An improved recombineering approach by adding RecA to λ Red recombination
Rent the article at a discountRent now
* Final gross prices may vary according to local VAT.Get Access
Recombineering is the use of homologous recombination in Escherichia coli for DNA engineering. Of several approaches, use of the λ phage Red operon is emerging as the most reliable and flexible. The Red operon includes three components: Redα, a 5′ to 3′ exonuclease, Redβ, an annealing protein, and Redλ, 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 thestabulity of cloned DNAs. However, loss of RecA also impairs general cellular integrity. Here we report that transient RecA co-expression enhances the total numer 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 pSC 101 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.
Hamilton, C. M., Aldea, M., Washburn, B. K., Babitzke, P., and Kushner, S. R. (1989) New method for generating deletions and gene replacements in Escherichia coli. J. Bacteriol. 171, 4617–4622.PubMed
Yang, X. W., Model, P., and Heintz, N. (1997) Homologous recombination based on modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat. Biotech. 15, 859–865.CrossRef
Zhang, Y., Muyrers, J. P. P., Testa, G., and Stewart, A. F. (2000) DNA cloning by homologous recombination in Escherichia coli. Nat. Biotech. 18, 1314–1317.CrossRef
Poteete, A. R. (2001) What makes the bacteriophage λ Red system useful for genetic engineering: molecular mechanism and biological function. FEMS Microbio. Lett. 201, 9–14.
Zhang, Y., Muyrers, J. P. P., Reintjes, J., and Stewart, A. F. (2003) Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells. BMC Mol. Biol. 16, 1–14.CrossRef
Murphy, K. C. (1991) Lambda Gam protein inhibits the helicase and chi-stimulated recombination activities of Escherichia coli RecBCD enzyme. J. Bacteriol. 173, 5808–5821.PubMed
Guzman, L. M., Belin, D., Carson, M. J., and Beck, J. (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose pBAD promoter. J. Bacteriol. 177, 4121–4130.PubMed
Hashimoto-Gotoh, T. and Sekiguchi, M. (1977) Mutations of temperature sensitivity in R plasmid pSC101, J. Bacteriol. 131, 405–412.PubMed
Muyrers, J. P. P., Zhang, Y., Buchholz, F., and Stewart, A. F. (2000) RecE/RecT and Redα/Redβ initiate doublestranded break repair by specifically interacting with their respective partners. Genes & Dev. 14, 1971–1982.
Testa, G., Zhang, Y., Vinteresten, K., et al. (2003) Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles. Nat. Biotech. 21, 443–447.CrossRef
Walker, G. C. (1996) The SOS response of Escherichia coli in Escherichia coli and Salmonella (Neidhardt, F. C., ed.). Washington, ASM Press.
Reyrat, J. M., Pelicic, V., Gicquel, B., and Rappuoli, R. (1998) Counterselectable markers: untapped tools for bacterial genetics and pathogenesis. Infect. Immunol. 66, 4011–4017.
- An improved recombineering approach by adding RecA to λ Red recombination
Volume 32, Issue 1 , pp 43-53
- Cover Date
- Print ISSN
- Online ISSN
- Humana Press
- Additional Links
- λ red
- counter selection
- Industry Sectors
- Author Affiliations
- 1. BiolnnovationsZentrum Dresden, Gene Bridges GmbH, Tatzber 47-51, 01307, Dresden, Germany
- 3. Biotec, Genomics, University of Technology Dresden, BrolnnovationsZentrum Dresden, Tatzberg 47-51, 01307, Dresden, Germany