Homologous Recombineering to Generate Chromosomal Deletions in Escherichia coli.

ABSTRACT

Homologous recombination methods enable modifications to be made to the bacterial chromosome. Commonly, the λ phage RED proteins are employed as a site-specific recombinase system, to facilitate recombination of linear DNA fragments with targeted regions of the chromosome. Here we describe methods for the efficient delivery of linear DNA segments containing homology to the chromosome into the cell as substrates for the λRED proteins. Combined with antibiotic selection and counterselection, we demonstrate that using this method facilitates accurate, rapid editing of the chromosome.