An efficient recombination system for chromosome engineering in Escherichia coli

A recombination system has been developed for efficient chromosome engineer ing in Escherichia coil by using electroporated linear DNA. A defective lam bda prophage supplies functions that protect and recombine an electroporate d linear DNA substrate in the bacterial cell. The use of recombination elim inates the requirement for standard cloning as all novel joints are enginee red by chemical synthesis in vitro and the linear DNA is efficiently recomb ined into place in vivo. The technology and manipulations required are simp le and straight forward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42 degrees C for 15 min. The efficient pr ophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defective lam bda prophage, The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of bo th the bacterial chromosome and bacterial plasmids are possible. This syste m will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries.