <li>Bands as expected (~4300 bp)</li>
 +
              </ul>
 +
                    </ul>
 +
                    <ul>
 +
              <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_can_csoS1-4_csoS1D</li>
 +
                    </ul>
 +
                    <ul>
 +
              <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>Not</i>I</a> </li>
 +
              <ul>
 +
        <li>Bands as expected (~2000 bp and ~4000 bp)</li>
 +
                        <div class="element" style="height:350px; width:120px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/5/55/Bielefeld_CeBiTec_2014-10-14_can_csoS1-4_csoS1D_Kontrollverdau_09_06.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/5/55/Bielefeld_CeBiTec_2014-10-14_can_csoS1-4_csoS1D_Kontrollverdau_09_06.png" height="230px"></a><br><font size="1">Agarose gel from restriction digestion. 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>
 +
                    </div>
 +
              </ul>
 +
                    </ul>
 +
 
 +
                  </ul>
 +
                </ul> <!-- /can_csoS1-4:csoS1D Fusion -->
 +
 
 +
                <br>
 +
 
 +
              <ul>
 +
<li><b><i>Hneap</i> and <i>T7</i></b></li>
          <ul>
          <ul>
-
    <li>This week we tried to assemble the <i>T7</i> promotor with the <i>Hneap</i>.</li>  
+
    <li>This week we tried to assemble the <i>T7</i> promoter with the <i>Hneap</i>.</li>  
                     <ul>
                     <ul>
                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</li>
              <ul>  
              <ul>  
-
        <li>Backbone (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" 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>
+
        <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>  
        <ul>  
  <li>pSB1A2_T7</li>  
  <li>pSB1A2_T7</li>  
        </ul>
        </ul>
-
        <li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" 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>
+
        <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>  
        <ul>  
  <li><i>Hneap</i></li>   
  <li><i>Hneap</i></li>   
        </ul>  
        </ul>  
              </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#Transformationviaheatshock" target="_blank">Transformation</a> with chemocompetent 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#VR-Primer" target="_blank">VR-Primer</a>)
+
                        
-
                      </li>
+
-
              <ul>
+
-
        <li>Annealing temperature: 55 °C</li>
+
-
        <li>Bands as expected (~2300 bp)</li>
+
-
              </ul>
+
                     </ul>
                     </ul>
Line 154: Line 286:
             </div>
             </div>
             <div class="content" style="margin-right:10%; margin-left:10%">
             <div class="content" style="margin-right:10%; margin-left:10%">
-
        </div>
+
              <ul>
-
      </div>
+
<li><b><i>glpX</i> and p<sub>tac</sub> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1465229" target="_blank">(BBa_K1465229)</a></b></li>
-
    </div>
+
                <ul>
 +
                  <li>This week we tried to bring <i>glpX</i> in the pSB1C3 backbone under the control of the p<sub>tac</sub> promoter.</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 (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_p<sub>tac</sub></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>glpX</i></li>
 +
      </ul>
 +
            </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/Primer#fw_pSB1_SBPase" target="_blank">fw_pSB1_SBPase</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1_SBPase" target="_blank">rv_pSB1_SBPase</a>)
 +
                      </li>
 +
              <ul>
 +
        <li>Annealing temperature: 54 °C</li>
 +
        <li>Bands as expected (~2100 bp)</li>
 +
              </ul>
 +
                    </ul>
 +
                    <ul>
 +
              <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_p<sub>tac</sub>_glpX</li>
 +
                    </ul>
 +
                    <ul>
 +
<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>Not</i>I</a></li>
 +
<ul>
 +
<li>Bands as expected (~2000 bp and ~2200 bp)</li>
 +
<div class="element" style="height:350px; width:150px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/6/64/Bielefeld_CeBiTec_2014-10-17_ptac_glpX_Kontrollverdau_09_09.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/6/64/Bielefeld_CeBiTec_2014-10-17_ptac_glpX_Kontrollverdau_09_09.png" height="230px"></a><br><font size="1">Agarose gel from restriction digestion. 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>
 +
                    </div>
 +
 
