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

From 2014.igem.org

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                       <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/2014/3/37/Bielefeld-CeBiTec_2014-10-14_PR6K_PCR_08_19.png" height="230px"></a><br><font size="1">Agarose gel from PCR amplification of pR6K. 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>
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                       <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/2014/3/37/Bielefeld-CeBiTec_2014-10-14_PR6K_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>
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                       <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/b/b3/Bielefeld-CeBiTec_2014-10-14_Knockout_deletion-cassette_PCR_08_09.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>
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                       <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/b/b3/Bielefeld-CeBiTec_2014-10-14_Knockout_deletion-cassette_PCR_08_09.png" height="230px"></a><br><font size="1">Agarose gel from PCR amplification of <i>pR6K</i> and <i>oprF</i>. As a Ladder we used <a href="http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/generuler-1-kb-dna-ladder-ready-to-use-250-to-10000-bp">GeneRuler™ 1 kb DNA Ladder from Thermo Scientific</a>. </font>
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<li><b><i>Fum A</i></b></li>
<li><b><i>Fum A</i></b></li>
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<li>This week we amplified <i>FumA</i> and assembled it with the T7-Promotor as well als several Anderson-Promotor using Biobrick-Assembly</li>
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<li>This week we assembled <i>FumA</i> it with the T7-Promotor as well as with several Anderson-Promotor using Biobrick-Assembly</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#FumA_fwd" target="_blank">FumA_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#FumA_rev" target="_blank">FumA_rev</a>)</li>
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<li>Annealing temperature: 55&deg;C</li>
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<li>Bands as expected (~1843 bp)</li>
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>FumA</i> and pSB1C3</li>
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<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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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of <i>Fum A</i> </li>
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<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>
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<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> of <i>pSB1C3_T7_Fum A</i> with electrocompotetent cells</li>
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<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>)
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<li>Annealing temperature: 55 °C</li>
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<li>Bands as expected (~2130 bp)</li>
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<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>
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<li><b>Cultivation of <i>E.coli</i> with neutral red and bromphenol blue</b></li>
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<ul><li>This week we tested if neutral red and bromphenol blue have an influence on the growth of <i>E. coli</i>. Therefore we cultivated <i>E.coli</i> wildtype in <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#M9medium" target="_blank">M9 medium</a> with glucose supplemented with 100 µM of neutral red or bromphenol blue. Samples were taken for OD<sub>600</sub> measurement to document the growth curve.</li>
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<div class="element" style="height:350px; width:120px; text-align:center">
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                       <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/f/f8/Bielefeld-CeBiTec_2014-10-14_Ccm_cassette_PCR_08_16.png" height="230px"></a><br><font size="1">Agarose gel from PCR amplification of <i>ccm</i>-cassette. 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>
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                       <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/f/f8/Bielefeld-CeBiTec_2014-10-14_Ccm_cassette_PCR_08_16.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>
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<div class="element" style="height:350px; width:120px; text-align:center">
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                       <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/2014/3/3d/Bielefeld-CeBiTec_2014-10-14_PccH_PCR_08_17.png" height="230px"></a><br><font size="1">Agarose gel from PCR amplification of <i>pccH</i>. As a Ladder we used <a href="http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/generuler-1-kb-dna-ladder-ready-to-use-250-to-10000-bp">GeneRuler™ 1 kb DNA Ladder from Thermo Scientific</a>. </font>
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                       <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/2014/3/3d/Bielefeld-CeBiTec_2014-10-14_PccH_PCR_08_17.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>
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Latest revision as of 19:52, 16 October 2014


August

Week 1     08/04 - 08/10
Week 1     08/04 - 08/10


  • Cultivation of E.coli with neutral red and bromphenol blue
    • This week we tested if neutral red and bromphenol blue have an influence on the growth of E. coli. Therefore we cultivated E.coli wildtype in M9 medium with glucose supplemented with 100 µM of neutral red or bromphenol blue. Samples were taken for OD600 measurement to document the growth curve.
Week 2     08/11 - 08/17
Week 2     08/11 - 08/17
  • Deletion of dcuB and integration of oprF into chromosome
    • This week we checked our first colonies to verify the deletion of dcuB
      • pR6K
        • The pR6K-cassette for the dcuB deletion was purified out of the gel
          • Transformation of pR6K-cassette for the deletion of dcuB with electrocompotetent cells
      • pRedET

Week 3     08/18 - 08/24
Week 3     08/18 - 08/24
  • Deletion of dcuB and integration of oprF into chromosome
    • This week we contninued to test colonies for the verification of the deletion if dcuB

  • Fum A


Week 4     08/25 - 08/31
Week 4     08/25 - 08/31
  • Deletion of dcuB and integration of oprF into chromosome
    • This week we tested the next colonies for the deletion of dcuB and performed the NPN-assay to verify the integration of oprF into chromosome
      • oprF
        • NPN-uptake assay for porine verification



  • ccm
    • This week we performed Eckhardt gels as a new method besides Colony PCR to identify positiv colonies
      • Eckhardt gel of ccm was performed two times
        • Bands not impeccable to identify (~6230 bp) and therefore a restriction digestion was done
          • Bands not as expected (~2070 bp and ~6239 bp)

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