Skip to main content
SpringerLink
Log in

An improved recombineering approach by adding RecA to λ Red recombination

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  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.

    Article  PubMed  CAS  Google Scholar 

  2. Zhao, S. (2001) A comprehensive BAC resource. Nucleic Acid Res. 29, 141–143.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  9. Murphy, K. C., Campellone, K. G., and Poteete, A. R. (2000) PCR-mediated gene replacement in Escherichia coli. Gene 246, 321–330.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  20. Hashimoto-Gotoh, T. and Sekiguchi, M. (1977) Mutations of temperature sensitivity in R plasmid pSC101, J. Bacteriol. 131, 405–412.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  27. Swaminathan, S., Ellis, H. M., Waters, L. S., et al. (2001) Rapid engineering of bacterial artificial chromosomes using oligonucleotides. Genesis 29, 14–21.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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. Court, D. L., Swaminathan, S., Yu, D., et al. (2003) Mini-lambda: a tractable system for chromosome and BAC engineering. Gene 315, 63–69.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Junping Wang

    Present address: The State Key Laboratory of Trauma, Burns, and Combined Injury, Institute of Combined Injury, The Third Military Medical University, Chongqing, 400038, China

  2. Yei Xie

    Present address: The State Education Ministry Laboratory of Development Genes and Human Diseases, Genetics Research Center, Southeast University Medical School, 210009, Nanjing, China

Authors and Affiliations

  1. BiolnnovationsZentrum Dresden, Gene Bridges GmbH, Tatzber 47-51, 01307, Dresden, Germany

    Junping Wang, Jeanette Rientjes, Jun Hu, Heike Hollak, Harald Kranz, Yei Xie & Youming Zhang

  2. Biotec, Genomics, University of Technology Dresden, BrolnnovationsZentrum Dresden, Tatzberg 47-51, 01307, Dresden, Germany

    Mihail Sarov & A. Francis Stewart

Authors
  1. Junping Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mihail Sarov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jeanette Rientjes
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jun Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Heike Hollak
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Harald Kranz
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yei Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. A. Francis Stewart
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Youming Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Youming Zhang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, J., Sarov, M., Rientjes, J. et al. An improved recombineering approach by adding RecA to λ Red recombination. Mol Biotechnol 32, 43–53 (2006). https://doi.org/10.1385/MB:32:1:043

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1385/MB:32:1:043

Index Entries

Search

Navigation