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
 
 +
 
 +
<br>
 +
 
 +
<li><b>Purification of the carboxysome</b></li>
 +
                <ul>
 +
                    <li>Protein expression is induced with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug#Week2" target="_blank"> labjournal</a>, but slightly modificated. The culture volumen was upscaled to 1 L. For a more effective cell lysis, the sonification protocol was modified using longer intervalls for sonication and for cooling. Furthermore, the ultracentrifugation was carried out for a longer time (45 min) to obtain a better concentration and seperation. </li>
 +
                   
 +
&rarr; The purification was not succesful, as you could not recognize a visible band in the gradient.
 +
                    </ul>
 +
                   
 +
               
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
                  </ul>
 +
                </ul>
 +
              </ul>
 +
            </div>
 +
          </div>
 +
        </div>
     </div>
     </div>
Line 170: Line 365:
             </div>
             </div>
             <div class="content" style="margin-right:10%; margin-left:10%">
             <div class="content" style="margin-right:10%; margin-left:10%">
-
        </div>
+
            <ul><!-- T7 Hneap -->
 +
              <li><b><i>Hneap</i> and T7 </b></li>
 +
              <ul>
 +
                <li> We tried to find positiv clones of pSB3A2_T7_Hneap.</li>
 +
                  <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#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR-Primer</a>)
 +
                  </li>
 +
            <ul>
 +
    <li>Annealing temperature: 55 °C</li>
 +
    <li>Bands as expected (~2000 bp)</li>
 +
          </ul>
 +
                </ul>
 +
                <ul>
 +
          <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1A2_T7_Hneap</li>
 +
                </ul>
 +
                <ul>
 +
          <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 (~2000 bp and ~1800 bp)</li>
 +
                    <div class="element" style="height:350px; width:130px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/e/e9/Bielefeld_CeBiTec_2014-10-14_T7_Hneap_Kontrollverdau_09_18.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/e/e9/Bielefeld_CeBiTec_2014-10-14_T7_Hneap_Kontrollverdau_09_18.png" height="230px"></a><br><font size="1">Agarose gel from restriction digestion. 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>
 +
                    </div>
 +
 
 +
          </ul>
 +
                </ul>               
 +
 
 +
              </ul>
 +
            </ul><!-- /T7 Hneap -->
 +
<br>
 +
            <ul><!-- sap -->
 +
              <li><b><i>sap</i> <a href="parts.igem.org/wiki/index.php?title=Part:BBa_K1465204" target="_blank">(BBa_K1465204)</a></b></li>
 +
                <ul>
 +
                  <li>We tried to assemble both parts of <i>sap</i>.</li>
 +
                  <ul>
 +
            <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> on the pJet plasmid for <i>sap_2</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#sap_2_fwd" target="_blank">sap_2_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#sap_2_rev" target="_blank">sap_2_rev</a>)</li>
 +
            <ul>
 +
      <li>Annealing temperature: 55 &deg;C</li>
 +
      <li>Bands as expected (~1500 bp)</li>
 +
            </ul>
 +
                  </ul>
 +
                  <ul>
 +
            <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>sap_1</i>, <i>sap_2</i> and pSB1C3</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/Primer#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR-Primer</a>)
 +
                    </li>
 +
            <ul>
 +
      <li>Annealing temperature: 55 °C</li>
 +
      <li>Bands as expected (~3000 bp)</li>
 +
            </ul>
 +
                  </ul>
 +
                  <ul>
 +
            <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 (~2000 bp (backbone) and ~2700 bp (insert))</li>
 +
            </ul>
 +
                  </ul>
 +
                  <ul>
 +
            <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>V</a></li>
 +
            <ul>
 +
      <li>Bands as expected (~1500 bp and ~3200 bp)</li>
 +
                      <div class="element" style="height:350px; width:130px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/8/8f/Bielefeld_CeBiTec_2014-10-14_sap_Kontrollverdau_09_21.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/8/8f/Bielefeld_CeBiTec_2014-10-14_sap_Kontrollverdau_09_21.png" height="230px"></a><br><font size="1">Agarose gel from restriction digestion. 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>
 +
                    </div>
 +
 
