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Modifying Bacteriophage \(\lambda\) with Recombineering

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Bacteriophages

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 501))

Abstract

Recombineering is a recently developed method of in vivo genetic engineering used in Escherichia coli and other Gram-negative bacteria. Recombineering can be used to create single-base changes, small and large deletions, and small insertions in phage \(\lambda\) as well as in bacterial chromosomes, plasmids, and bacterial artificial chromosomes (BACS). This technique uses the bacteriophage \(\lambda\) generalized recombination system, Red, to catalyze homologous recombination between linear DNA and a replicon using short homologies of 50 base pairs. With recombineering, single-stranded oligonucleotides or double-stranded PCR products can be used to directly modify the phage \(\lambda\) genome in vivo. It may also be possible to modify the genomes of other bacteriophages with recombineering.

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References

  1. Georgeopolous, C., Tilly, K. and Casjens, S. 1983. Lambdoid phage head assembly. In Lambda II (R. Hendrix, J. Roberts, F. Stahl, and R. Weisberg, eds.) pp. 279–304. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

    Google Scholar 

  2. Katsura, I. 1983. Tail assembly and injection. In Lambda II (R. Hendrix, J. Roberts, F. Stahl, and R. Weisberg, eds.) pp. 331–346. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

    Google Scholar 

  3. Yu, D., Ellis, H. M., Lee, E. C., Jenkins, N. A., Copeland, N. G., and D. L. Court. 2000. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 97, 5978–5983.

    Article  CAS  PubMed  Google Scholar 

  4. 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. U.S.A. 98, 6742–6746.

    Article  CAS  PubMed  Google Scholar 

  5. Court, D.L., Sawitzke, J.A., and Thomason L.C. 2002. Genetic engineering using homologous recombination. Annu. Rev. Genet. 36, 361–388.

    Article  CAS  PubMed  Google Scholar 

  6. Thomason L.C., Myers, R.S., Oppenheim, A., Costantino, N., Sawitzke, J.A. Datta, S., Bubenenko, M. and Court D.L. (2005). Recombineering in prokaryotes. In Phages: Their Role in Bacterial Pathogenesis and Biotechnology. pp. 383–399. ASM Press, Herndon, Va.

    Google Scholar 

  7. Smith, G.R. General recombination. 1983. In Lambda II (R. Hendrix, J. Roberts, F. Stahl, and R. Weisberg, eds.) pp. 175–209. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

    Google Scholar 

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

    CAS  Google Scholar 

  9. Datta, S, Costantino, N., and Court, D.L. 2006. A set of recombineering plasmids for gram-negative bacteria. Gene. 379, 109–115.

    Article  CAS  PubMed  Google Scholar 

  10. Zhang, Y., Buchholz, F., Muyrers, J. P. P., and Stewart, F. 1998. A new logic for DNA engineering using recombination in Escherichia coli. Nature Genetics. 20, 123–128.

    Article  CAS  PubMed  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. U.S.A. 97, 6640–6645.

    Article  CAS  PubMed  Google Scholar 

  12. Court, D.L., Swaminathan, S., Yu, D., Wilson, H., Baker, T., Bubunenko, M., Sawitzke, J., and Sharan, S.K. 2003. Mini-lambda: a tractable system for chromosome and BAC engineering. Gene. 315, 63–69.

    Article  CAS  PubMed  Google Scholar 

  13. Oppenheim, A.B., Rattray, A.J., Bubunenko, M., Thomason, L.C., and Court, D.L. 2004. In vivo recombineering of bacteriophage \(\lambda\) by PCR fragments and single-strand oligonucleotides. Virology. 319, 185–189.

    Article  CAS  PubMed  Google Scholar 

  14. Arber, W., Enquist, L., Hohn, B., Murray, N.E., and Murray, K. 1983. Experimental methods for use with lambda. In Lambda II (R. Hendrix, J. Roberts, F. Stahl, and R. Weisberg, eds.) pp. 433–471. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

    Google Scholar 

  15. Sambrook, J. and Russell, D. W. 2001. Bacteriophage \(\lambda\) and its vectors. In Molecular Cloning: A Laboratory Manual (Third Edition) pp. 2.25–2.110. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

    Google Scholar 

  16. Campbell, A. 1971. Genetic structure. In: The Bacteriophage Lambda (A.D. Hershey, ed.) pp. 13–44. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

    Google Scholar 

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Thomason, L.C., Oppenheim, A.B., Court, D.L. (2009). Modifying Bacteriophage \(\lambda\) with Recombineering. In: Clokie, M.R., Kropinski, A.M. (eds) Bacteriophages. Methods in Molecular Biology™, vol 501. Humana Press. https://doi.org/10.1007/978-1-60327-164-6_21

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  • DOI: https://doi.org/10.1007/978-1-60327-164-6_21

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-682-5

  • Online ISBN: 978-1-60327-164-6

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