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

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                 <a  style="font-size:24px" href="#"><p style="margin-left:42%">Week 1 &nbsp;&nbsp;&nbsp; 08/04 - 08/10</p></a>
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                 <h6><a  style="font-size:24px" href="#">Week 1 &nbsp;&nbsp;&nbsp; 08/04 - 08/10</a></h6>
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            <ul>
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        <li><b>psB1C3-<i>alsS</i>-<i>ilvC</i>-<i>ilvD</i></b></li>
 +
        <ul>
 +
  <li>This week we wanted to identify positive colonies.</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#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>)</li>
 +
              <ul>
 +
        <li>Annealing temperature: 65 &deg;C</li>
 +
        <li>Bands not as expected (~ 3450 bp) </li>
 +
              </ul>
 +
                  </ul>
 +
                  <li>New try of the combination of the four CDS's with the pSB1C3 backbone.</li>
 +
                  <ul>              
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>kivD</i>, <i>alsS</i>, <i>ilvC</i> and <i>ilvD</i> and pSB1C3</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>DpnI</i></a></li>
 +
                      <li>Transformation with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">electrocompetent</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaheatshock" target="_blank"> chemocompetent</a> 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_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>)</li>
 +
              <ul>
 +
        <li>Annealing temperature: 65 &deg;C</li>
 +
        <li>Bands not as expected (~ 3450 bp) </li>
 +
              </ul>
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                  <ul>
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        </ul>
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                 <a  style="font-size:24px" href="#"><p style="margin-left:42%">Week 2 &nbsp;&nbsp;&nbsp; 08/11 - 08/17</p></a>
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                 <h6><a  style="font-size:24px" href="#">Week 2 &nbsp;&nbsp;&nbsp; 08/11 - 08/17</a></h6>
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            <ul>
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        <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and backbone of pSB1C3</b></li>
 +
        <ul>
 +
  <li>We started again back by zero to solve our problems.</li>
 +
                  <ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (as described before)</li>
 +
              <ul>
 +
                        <li>We used pSB1C3-RFP (<a href="http://parts.igem.org/Part:BBa_J04450" target="_blank">J04450</a>) instead of pSB1C3-CFP as template for backbone amplification</li>
 +
        <li>Annealing temperature: 65 °C</li>
 +
        <li>Bands as expected (~ 2200 bp)</li>
 +
              </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" target="_blank"><i>DpnI</i></a></li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>alsS</i>, <i>ilvC</i>, <i>ilvD</i> and <i>kivD</i> on pSB1C3</li>
 +
                      <li>Transformation with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">electrocompotetent cells</a></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_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>)</li>
 +
              <ul>
 +
        <li>Annealing temperature: 65 &deg;C</li>
 +
        <li>Bands not as expected (~ 3450 bp)</li>
 +
              </ul>
 +
                  </ul>
 +
                </ul>
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                 <a  style="font-size:24px" href="#"><p style="margin-left:42%">Week 3 &nbsp;&nbsp;&nbsp; 08/18 - 08/24</p></a>
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                 <h6><a  style="font-size:24px" href="#">Week 3 &nbsp;&nbsp;&nbsp; 08/18 - 08/24</a></h6>
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             <div class="content" style="margin-right:10%; margin-left:10%">
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              <ul>
 +
        <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and backbone pSB1K3</b></li>
 +
        <ul>
 +
    <li>This week we tried to begin from zero again with optimized conditions and new competent cells. <br>Therefor we prepared new plasmids from <i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and the backbone pSB1K3-RFP</li>
 +
                    <ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a></li>
 +
              <ul>
 +
                          <li>Primer were used as described before</li>
 +
                          <li>pSB1K3-RFP was used as template for backbone amplification</li>
 +
          <li>Annealing temperature: 53 &deg;C</li>
 +
          <li>Bands as expected for <br><i>alsS</i>: about 1800 bp, <br><i>ilvC</i>: about  1550 bp, <br><i>ilvD</i>: about 1950 bp, <br><i>kivD</i>: about 1700 bp, <br>but not for pSB1K3-RFP</li>
 +
              </ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a></li> with Q5 for pSB1K3-RFP
 +
                      <ul>
 +
                          <li>Primer were used as described before</li>
 +
          <li>Annealing temperature: 53 &deg;C</li>
 +
          <li>Bands as expected (~ 2200 bp)</li>
 +
              </ul>
 +
                     
 +
              <li>PCR products were <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a></li>
 +
 