 +
            </ul>
 +
                  </ul>               
 +
 
 +
              </ul>
 +
            </ul>
 +
<br>
 +
<ul>
 +
<li><b><i>can_csoS1-4</i> respectively <i>can_csoS1-4_csoS1D</i> and <i>sap</i></b></li>
 +
          <ul>
 +
            <li>We tried to finish our carboxysome with and without <i>csoS1D</i> but we could not find a correct clone until the begining of october. We suggested that our colony PCR did not work but also with a restriction digestion we got no result for this construct.</li>
 +
          </ul>
 +
 
 +
 
 +
 
 +
<br>
 +
 
 +
<li><b>Purification of the carboxysome</b></li>
 +
                <ul>
 +
                    <li>Protein expression is still induced with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug#Week2" target="_blank"> labjournal</a>, but slightly modificated. A larger culture volume of 1,6 L was used. The resultion pellet was resuspended in 20 mL TEMB buffer. The cell lysis was carried out this time using a French pressure cell (Aminco SLM Instrument) with a pressure of 5000 psig (around 345 bar). The cell suspension was homogenisated two times in the French pressure cell. Before the step of ultracentrifugation, the resulting pellet was resuspended in only 1,5 mL TEMB buffer.  </li>
 +
                   
 +
&rarr; The purification was not succesful, as you could not recognize a visible band in the gradient.
 +
                    </ul>
 +
 
 +
                   
 +
                </ul>
 +
          </div>
 +
        </div>
       </div>
       </div>
-
    </div>
 
     </div>
     </div>
 +
 +
 +
 +
 +
 +
 +
 +
Line 186: Line 482:
             </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>T7 and <i>prkA</i></b></li>
 +
              <ul>
 +
              <li>This week we wanted to bring the <i>prkA</i> under the control of the T7 promoter.</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 (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 (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>prkA</i></li>
 +
</ul>
 +
</ul>
 +
              </ul>
 +
              <ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaheatshock" target="_blank">Transformation</a> with chemocompetent 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#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR-Primer</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 55 °C</li>
 +
<li>Bands as expected (~1400 bp)</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1A2_T7_prkA</li>
 +
</ul>
 +
<ul>
 +
<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> </li>
 +
<ul>
 +
<li>Bands as expected (~2000 bp and ~1100 bp)</li>
 +
<div class="element" style="height:350px; width:140px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/d/dc/Bielefeld_CeBiTec_2014-10-17_T7_prkA_Kontrollverdau_09_26.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/d/dc/Bielefeld_CeBiTec_2014-10-17_T7_prkA_Kontrollverdau_09_26.png" height="230px"></a><br><font size="1">Agarose gel from restriction digestion. 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>
 +
                    </div>
 +
 +
</ul>
 +
</ul>
 +
 +
 +
 +
            </ul>
 +
 +
<br>
 +
 +
<ul>
 +
<li><b>T7 and <i>sap</i></b></li>
 +
              <ul>
 +
              <li>We tried to bring the <i>sap</i> under the control of the T7 promoter.</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 (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 (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>sap</i></li>
 +
</ul>
 +
</ul>
 +
              </ul>
 +
              <ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaheatshock" target="_blank">Transformation</a> with chemocompetent 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#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR-Primer</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 55 °C</li>
 +
<li>Bands as expected (~3000 bp)</li>
 +
</ul>
 +
</ul>
 +
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1A2_T7_sap</li>
 +
</ul>
 +
<ul>
 +
<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> </li>
 +
<ul>
 +
<li>Bands as expected (~2800 bp and ~2000 bp)</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
 +
<br>
 +
 +
<ul>
 +
<li><b><i>csoS1-4_GFP</i> and T7_sap</b></li>
 +
<ul>
 +
        <li>We tried to assemble our GFP construct with the shell associated protein <i>sap</i> and the T7 promoter.</li> 
 +
        <ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Prefix)</li>
 +
<ul>
 +
<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>pSB1C3_csoS1-4_GFP</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>T7_sap</li>
 +
</ul>
 +
</ul>
 +
</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#Transformationviaheatshock" target="_blank">Transformation</a> with chemocompetent cells</li>
 +
</ul>
 +
 +
 +
 +
</ul>
 +
 +
 +
<br>
 +
 +
<li><b>Purification of the carboxysome</b></li>
 +
                <ul>
 +
                    <li>Protein expression is still induced with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/CO2-fixation/Aug#Week2" target="_blank"> labjournal</a>, but slightly modificated. A larger culture volume of 1,6 L was used. The resultion pellet was resuspended in 20 mL TEMB buffer. The cell lysis was carried out this time using a French pressure cell (Aminco SLM Instrument) with a pressure of 5000 psig (around 345 bar). The cell suspension was homogenisated two times in the French pressure cell. Before the step of ultracentrifugation, the resulting pellet was resuspended in only 1,5 mL TEMB buffer. With the resuspended pellet two different ultracentrifugations were done. One centrifugation for 30 min, one further centrifugation for 60 min.  </li>
 +
                   
