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

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<td width="100px"><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/May"><img src="https://static.igem.org/mediawiki/2014/b/b9/Bielefeld-CeBiTec_2014-08-14_Arrrow-left.png" width="50px"> </a></td>
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<td style="padding:30px; width:400px"><h1> June </h1></td>
<td style="padding:30px; width:400px"><h1> June </h1></td>
<td width="100px" align="right"><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jul"><img src="https://static.igem.org/mediawiki/2014/9/9b/Bielefeld-CeBiTec_2014-08-14_Arrow-right.png" width="50px"> </a>      </td>
<td width="100px" align="right"><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jul"><img src="https://static.igem.org/mediawiki/2014/9/9b/Bielefeld-CeBiTec_2014-08-14_Arrow-right.png" width="50px"> </a>      </td>
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                <a href="#Week1">Week 1 &nbsp;&nbsp;&nbsp; 06/02 - 06/08</a>
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                <a href="#Week2">Week 2 &nbsp;&nbsp;&nbsp; 06/09 - 06/15</a>
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                <a  style="font-size:24px" href="#"><p style="margin-left:42%">Week 2 &nbsp;&nbsp;&nbsp; 06/09 - 06/15</p></a>
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                <a href="#Week3">Week 3 &nbsp;&nbsp;&nbsp; 06/16 - 06/22</a>
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                <a  style="font-size:24px" href="#"><p style="margin-left:42%">Week 3 &nbsp;&nbsp;&nbsp; 06/16 - 06/22</p></a>
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                 <a  style="font-size:24px" href="#"><p style="margin-left:42%">Week 4 &nbsp;&nbsp;&nbsp; 06/23 - 06/29</p></a>
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                 <h6><a  style="font-size:24px" href="#">Week 4 &nbsp;&nbsp;&nbsp; 06/23 - 06/29</a></h6>
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                  <li><b><i>alsS</i></b></li>
 +
          <ul>
 +
    <li>This week we aim to transform the construct from the parts distribution.</li>
 +
                    <ul>
 +
                <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
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                        <ul>
 +
                          <li><a href="http://parts.igem.org/Part:BBa_K539627" target="_blank">K539627</a>: pSB1C3-<i>alsS</i> (parts distribution from 2013, plate 1, 24H)</li>
 +
                        </ul>
 +
                        <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of <i>alsS</i></li>
 +
                        <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sequencing" target="_blank">Sequencing</a> confirmed the identity of pSB1C3-<i>alsS</i>.</li>
 +
                        <li>Glycerin stock created: <i>E. coli</i> KRX pSB1C3-<i>alsS</i></li>
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                    </ul>
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          </ul>
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                  <li><b><i>ilvC</i></b></li>
 +
          <ul>
 +
    <li>This week we aim to transform the construct from the parts distribution.</li>
 +
                    <ul>
 +
                <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
 +
                        <ul>
 +
                          <li><a href="http://parts.igem.org/Part:BBa_K539621" target="_blank">K539621</a>: pSB1C3-<i>ilvC</i> (parts distribution from 2013, plate 1, 24F)</li>
 +
                        </ul>
 +
                        <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of <i>ilC</i></li>
 +
                        <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sequencing" target="_blank">Sequencing</a> confirmed the identity of pSB1C3-<i>ilvC</i>.</li>
 +
                        <li>Glycerin stock created: <i>E. coli</i> KRX pSB1C3-<i>ilvC</i></li>
 +
                    </ul>
 +
          </ul>
 +
                  <li><b><i>ilvD</i></b></li>
 +
          <ul>
 +
    <li>This week we aim to transform the construct from the parts distribution.</li>
 +
                    <ul>
 +
                <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
 +
                        <ul>
 +
                          <li><a href="http://parts.igem.org/Part:BBa_K539626" target="_blank">K539626</a>: pSB1C3-<i>ilvD</i> (parts distribution from 2013, plate 1, 22J)</li>
 +
                        </ul>
 +
                        <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of <i>ilvD</i></li>
 +
                        <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sequencing" target="_blank">Sequencing</a> confirmed the identity of pSB1C3-<i>ilvD</i>.</li>
 +
                        <li>Glycerin stock created: <i>E. coli</i> KRX pSB1C3-<i>ilvD</i></li>
 +
                    </ul>
 +
          </ul>
 +
                  <li><b><i>kivD</i></b></li>
 +
          <ul>
 +
    <li>This week we aim to transform the construct from the parts distribution.</li>
 +
                    <ul>
 +
                <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
 +
                        <ul>
 +
                          <li><a href="http://parts.igem.org/Part:BBa_K539742" target="_blank">K539742</a>: pSB1C3-<i>kivD</i> (parts distribution from 2013, plate 1, 24L)</li>
 +
                        </ul>
 +
                        <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of <i>kivD</i></li>
 +
                        <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sequencing" target="_blank">Sequencing</a> confirmed the identity of pSB1C3-<i>kivD</i>.</li>
 +
                        <li>Glycerin stock created: <i>E. coli</i> KRX pSB1C3-<i>kivD</i></li>
 +
                    </ul>
 +
          </ul>
 +
              </ul>
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation of electrocompotetent <i> E.coli </i> KRX cells:</a></li>
 
