Molecular Biotechnology

, Volume 32, Issue 1, pp 43–53 | Cite as

An improved recombineering approach by adding RecA to λ Red recombination

  • Junping Wang
  • Mihail Sarov
  • Jeanette Rientjes
  • Jun Hu
  • Heike Hollak
  • Harald Kranz
  • Yei Xie
  • A. Francis Stewart
  • Youming ZhangEmail author


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.

Index Entries

Recombineering Red/ET recA λ red counter selection BAC 


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  1. 1.
    Shizuya H., Birren, B., Kim, U. J., et al.. (1992) Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl. Acad. Sci. USA 89, 8794–8797.PubMedCrossRefGoogle Scholar
  2. 2.
    Zhao, S. (2001) A comprehensive BAC resource. Nucleic Acid Res. 29, 141–143.PubMedCrossRefGoogle Scholar
  3. 3.
    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.PubMedGoogle Scholar
  4. 4.
    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.CrossRefGoogle Scholar
  5. 5.
    Gong, S., Yang, X. W., Li, C., and Heintz, N. (2002) Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6K · origin of replication. Genome Res. 12, 1992–1998.PubMedCrossRefGoogle Scholar
  6. 6.
    Zhang, Y., Buchholz, F., Muyrers, J. P. P., and Stewart, A. F. (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20, 123–128.PubMedCrossRefGoogle Scholar
  7. 7.
    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.CrossRefGoogle Scholar
  8. 8.
    Muyrers, J. P. P., Zhang, Y., Testa, G., and Stewart, A. F. (1999) Rapid modification of bacterial artificial chromosome by ET-recombination. Nucleic Acids Res. 27, 1555–1557.PubMedCrossRefGoogle Scholar
  9. 9.
    Murphy, K. C., Campellone, K. G., and Poteete, A. R. (2000) PCR-mediated gene replacement in Escherichia coli. Gene 246, 321–330.PubMedCrossRefGoogle Scholar
  10. 10.
    Yu, D., Ellis, H. M., Lee, E. C., Jenkins, N. A., Copeland, N. G., and Court, D. L. (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. USA 97, 5978–5983.PubMedCrossRefGoogle Scholar
  11. 11.
    Datsenko, K. A. and Wanner, B. L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640–6645.PubMedCrossRefGoogle Scholar
  12. 12.
    Zhang, P., Li, M. Z., and Elledge, S. J. (2002) Towards genetic genome projects: genomic library screening and gene-targeting vector construction in a single step. Nat. Genet. 30, 31–39.PubMedCrossRefGoogle Scholar
  13. 13.
    Copeland, N. G., Jenkins, N. A., and Court, D. L. (2001) Recombineering: a powerful new tool for mouse functional genomics. Nat. Rev. Genet. 2, 769–779.PubMedCrossRefGoogle Scholar
  14. 14.
    Muyrers, J. P. P., Zhang, Y., and Stewart, A. F. (2001) Techniques: recombinogenic engineering—new options for cloning and manipulating DNA. Trends Biochem. Sci. 26, 325–331.PubMedCrossRefGoogle Scholar
  15. 15.
    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.Google Scholar
  16. 16.
    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.CrossRefGoogle Scholar
  17. 17.
    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.PubMedGoogle Scholar
  18. 18.
    Lee, E. C., Yu, D., Martinez de Velssco, J., et al. (2001) A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73, 56–65.PubMedCrossRefGoogle Scholar
  19. 19.
    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.PubMedGoogle Scholar
  20. 20.
    Hashimoto-Gotoh, T. and Sekiguchi, M. (1977) Mutations of temperature sensitivity in R plasmid pSC101, J. Bacteriol. 131, 405–412.PubMedGoogle Scholar
  21. 21.
    Penfold, R. J. and Pemberton, J. M. (1992) An improved suicide vector for construction of chromosomal insertion mutations in bacteria. Gene. 118, 145–146.PubMedCrossRefGoogle Scholar
  22. 22.
    Buchholz, F., Ringrose, L., Angrand, P. O., Rossi, F., and Stewart A. F. (1996) Different thermostabilities of FLP and Cre recombinases: implications for applied site-specific recombination. Nucleic Acid Res. 24, 4256–4262.PubMedCrossRefGoogle Scholar
  23. 23.
    Gasparich, G. E., Hackett, K. J., Stamburski, C., Renaudin, J., and Bove, J. M. (1993) Optimization of methods for transfecting Spiroplasm Citri strain R8A2HP with the spiroplasma virus SpV1 replicate form. Plasmid 29, 193–205.PubMedCrossRefGoogle Scholar
  24. 24.
    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.Google Scholar
  25. 25.
    Ellis, H. M., Yu, D., DiTizio, T., and Court, D. L. (2001) High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc. Natl. Acad. Sci. USA 98, 6742–6746.PubMedCrossRefGoogle Scholar
  26. 26.
    Yu, D., Sawitzke, J. A., Ellis, H. M., and Court, D. L. (2003) Recombineering with overlapping singlestranded DNA oligonucleotides: testing a recombination intermediate. Proc. Natl. Acad. Sci. USA 100, 7207–7212.PubMedCrossRefGoogle Scholar
  27. 27.
    Swaminathan, S., Ellis, H. M., Waters, L. S., et al. (2001) Rapid engineering of bacterial artificial chromosomes using oligonucleotides. Genesis 29, 14–21.PubMedCrossRefGoogle Scholar
  28. 28.
    Li, X. T., Costantino, N., Lu, L. Y., et al. (2003) Identification of factors influencing strand bias in oligonucleotide-mediated recombination in Escherichia coli. Nucleic Acids Res. 31, 6674–6687.PubMedCrossRefGoogle Scholar
  29. 29.
    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.CrossRefGoogle Scholar
  30. 30.
    Walker, G. C. (1996) The SOS response of Escherichia coli in Escherichia coli and Salmonella (Neidhardt, F. C., ed.). Washington, ASM Press.Google Scholar
  31. 31.
    Court, D. L., Swaminathan, S., Yu, D., et al. (2003) Mini-lambda: a tractable system for chromosome and BAC engineering. Gene 315, 63–69.PubMedCrossRefGoogle Scholar
  32. 32.
    Muyrers, J. P. P., Zhang, Y., Benes, V., Testa, G., Ansorge, W., and Stewart, A. F. (2000) Point mutation of bacterial artificial chromosomes by ET recombination. EMBO Rep. 1, 239–243.PubMedCrossRefGoogle Scholar
  33. 33.
    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.Google Scholar
  34. 34.
    Filutowicz, M. and Rakowski, S. A. (1998) Regulatory implications of protein assemblies at the origin of plasmid R6K: a review. Gene 223, 195–204.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 2006

Authors and Affiliations

  • Junping Wang
    • 1
  • Mihail Sarov
    • 2
  • Jeanette Rientjes
    • 1
  • Jun Hu
    • 1
  • Heike Hollak
    • 1
  • Harald Kranz
    • 1
  • Yei Xie
    • 1
  • A. Francis Stewart
    • 2
  • Youming Zhang
    • 1
    Email author
  1. 1.BiolnnovationsZentrum DresdenGene Bridges GmbHDresdenGermany
  2. 2.Biotec, Genomics, University of Technology DresdenBrolnnovationsZentrum DresdenDresdenGermany

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