Directed homologous recombination for genome engineering inEscherichia coli

Abstract

E. coli K-12 is the workhorse of molecular biology and the platform of choice for production of DNA, metabolites and proteins of industrial interest. To construct strains for the multitude of purposes, efficient genome manipulation methods are required. The suicide plasmid-mediated, homologous recombination-based gene replacement method is a convenient genome engineering tool. In consecutive recombination events, genomic integration of the plasmid, carrying the modified allele, is followed by its excision, resulting in either a modified genome or the original wild-type chromosome. Using the lac operon as a chromosomal target, we systematically investigated the effects of several factors influencing the outcome of the procedure. Recombinogenic activity was proportional to the length of the targeting homologous fragments. Presence of a properly oriented Chi site stabilized broken chromosomal ends and stimulated recombination in the downstream genomic region. Introduction of a double-stranded break in the chromosome had a profound stimulatory effect on recombination of the free DNA ends. These results shed light on some details of the complex events of intra- and intermolecular homologous recombination in the E. coli genome. Taking into account these findings at the assembly of the targeting plasmid constructs, serial genomic modifications can be created with enhanced efficiency and speed.