-
<ul>
 
-
<li>K538742: <i>pSB1C3-kivD</i> (parts distribution from 2013, plate 1, 24L)</li>
 
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<li>K539621: <i>pSB1C3-ilvC</i> (parts distribution from 2013, plate 1, 24F)</li>
 
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<li>K539626: <i>pSB1C3-ilvD</i> (parts distribution from 2013, plate 1, 22J)</li>
 
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<li>K539627: <i>pSB1C3-alsS</i> (parts distribution from 2013, plate 1, 24H)</li>
 
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</ul>
 
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<li> Resulting colonies were selected to start liquid cultures: </li>
 
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<ul>
 
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<li>5 ml of LB in 20 ml reaction tube</li>
 
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<li>incubation over night at 37°C and 250 rpm</li>
 
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</ul>
 
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<li>Plasmid isolation by ThermoScientific MiniPrep</li>
 
-
<li>Sanger sequencing using <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VF-Primer" target="_blank">VF</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR</a> as sequencing primers</li>
 
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<ul>
 
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<li>Identities of all parts were confirmed, but not whole sequence of the parts was covered</li>
 
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</ul>
 
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<li>Glycerin stocks were created for all four strains:</li>
 
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<ul>
 
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<li><i>E. coli</i> KRX <i>pSB1C3-alsS</i></li>
 
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<li><i>E. coli</i> KRX <i>pSB1C3-ilvC</i></li>
 
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<li><i>E. coli</i> KRX <i>pSB1C3-ilvD</i></li>
 
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<li><i>E. coli</i> KRX <i>pSB1C3-kivD</i></li>
 
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                 <a  style="font-size:24px" href="#"><p style="margin-left:42%">Week 5 06/30 - 07/06</p></a>
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                 <h6><a  style="font-size:24px" href="#">Week 5 06/30 - 07/06</a></h6>
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              <li><b><i>alsS</i></b></li>
 +
      <ul>
 +
  <li>This week we tried to amplify <i>alsS</i> to use it for the Gibson Assembly.</li>
 +
                  <ul>
 +
                    <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_pSB1C3" target="_blank">fw_alsS_pSB1C3</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: 55 &deg;C</li>
 +
        <li>Bands not as expected (~ 1800 bp)</li>
 +
            </ul>
 +
                  </ul>
 +
      </ul>
 +
              <li><b><i>ilvC</i></b></li>
 +
      <ul>
 +
  <li>This week we tried to amplify <i>ilvC</i> to use it for the Gibson Assembly.</li>
 +
                  <ul>
 +
                    <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</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: 55 &deg;C</li>
 +
        <li>Bands not as expected (~ 1550 bp)</li>
 +
            </ul>
 +
                  </ul>
 +
      </ul>
 +
              <li><b><i>ilvD</i></b></li>
 +
      <ul>
 +
  <li>This week we tried to amplify <i>ilvD</i> to use it for the Gibson Assembly.</li>
 +
                  <ul>
 +
                    <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</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_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>)</li>
 +
            <ul>
 +
        <li>Annealing temperature: 55 &deg;C</li>
 +
        <li>Bands not as expected (~ 1950 bp)</li>
 +
            </ul>
 +
                  </ul>
 +
      </ul>
 +
              <li><b><i>kivD</i></b></li>
 +
      <ul>
 +
  <li>This week we tried to amplify <i>kivD</i> to use it for the Gibson Assembly.</li>
 +
                  <ul>
 +
                    <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</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: 55 &deg;C</li>
 +
        <li>Bands not as expected (~ 1750 bp)</li>
 +
            </ul>
 +
                  </ul>
 +
      </ul>
 +
              <li><b>Backbone</b></li>
 +
      <ul>
 +
  <li>This week we tried to amplify the backbone pSB1C3 to use it for the Gibson Assembly.</li>
 +
                  <ul>
 +
                    <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<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>)</li>
 +
            <ul>
 +
        <li>Annealing temperature: 55 &deg;C</li>
 +
        <li>Bands not as expected (~ 2.200 kb)</li>
 +
            </ul>
 +
                  </ul>
 +
      </ul>
 +
            <li>PCR conditions need to be optimized due to low product quality and missing products</li>
 +
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Latest revision as of 09:12, 17 October 2014


June

Week 4     06/23 - 06/29
Week 4     06/23 - 06/29
  • alsS
    • This week we aim to transform the construct from the parts distribution.
  • ilvC
    • This week we aim to transform the construct from the parts distribution.
  • ilvD
    • This week we aim to transform the construct from the parts distribution.
  • kivD
    • This week we aim to transform the construct from the parts distribution.
Week 5     06/30 - 07/06
Week 5 06/30 - 07/06

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