Team:Bielefeld-CeBiTec/Notebook/Journal/rMFC/Jul

From 2014.igem.org

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<ul>
<ul>
-
<li><b> Amplification of pSB1C3 backbone for FumA and FumBCD </b></li>
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<li><b><i> FumA </i></b></li>
<ul>
<ul>
-
<li> This week we amplified the pSB1C3 backbone of FumA and FumBCD to use it for Gibson Assembly afterwards </li>
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<li> This week we amplified the pSB1C3 backbone of FumA to use it for <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> afterwards </li>
<ul>
<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#pSB1C3_pre_Fum_A" target="_blank"> pSB1C3_pre_Fum_A</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_suf_Fum_A " target="_blank"> pSB1C3_suf_Fum_A </a>)</li>  
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification </a>of <i>FumA</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_pre_Fum_A" target="_blank"> pSB1C3_pre_Fum_A</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_suf_Fum_A " target="_blank"> pSB1C3_suf_Fum_A </a>)</li>  
<ul>  
<ul>  
-
<li>Annealing temperature: -- &deg;C</li>
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<li>Annealing temperature: 55 &deg;C</li>
-
<li>Bands are as expected (2,07 kb)</li>
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<li>Bands are as expected (2070 bp)</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<br>
 +
<ul>
 +
<li><i><b>FumBCD</b></i></li>
 +
<ul>
 +
<li>This week we amplified the pSB1C3 backbone of FumBCD to use it for <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> afterwards </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#pSB1C3_pre_Fum_BCD" target="_blank"> pSB1C3_pre_Fum_BCD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_suf_Fum_BCD " target="_blank"> pSB1C3_suf_Fum_BCD </a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 55 &deg;C</li>
 +
<li>Bands are as expected (2070 bp)</li>
 +
</ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">PCR purification</a> of backbones</li>
 +
</ul>
 +
</ul>
 +
</ul>
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 +
 +
<ul>
 +
<li><b> Deletion of <i>dcuB</i> and integration of <i>oprF</i> into chromosome </b></li>
 +
<ul>
 +
<li> This week we started the deletion of the fumarate/succinate antiporter <i>dcuB</i> and the integration of outer membrane porin <i> oprF</i> into chromosome using the <a href="http://www.genebridges.com/storage/Manuals_PDF/K006%20Ecoli%20Gene%20Deletion%20Kit-version2.3-2012.pdf" target="_blank">Genebridge Red/ET-System</a>
 +
<ul>
 +
<li>pRedET plasmid</li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pRedET plasmid </li>
 +
</ul>
 +
<li> oprF (<a href="http://parts.igem.org/wiki/index.php/Part:http://parts.igem.org/wiki/index.php/Part:BBa_K1172507" target="_blank">BBa_K1172507</a>)</li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_BBa_K1172507 </li>
 +
</ul>
 +
                  </ul>
 +
</ul>
 +
</ul>
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 +
<ul>
 +
<li><b> Deletion of <i>dcuB</i> and integration of <i>oprF</i> into chromosome</b></li>
 +
<ul> <li> This week we amplified the <i>pR6K</i>-cassette and the <i>oprF</i> gene to connect them to each other. Additionally we transform the RedET plasmid into the <i> E. coli </i> strain KRX.
 +
</ul>
 +
<ul>
 +
<ul>
 +
<li> RedET plasmid </li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells of the RedET plasmid into <i> E. coli KRX</i>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><i>pR6K</i>
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<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of <i>pR6K</i>-cassette (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pR6K_dcuB_fwd" target="_blank">pR6K_dcuB_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pR6K_dcuB_rev" target="_blank">pR6K_dcuB_rev</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 55 &deg;C</li>
 +
<li>Bands are as expected (~1600 bp) but slightly visible </li>
 +
<li> <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of <i>pR6K</i>-cassette is repeated two times </i>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li>PCR product <i>pR6K</i> was <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a> out of the gel</li>
 +
</ul>
 +
</ul>
 +
<ul>
 +
<li><i>oprF</i> (<a href="http://parts.igem.org/wiki/index.php/Part:http://parts.igem.org/wiki/index.php/Part:BBa_K1172507" target="_blank">BBa_K1172507</a>)</li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification </a> of <i>oprF</i> for integration into chromosome (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#porine_fwd_FRT" target="_blank"> porine_fwd_FRT</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#porine_rev_FRT " target="_blank"> porine_rev_FRT </a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 55 &deg;C</li>
 +
<li>bands are as expected (~1359 bp) </li>
 +
                  </ul>
 +
<ul>
 +
<li>PCR product <i>oprF</i> was <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a> out of the gel</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
<div class="element" style="height:350px; width:120px; text-align:center">
 +
                      <a href="https://static.igem.org/mediawiki/2014/3/33/Bielefeld_CeBiTec_2014-09-24_csoS1-4_cPCR_08_22.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/c/c7/Bielefeld-CeBiTec_2014-10-14_OprF_PCR_08_19.png" height="230px"></a><br><font size="1">Agarose gel from colony PCR. 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>
 +
 +
<br>
 +
<ul>
 +
<li>Connection of <i>pR6K</i> and <i>oprF</i>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> to connect the <i>pR6K</i>-cassette and <i>oprF</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#porine_fwd_FRT" target="_blank">porine_fwd_FRT</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pR6K_dcuB_rev" target="_blank">pR6K_dcuB_rev</a>)</li>
 +
<ul>
 +
<li>Annealing temperature: 55 &deg;C</li>
 +
<li>Bands not as expected because of too much template (~2900 bp)</li>
 +
<li> Next time the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> was planned with a different usage of primer
 +
<li> additionally a new <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (as described further up) of <i>pR6K</i>-cassette and a new <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purification</a> of it and <i>oprF</i> out of the gel was done</li>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> to connect the <i>pR6K</i>-cassette and <i>oprF</i> was repeated with the new <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a> <i>pR6K</i>-cassette and <i>oprF</i> and different primer usage</li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> was initially performed 15 cycles without primer and afterwards 30 cycles with addition of primer</li>
 +
</ul>
 +
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
</ul>
 +
 +
</ul>
 +
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             <div class="content" style="margin-right:10%; margin-left:10%">
             <div class="content" style="margin-right:10%; margin-left:10%">
 +
<ul>
 +
<li><b> Deletion of <i>dcuB</i> and integration of <i>oprF</i> into chromosome</b></li>
 +
<ul><li>This week we connected the pR6K-cassette to <i>oprF</i> and amplified and purified both again seperatly
 +
<ul>
 +
<li>Connection of pR6K-cassette and <i>oprF</i></li>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> to connect the <i>pR6K</i>-cassette and <i>oprF</i>
 +
</ul>
 +
<ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purification</a> of <i>pR6K</i>-cassette and <i>oprF</i> seperatly</li>
 +
</ul>
         </div>
         </div>
       </div>
       </div>

Latest revision as of 22:13, 15 October 2014


July

Week 1     07/07 - 07/13
Week 1     07/07 - 07/13

Week 2     07/14 - 07/20
Week 2     07/14 - 07/20
Week 3     07/21 - 07/27
Week 3     07/21 - 07/27
Week 4     07/28 - 08/03
Week 4     07/28 - 08/03
  • Deletion of dcuB and integration of oprF into chromosome
    • This week we connected the pR6K-cassette to oprF and amplified and purified both again seperatly

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