title: An improved recombineering approach by adding RecA to lambda Red recombination.
abstract: Recombineering is the use of homologous recombination in Escherichia coli for DNA engineering. Of several approaches, use of the lambda phage Red operon is emerging as the most reliable and flexible. The Red operon includes three components: Redalpha, a 5' to 3' exonuclease, Redbeta, an annealing protein, and Redgamma, an inhibitor of the major E. coli exonuclease and recombination complex, RecBCD. Most E. coli cloning hosts are recA deficient to eliminate recombination and therefore enhance the stability of cloned DNAs. However, loss of RecA also impairs general cellular integrity. Here we report that transient RecA co-expression enhances the total number of successful recombinations in bacterial artificial chromosomes (BACs), mostly because the E. coli host is more able to survive the stresses of DNA transformation procedures. We combined this practical improvement with the advantages of a temperature-sensitive version of the low copy pSC101 plasmid to develop a protocol that is convenient and more efficient than any recombineering procedure, for use of either double- or single-stranded DNA, published to date.
authors.affiliation: Gene Bridges GmbH, BioInnovationsZentrum Dresden, Tatzberg 47-51, 01307 Dresden, Germany.
authors.forename: Junping
authors.initials: J
authors.lastname: Wang
authors.forename: Mihail
authors.initials: M
authors.lastname: Sarov
authors.forename: Jeanette
authors.initials: J
authors.lastname: Rientjes
authors.forename: Jun
authors.initials: J
authors.lastname: Fu
authors.forename: Heike
authors.initials: H
authors.lastname: Hollak
authors.forename: Harald
authors.initials: H
authors.lastname: Kranz
authors.forename: Wei
authors.initials: W
authors.lastname: Xie
authors.forename: A Francis
authors.initials: AF
authors.lastname: Stewart
authors.forename: Youming
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001089:Arabinose / Q000494:pharmacology; D010582:Bacteriophage lambda / Q000235:genetics*; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D002876:Chromosomes, Bacterial / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D011495:Histone-Lysine N-Methyltransferase; D051379:Mice; D009154:Mutation / Q000235:genetics; D051788:Myeloid-Lymphoid Leukemia Protein / Q000235:genetics; D009876:Operon / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011938:Rec A Recombinases / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*; D014169:Transformation, Bacterial / Q000235:genetics
chemical_list: D051788:Myeloid-Lymphoid Leukemia Protein; D001089:Arabinose; D011495:Histone-Lysine N-Methyltransferase; C497423:Kmt2a protein, mouse; D011938:Rec A Recombinases