 +
&rarr; The purification was not succesful, as you could not recognize a visible band in the gradient.
 +
                    </ul>
 +
 +
                   
 +
                </ul>
 +
</ul>
 +
 +
         </div>
         </div>
       </div>
       </div>
Line 202: Line 633:
             </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>T7_sap_csoS1-4_GFP</i> <a href="parts.igem.org/wiki/index.php?title=Part:BBa_K1465223" target="_blank">(BBa_K1465223)</a></b></li>
 +
<ul>
 +
        <li>We tried find the construct to use it for the microscopy.</li>
 +
          <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#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Primer2" target="_blank">Primer2</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 55 °C</li>
 +
<li>Bands as expected (~3000 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_T7_sap_csoS1-4_GFP</li>
 +
</ul>
 +
<ul>
 +
<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>Pst</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> </li>
 +
<ul>
 +
<li>Bands as expected (~2000 bp and ~4600 bp)</li>
 +
<div class="element" style="height:350px; width:120px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/c/c2/Bielefeld_CeBiTec_2014-10-17_T7_sap_csoS1-4_gfp_Kontrollverdau_10_02.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/c/c2/Bielefeld_CeBiTec_2014-10-17_T7_sap_csoS1-4_gfp_Kontrollverdau_10_02.png" height="230px"></a><br><font size="1">Agarose gel from restriction digestion. As a Ladder we used <a href="http://www.neb.com/products/n3232-1-kb-dna-ladder">1 kb DNA Ladder from NEB</a>. </font>
 +
                    </div>
 +
 +
</ul>
 +
</ul>
 +
 +
 +
 +
</ul>
 +
</ul>
 +
 +
 +
 +
<br>
 +
              <ul>
 +
                <li><b><i>tkt</i></b></li>
 +
                <ul>
 +
                    <li>This week we wanted to purify the enzyme of <i>tkt</i> for the SBPase assay.</li>
 +
                    <ul>
 +
                      <li>Cultivation of pet16b_tkt in 250 ml <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#LBmedium" target="_blank">LB medium</a></li>
 +
                      <li>Induction with 1 mM IPTG at OD 0.8. Taking samples:t<sub>0</sub>, t<sub>1</sub>, t<sub>2</sub>, t<sub>3</sub>, t<sub>15</sub>, t<sub>17</sub></li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis (SDS-PAGE)" target="_blank">SDS-Page</a> of cultivation result in correct bands at ~100kD</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#HisTrapFFPurification" target="_blank">His-Tag purification</a> of tkt</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis (SDS-PAGE)" target="_blank">SDS-Page</a> of His-Tag purification result in correct bands at imidazol concentration of ...</li>
 +
                    </ul>
 +
                </ul>
 +
              </ul>
 +
 +
<br>
 +
 +
              <ul>
 +
                <li><b><i>fba</i></b></li>
 +
                <ul>
 +
                    <li>This week we wanted to purify the enzyme of <i>fba</i> for the SBPase assay.</li>
 +
                    <ul>
 +
                      <li>Cultivation of pet16b_fba in 250 ml <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#LBmedium" target="_blank">LB medium</a></li>
 +
                      <li>Induction with 1 mM IPTG at OD 0.8. Taking samples:t<sub>0</sub>, t<sub>1</sub>, t<sub>2</sub>, t<sub>3</sub>, t<sub>15</sub>, t<sub>17</sub></li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis (SDS-PAGE)" target="_blank">SDS-Page</a> of cultivation result in correct bands at ~40kD</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#HisTrapFFPurification" target="_blank">His-Tag purification</a> of fba</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis (SDS-PAGE)" target="_blank">SDS-Page</a> of His-Tag purification result in correct bands at imidazol concentration of ...</li>
 +
                    </ul>
 +
                </ul>
 +
              </ul>
 +
 +
<br>
 +
 +
              <ul>
 +
                <li><b><i>glpX</i> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1465228" target="_blank">(BBa_K1465228 (pSB1C3_glpX))</a></b></li>
 +
                <ul>
 +
                    <li>This week we wanted to purify the enzyme of <glpX</i> for the SBPase assay.</li>
 +
                    <ul>
 +
                      <li>Cultivation of pet16b_glpX in 250 ml <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#LBmedium" target="_blank">LB medium</a></li>
 +
