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Team:Bielefeld-CeBiTec/Notebook/Journal/Biosafety/Jun
From 2014.igem.org
June |
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- Design of the Primer for the deletion of the whole coding sequence of the constitutive alanine racemase (alr) and the catabolic alanine racemase (dadX) from E. coli using the Genebridge Red/ET-System.
- Design of the Primer for the integration of the konstitutive alanine racemase alr into the pSB1C3-Backbone.
- Transformation of the E. coli strains KRX (Promega) and DH5alpha (Invitrogen) with the plasmid pRedET containg the Recombinase using the Genebridge RedET-System protocol.
- 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.
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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.
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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.
