Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination

Abstract

AbstractThe bacterial chromosome and plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage‐encoded recombination functions efficiently recombine sequences with homologies as short as 35 to 40 bases. This recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with double‐stranded DNA (dsDNA) or single‐stranded DNA (ssDNA). Support protocols describe a two‐step method of making genetic alterations without leaving any unwanted changes, and a method for retrieving a genetic marker (cloning) from the E. coli chromosome or a coelectroporated DNA fragment and moving it onto a plasmid. A method is also given to screen for unselected mutations. Additional protocols describe removal of defective prophage, methods for recombineering.