                      <li>Induction with 1 mM IPTG at OD 0.8. Taking samples:t<sub>0</sub>, t<sub>1</sub>, t<sub>2</sub>, t<sub>3</sub>, t<sub>15</sub>, t<sub>17</sub></li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis (SDS-PAGE)" target="_blank">SDS-Page</a> of cultivation result in correct bands at ~38kD</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#HisTrapFFPurification" target="_blank">His-Tag purification</a> of glpX</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis (SDS-PAGE)" target="_blank">SDS-Page</a> of His-Tag purification result in correct bands at imidazol concentration of ...</li>
 +
                    </ul>
 +
<br>
 +
<li>We also tried to bring <i>glpX</i> in the right vector pSB1C3.</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 (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</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>glpX</i></li>
 +
</ul>
 +
</ul>
 +
</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#Transformationviaheatshock" target="_blank">Transformation</a> with chemocompetent 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_pSB1_SBPase" target="_blank">fw_pSB1_SBPase</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1_SBPase" target="_blank">rv_pSB1_SBPase</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 54 °C</li>
 +
<li>Bands as expected (~1300 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_glpX</li>
 +
</ul>
 +
<ul>
 +
<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>Pst</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> </li>
 +
<ul>
 +
<li>Bands as expected (~2000 bp and ~1000 bp)</li>
 +
<div class="element" style="height:350px; width:120px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/3/3b/Bielefeld_CeBiTec_2014-10-17_glpX_Kontrollverdau_10_06.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/3/3b/Bielefeld_CeBiTec_2014-10-17_glpX_Kontrollverdau_10_06.png" height="230px"></a><br><font size="1">Agarose gel from restriction digestion. As a Ladder we used <a href="http://www.neb.com/products/n3232-1-kb-dna-ladder">1 kb DNA Ladder from NEB</a>. </font>
 +
                    </div>
 +
</ul>
 +
</ul>
 +
 +
<br>
 +
 +
<ul>
 +
<li><b><i>prkA</i> and RBS BioBrick<a href="http://parts.igem.org/Part:BBa_B0034" target="_blank"> (BBa_B0034)</a></b></li>
 +
      <ul>
 +
        <li>We recognized that our pSB1C_prkA construct did not have the desired RBS so we tried to take the RBS (pSB1A2_RBS) out of the parts distrubution 2014 and clone it in front of our <i>prkA</i>.</li>
 +
        <ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1A2_RBS</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick Assembly</a> (Suffix)</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>pSB1A2_RBS</li>
 +
</ul>
 +
<li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</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>prkA</i></li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaheatshock" target="_blank">Transformation</a> with chemocompetent 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#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR-Primer</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 55 °C</li>
 +
<li>Bands as expected (~1500 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1A2_prkA</li>
 +
</ul>
 +
 +
      </ul>
 +
</ul>
 +
 +
<br>
 +
 +
 +
 +
 +
 +
 +
 +
<ul>
 +
<li><b><i>prkA</i></b></li>
 +
      <ul>
 +
        <li>After recognation of the fact, that our pSB1C_prkA construct did not have the desired RBS, we wanted to see whether there is protein expression after induction of the construct p<sub>tac</sub>_prkA. Cultivation was carried out using the method of <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Cultivation%20for%20Expression%20of%20recombinant%20proteins" target="_blank">Cultivation for Expression of recombinant proteins</a>. Protein expression was induced with 0,5 mM IPTG (final concentration). To verify the expression of the RuBisCo through <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis%20%28SDS-PAGE%29" target="_blank">SDS-PAGE </a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Matrix-assistedLaserDesorption/Ionization%E2%80%93Timeofflight%20%28MALDI-TOF%29" target="_blank">MALDI-TOF</a>, samples were generated using the protocol for <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#FastCellLysisforSDS-PAGE" target="_blank">Fast Cell Lysis for SDS-PAGE. </a></</li>
 +
 +
<center>
 +
<div class="element" style="width:350px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/e/e0/Bielefeld-CeBiTec_14-10-16_ptac_prkA.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/e/e0/Bielefeld-CeBiTec_14-10-16_ptac_prkA.jpg" width="350"></a><br>
 +
<font size="2">Proteinexpression of p<sub>tac</sub>_prkA induced with 0.5 mM IPTG. </font>
 +
                    </div> </center>
 +
 +
 +
       
