Recombineering: Genetic Engineering in Escherichia coli Using Homologous Recombination

Abstract

AbstractThe bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using either PCR products or synthetic double‐stranded DNA (dsDNA) or single‐stranded DNA as substrates. Multiple linear dsDNA molecules can be assembled into an intact plasmid. The technology of recombineering is possible because bacteriophage‐encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to the location of restriction sites and can also be used in combination with CRISPR/Cas targeting systems. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.Basic Protocol: Making electrocompetent cells and transforming with linear DNASupport Protocol 1: Selection/counter‐selections for genome engineeringSupport Protocol 2: Creating and screening for oligo recombinants by PCRSupport Protocol 3: Other methods of screening for unselected recombinantsSupport Protocol 4: Curing recombineering plasmids containing a temperature‐sensitive replication functionSupport Protocol 5: Removal of the prophage by recombineeringAlternate Protocol 1: Using CRISPR/Cas9 as a counter‐selection following recombineeringAlternate Protocol 2: Assembly of linear dsDNA fragments into functional plasmidsAlternate Protocol 3: Retrieval of alleles onto a plasmid by gap repairAlternate Protocol 4: Modifying multicopy plasmids with recombineeringSupport Protocol 6: Screening for unselected plasmid recombinantsAlternate Protocol 5: Recombineering with an intact λ prophageAlternate Protocol 6: Targeting an infecting λ phage with the defective prophage strains