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>kivD</i>, <i>alsS</i>, <i>ilvC</i> and  <i>ilvD</i> on pSB1K3</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>DpnI</i></a></li>
 +
              <ul>
 +
                          <li>Aberation from protocol: Incubation for about 10 hours.</li>
 +
              </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_ilvC_ilvD" target="_blank">fw_ilvC_ilvD</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 &deg;C</li>
 +
          <li>Bands as expected (~ 3600 bp)</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#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvD_ilvC" target="_blank">rv_ilvD_ilvC</a>)</li>
 +
              <ul>
 +
          <li>Annealing temperature: 65 &deg;C</li>
 +
          <li>Bands as expected (~ 3300 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 pSB1K3-<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>NotI</i></a></li>
 +
              <ul>
 +
                  <li>Bands as expected (backbone:~ 2200 bp and insert: ~ 7000 bp)</li>
 +
              </ul>
 +
                    </ul>
 +
        </ul>
 +
              </ul>
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                 <a  style="font-size:24px" href="#"><p style="margin-left:42%">Week 4 &nbsp;&nbsp;&nbsp; 08/25 - 08/31</p></a>
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                 <h6><a  style="font-size:24px" href="#">Week 4 &nbsp;&nbsp;&nbsp; 08/25 - 08/31</a></h6>
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 +
<ul>
 +
        <li><b><i>adhA</i></b></li>
 +
        <ul>
 +
  <li>This week the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#L.lactis" target="_blank"> <i>Lactococcus lactis</i></a> arrived. We tried to amplify it's <i>adhA</i>.</li>
 +
                  <ul>
 +
                  <ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAisolation" target="_blank">DNA isolation</a> of <i>Lactococcus lactis</i></li>
 +
</ul>
 +
 
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of the <i>adhA</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev-pBS1C3-adhA" target="_blank">rev-pBS1C3-adhA</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw-pSB1C3_adhA" target="_blank">fw-pSB1C3_adhA</a>) and the Backbone out of psB1K3-<i>RFP</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev-adhA_pSB1C3" target="_blank">rev-adhA_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw-adhA-pSB1C3" target="_blank">fw-adhA-pSB1C3</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 54 &deg;C</li>
 +
<li>Bands as expected (~1200 bp for the <i>adhA</i> and ~ 2200 bp for the Backbone)</li>
 +
</ul>
 +
       
 +
</ul>
 +
<ul>
 +
<li>PCR products were extracted out of the gel and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a>.</li>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>adhA</i> and pSB1K3</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>Dpn1</i></a> <ul/>
 +
 
 +
 
 +
 
 +
                  </ul>
 +
 
 +
        </ul>
 +
<ul>
 +
<li> Transformation  <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaheatshock" target="_blank"> 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#rev-pBS1C3-adhA target="_blank">rev-pBS1C3-adhArev-pBS1C3-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>
 +
</ul>
 +
<ul>
 +
</ul>
 +
            </ul>
 +
</ul>
 +
<li><b><i>alsS</i>-<i>ilvC</i>-<i>ilvD</i>-<i>kivD</i> with T7-Promotor</b></li>
 +
        <ul>
 +
<li> BioBrick Assembly:
 +
<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>alsS</i>-<i>ilvC</i>-<i>ilvD</i>-<i>kivD</i></li>
 +
</ul>
 +
</ul>
 +
 
 +
 
 +
 
 +
</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/Primer#fw-pSB1C3-alsS" target="_blank">fw-pSB1C3-alsS</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv-ilvC-alsS" target="_blank">rv-ilvC-alsS</a>)
 +
            </li>
 +
<ul>
 +
<li>Annealing temperature: 65 °C</li>
 +
<li>Bands as expected (~ 1800 bp)</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li>Liquid cultures for a restriction digest were prepared.</li>
 +
</ul>
 +
            </ul>
 +
<br>
 +
</div>
       </div>
       </div>
     </div>
     </div>

Latest revision as of 16:49, 16 October 2014


August

Week 1     08/04 - 08/10
Week 1     08/04 - 08/10
Week 2     08/11 - 08/17
Week 2     08/11 - 08/17
Week 3     08/18 - 08/24
Week 3     08/18 - 08/24
  • alsS, ilvC, ilvD, kivD and backbone pSB1K3
    • This week we tried to begin from zero again with optimized conditions and new competent cells.
      Therefor we prepared new plasmids from alsS, ilvC, ilvD, kivD and the backbone pSB1K3-RFP
Week 4     08/25 - 08/31
Week 4     08/25 - 08/31

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