 +
</ul>
 +
</ul>
 +
<br>
 +
 +
 +
<ul>
 +
<li><b>T7_sRNA:pfkA and p<sub>tac</sub>_sRNA:pfkA</b></li>
 +
      <ul>
 +
        <li>We made a cultivation with our two constructs to see if there is a difference between induced, not induced and the wild type of <i>E. coli</i>. The cultivations were arranged in shaking flasks with a volume of 30 ml in 250 ml flasks at 37°C and 250 rpm. The induction was with rhamnose for the T7 promoter and IPTG for the p<sub>tac</sub> at an OD<sub>600</sub> value of 0.6. The cultivation lasted 13 hours. We made 2 biological and 2 technical replica for each culture. We took two samples (1 ml) of each biological replica for <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#HPLC" target="_blank">HPLC</a> analysis that followed.</li>
 +
</ul>
 +
</ul>
 +
 +
         </div>
         </div>
       </div>
       </div>

Latest revision as of 00:35, 18 October 2014


September

Week 1     09/01 - 09/07
Week 1     09/01 - 09/07






Week 2     09/08 - 09/14
Week 2     09/08 - 09/14
  • glpX and ptac (BBa_K1465229)

  • Purification of the carboxysome
    • Protein expression is induced with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our labjournal, but slightly modificated. The culture volumen was upscaled to 1 L. For a more effective cell lysis, the sonification protocol was modified using longer intervalls for sonication and for cooling. Furthermore, the ultracentrifugation was carried out for a longer time (45 min) to obtain a better concentration and seperation.
      • → The purification was not succesful, as you could not recognize a visible band in the gradient.
Week 3     09/15 - 09/21
Week 3     09/15 - 09/21


  • can_csoS1-4 respectively can_csoS1-4_csoS1D and sap
    • We tried to finish our carboxysome with and without csoS1D but we could not find a correct clone until the begining of october. We suggested that our colony PCR did not work but also with a restriction digestion we got no result for this construct.

