An improved recombineering approach by adding RecA to lambda Red recombination.
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.
Wang
Junping
J
Gene Bridges GmbH, BioInnovationsZentrum Dresden, Tatzberg 47-51, 01307 Dresden, Germany.
Sarov
Mihail
M
Rientjes
Jeanette
J
Fu
Jun
J
Hollak
Heike
H
Kranz
Harald
H
Xie
Wei
W
Stewart
A Francis
AF
Zhang
Youming
Y
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
D051788:Myeloid-Lymphoid Leukemia Protein; D001089:Arabinose; D011495:Histone-Lysine N-Methyltransferase; C497423:Kmt2a protein, mouse; D011938:Rec A Recombinases