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.
Successful amplifikation of the oligonucleotide containing the flanking sites for the deletion of alr and purification using gel extraction clean-up kit from Promega (link)
Transformation of the oligonucleotide containing the flanking sites for the deletion of alr. The successful replacement of the constitutive alanine racemase was verified via
Colony PCR (alr_Ec_control1, alr_Ec_control2)
Annealing temperature: 55 °C
Bands as expected (10 bp)
and kanamycin selection. Resulting in the genotype KRX kan:alr, DH5aplha kan:alr repsectivly.