Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome.
An efficient method for moving mutations in cloned Escherichia coli DNA from plasmid vectors to the bacterial chromosome was developed. Cells carrying plasmids that had been mutated by the insertion of a resistance gene were infected with lambda phage containing homologous cloned DNA, and resulting lysates were used for transduction. Chromosomal transductants (recombinants) were distinguished from plasmid transductants by their ampicillin-sensitive phenotype, or plasmid transductants were avoided by using a recBC sbcB E. coli strain as recipient. Chromosomal transductants were usually haploid when obtained in a nonlysogen because of selection against the lambda vector and partially diploid when obtained in a lysogen. Pure stocks of phage that carry the resistance marker and transduce it at high frequency were obtained from transductant bacteria. The lambda-based method for moving mutant alleles into the bacterial chromosome described here should be useful for diverse analyses of gene function and genome structure.
Kulakauskas
S
S
Department of Molecular Microbiology, Washington University Medical School, St. Louis, Missouri 63110.
Wikström
P M
PM
Berg
D E
DE
D000483:Alleles; D010582:Bacteriophage lambda; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D016254:Mutagenesis, Insertional; D010957:Plasmids; D014161:Transduction, Genetic