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

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Design of the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Deletion_alanine_racemase" target="_blank">Primer</a> for the deletion of the whole coding sequence of the constitutive alanine racemase (<i>alr</i>) and the catabolic alanine racemase (<i>dadX</i>) from <i>E. coli</i> 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>.
Design of the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Deletion_alanine_racemase" target="_blank">Primer</a> for the deletion of the whole coding sequence of the constitutive alanine racemase (<i>alr</i>) and the catabolic alanine racemase (<i>dadX</i>) from <i>E. coli</i> 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>.
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<li>alr: <a href="http://parts.igem.org/Part:BBa_K1465403" target="_blank">BBa_K1465403</a> and <a href="http://parts.igem.org/Part:BBa_K1465404" target="_blank">BBa_K1465404</a></li>
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<li>dadX: <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></li>
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Design of the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Integration_alr" target="_blank">Primer</a> for the integration of the konstitutive alanine racemase <i>alr</i> into the pSB1C3-Backbone.
Design of the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Integration_alr" target="_blank">Primer</a> for the integration of the konstitutive alanine racemase <i>alr</i> into the pSB1C3-Backbone.
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Successful amplifikation of the oligonucleotide containing the flanking sites for the deletion of <i>alr</i> and 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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Successful amplifikation of the oligonucleotide containing the flanking sites for the deletion of <i>alr</i> using the primer <a href="http://parts.igem.org/Part:BBa_K1465403" target="_blank">BBa_K1465403</a> and <a href="http://parts.igem.org/Part:BBa_K1465404" target="_blank">BBa_K1465404</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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The successfull deletion of the chromosomal kanamycin resistance was established by the plasmid Flp705 as described in 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>.<br>
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The plasmid is removed by a temperature shift and the removal of the kanamycin resistance is verified by streaking colonies in parallel on LB-kanamycin and LB agar.
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Resulting in the genotype KRX <i>∆ alr</i>, DH5aplha <i>∆ alr</i> repsectivly.
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Latest revision as of 02:36, 9 October 2014


June

  • Design of the Primer for the integration of the konstitutive alanine racemase alr into the pSB1C3-Backbone.
  • Successful amplifikation of the oligonucleotide containing the flanking sites for the deletion of alr using the primer BBa_K1465403 and BBa_K1465404
  • Transformation of the oligonucleotide containing the flanking sites for the deletion of alr. The successful replacement of the constitutive alanine racemase was verified via kanamycin selection and Colony PCR (alr_Ec_control1, alr_Ec_control2)
    • Annealing temperature: 55 °C
    • Bands as expected (3004 bp)
  • Resulting in the genotype KRX kan:alr, DH5aplha kan:alr repsectivly.
  • The successfull deletion of the chromosomal kanamycin resistance was established by the plasmid Flp705 as described in the Genebridge RedET-System protocol.
    The plasmid is removed by a temperature shift and the removal of the kanamycin resistance is verified by streaking colonies in parallel on LB-kanamycin and LB agar.
  • Resulting in the genotype KRX ∆ alr, DH5aplha ∆ alr repsectivly.