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

From 2014.igem.org

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Transformation of the single deletions strains <i>E. coli</i> strains KRX ∆<i>alr</i> and DH5alpha ∆<i>alr</i> with the plasmid pRedET containg the Recombinase using the <a href="http://www.genebridges.com/storage/Manuals_PDF/K006%20Ecoli%20Gene%20Deletion%20Kit-version2.3-2012.pdf" target="_blank">Genebridge RedET-System protocol</a>.
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Successful amplifikation of the oligonucleotide containing the flanking sites for the deletion of <i>dadX</i> using the primer <a href="http://parts.igem.org/Part:BBa_K1465405" target="_blank">BBa_K1465405</a> and <a href="http://parts.igem.org/Part:BBa_K1465406" target="_blank">BBa_K1465406</a>
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Purification using the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugationgel" target="_blank">gel extraction clean-up kit</a> from Promega.
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Transformation of the oligonucleotide containing the flanking sites for the deletion of <i>dadX</i>. The successful replacement of the catabolic alanine racemase was verified via kanamycin selection and
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<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#dadX_Ec_control1" target="_blank">dadX_Ec_control1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#dadX_Ec_control2" target="_blank">dadX_Ec_control2</a>)
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<li>Annealing temperature: 55 °C</li>
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<li>Bands as expected (3004 bp)</li>
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Resulting in the genotype DH5aplha ∆<i>alr</i> <i>kan:dadX</i>, while the dadX deletion in the KRX strain was not successful.
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<ul>
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<li>
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Transformation of the oligonucleotide containing the flanking sites for the deletion of <i>dadX</i> into the KRX ∆<i>alr</i>. The successful replacement of the katabolic alanine racemase was verified via kanamycin selection and
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<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#dadX_Ec_control1" target="_blank">dadX_Ec_control1</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#dadX_Ec_control2" target="_blank">dadX_Ec_control2</a>)
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<ul>
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<li>Annealing temperature: 55 °C</li>
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<li>Bands as expected (3004 bp)</li>
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</ul>
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</li>
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<li>
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Resulting in the genotype KRX ∆<i>alr</i> <i>kan:dadX</i>.
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</ul>
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Latest revision as of 02:34, 9 October 2014


July

Week 1     07/07 - 07/13
Week 1     07/07 - 07/13
  • Transformation of the single deletions strains E. coli strains KRX ∆alr and DH5alpha ∆alr with the plasmid pRedET containg the Recombinase using the Genebridge RedET-System protocol.
Week 2     07/14 - 07/20
Week 2     07/14 - 07/20
  • Successful amplifikation of the oligonucleotide containing the flanking sites for the deletion of dadX using the primer BBa_K1465405 and BBa_K1465406
  • Transformation of the oligonucleotide containing the flanking sites for the deletion of dadX. The successful replacement of the catabolic alanine racemase was verified via kanamycin selection and Colony PCR (dadX_Ec_control1, dadX_Ec_control2)
    • Annealing temperature: 55 °C
    • Bands as expected (3004 bp)
  • Resulting in the genotype DH5aplha ∆alr kan:dadX, while the dadX deletion in the KRX strain was not successful.
Week 4     07/28 - 08/03
Week 4     07/28 - 08/03
  • Transformation of the oligonucleotide containing the flanking sites for the deletion of dadX into the KRX ∆alr. The successful replacement of the katabolic alanine racemase was verified via kanamycin selection and Colony PCR (dadX_Ec_control1, dadX_Ec_control2)
    • Annealing temperature: 55 °C
    • Bands as expected (3004 bp)
  • Resulting in the genotype KRX ∆alr kan:dadX.

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