Strategy for chromosomal gene targeting in RecA-deficient Escherichia coli strains.
Reengineering DNA by homologous recombination in Escherichia coli often depends on helper functions provided on a temporarily introduced replicon that is subsequently cured from the cells. The suicide vector pKSS offers a new curing strategy. pKSS specifies a variant of phenylalanyl-transfer RNA (tRNA) synthetase conferring relaxed substrate specificity towards phenylalanine analogs that results in their lethal incorporation into cellular proteins. Consequently, the presence of p-chlorophenylalanine selects for strains that have lost pKSS. This principle, in conjunction with a plasmid-borne recA gene, was exploited for targeted chromosomal mutagenesis by double homologous recombination in RecA-negative E. coli strains. Gene replacement with a kanamycin-resistance cassette was possible in a single step by plating on kanamycin and p-chlorophenylalanine agar plates and incubating at 37 degrees C. The presence of the correct chromosomal mutation and the absence of the plasmid were established by several control experiments. A simple screen confirmed the desired resistance phenotype in 44% of the initially selected clones, and 75% of these had the correct genotype.
Gamper
Marianne
M
Swiss Federal Institute of Technology (ETH), Zürich, Switzerland.
Kast
Peter
P
D002876:Chromosomes, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D011938:Rec A Recombinases / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic
D011938:Rec A Recombinases