  • Purification of the carboxysome
    • Protein expression is still induced with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our labjournal, but slightly modificated. A larger culture volume of 1,6 L was used. The resultion pellet was resuspended in 20 mL TEMB buffer. The cell lysis was carried out this time using a French pressure cell (Aminco SLM Instrument) with a pressure of 5000 psig (around 345 bar). The cell suspension was homogenisated two times in the French pressure cell. Before the step of ultracentrifugation, the resulting pellet was resuspended in only 1,5 mL TEMB buffer.
      • → The purification was not succesful, as you could not recognize a visible band in the gradient.
Week 4     09/22 - 09/28
Week 4     09/22 - 09/28


  • csoS1-4_GFP and T7_sap

  • Purification of the carboxysome
    • Protein expression is still induced with a final IPTG concentration of 0,5 mM. The purification was carried out using the protocol as described in our labjournal, but slightly modificated. A larger culture volume of 1,6 L was used. The resultion pellet was resuspended in 20 mL TEMB buffer. The cell lysis was carried out this time using a French pressure cell (Aminco SLM Instrument) with a pressure of 5000 psig (around 345 bar). The cell suspension was homogenisated two times in the French pressure cell. Before the step of ultracentrifugation, the resulting pellet was resuspended in only 1,5 mL TEMB buffer. With the resuspended pellet two different ultracentrifugations were done. One centrifugation for 30 min, one further centrifugation for 60 min.
      • → The purification was not succesful, as you could not recognize a visible band in the gradient.
Week 5     09/29 - 10/05
Week 5 09/29 - 10/05

  • tkt
    • This week we wanted to purify the enzyme of tkt for the SBPase assay.
      • Cultivation of pet16b_tkt in 250 ml LB medium
      • Induction with 1 mM IPTG at OD 0.8. Taking samples:t0, t1, t2, t3, t15, t17
      • SDS-Page of cultivation result in correct bands at ~100kD
      • His-Tag purification of tkt
      • SDS-Page of His-Tag purification result in correct bands at imidazol concentration of ...

  • fba
    • This week we wanted to purify the enzyme of fba for the SBPase assay.
      • Cultivation of pet16b_fba in 250 ml LB medium
      • Induction with 1 mM IPTG at OD 0.8. Taking samples:t0, t1, t2, t3, t15, t17
      • SDS-Page of cultivation result in correct bands at ~40kD
      • His-Tag purification of fba
      • SDS-Page of His-Tag purification result in correct bands at imidazol concentration of ...

  • glpX (BBa_K1465228 (pSB1C3_glpX))
    • This week we wanted to purify the enzyme of for the SBPase assay.
      • Cultivation of pet16b_glpX in 250 ml LB medium
      • Induction with 1 mM IPTG at OD 0.8. Taking samples:t0, t1, t2, t3, t15, t17
      • SDS-Page of cultivation result in correct bands at ~38kD
      • His-Tag purification of glpX
      • SDS-Page of His-Tag purification result in correct bands at imidazol concentration of ...

    • We also tried to bring glpX in the right vector pSB1C3.


      • prkA
        • After recognation of the fact, that our pSB1C_prkA construct did not have the desired RBS, we wanted to see whether there is protein expression after induction of the construct ptac_prkA. Cultivation was carried out using the method of Cultivation for Expression of recombinant proteins. Protein expression was induced with 0,5 mM IPTG (final concentration). To verify the expression of the RuBisCo through SDS-PAGE and MALDI-TOF, samples were generated using the protocol for Fast Cell Lysis for SDS-PAGE.

          Proteinexpression of ptac_prkA induced with 0.5 mM IPTG.

      • T7_sRNA:pfkA and ptac_sRNA:pfkA
        • We made a cultivation with our two constructs to see if there is a difference between induced, not induced and the wild type of E. coli. The cultivations were arranged in shaking flasks with a volume of 30 ml in 250 ml flasks at 37°C and 250 rpm. The induction was with rhamnose for the T7 promoter and IPTG for the ptac at an OD600 value of 0.6. The cultivation lasted 13 hours. We made 2 biological and 2 technical replica for each culture. We took two samples (1 ml) of each biological replica for HPLC analysis that followed.

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