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

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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 and purification using the gel extraction clean-up kit from Promega.
  • 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.