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Multipart DNA Assembly Using Site-Specific Recombinases from the Large Serine Integrase Family

  • Femi J. Olorunniji
  • Christine Merrick
  • Susan J. Rosser
  • Margaret C. M. Smith
  • W. Marshall Stark
  • Sean D. CollomsEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1642)

Abstract

Assembling multiple DNA fragments into functional plasmids is an important and often rate-limiting step in engineering new functions in living systems. Bacteriophage integrases are enzymes that carry out efficient recombination reactions between short, defined DNA sequences known as att sites. These DNA splicing reactions can be used to assemble large numbers of DNA fragments into a functional circular plasmid in a method termed serine integrase recombinational assembly (SIRA). The resulting DNA assemblies can easily be modified by further recombination reactions catalyzed by the same integrase in the presence of its recombination directionality factor (RDF). Here we present a set of protocols for the overexpression and purification of bacteriophage ϕC31 and Bxb1 integrase and RDF proteins, their use in DNA assembly reactions, and subsequent modification of the resulting DNA assemblies.

Key words

Site-specific recombination DNA assembly Large serine integrase Bacteriophage ϕC31 Synthetic biology Metabolic engineering 

References

  1. 1.
    Craig NL (2015) A moveable feast: an introduction to mobile DNA. In: Craig NL, Chandler M, Gellert M, Lambowitz A, Rice PA, Sandmeyer SB (eds) Mobile DNA III. ASM Press, Washington, DC, pp 3–39CrossRefGoogle Scholar
  2. 2.
    Grindley ND, Whiteson KL, Rice PA (2006) Mechanisms of site-specific recombination. Annu Rev Biochem 75:567–605CrossRefPubMedGoogle Scholar
  3. 3.
    Jayaram M (2015) An overview of tyrosine site-specific recombination: from an Flp perspective. In: Craig NL, Chandler M, Gellert M, Lambowitz A, Rice PA, Sandmeyer SB (eds) Mobile DNA III. ASM Press, Washington, DC, pp 43–71Google Scholar
  4. 4.
    Stark WM (2015) The serine recombinases. In: Craig NL, Chandler M, Gellert M, Lambowitz A, Rice PA, Sandmeyer SB (eds) Mobile DNA III. ASM Press, Washington, DC, pp 73–89Google Scholar
  5. 5.
    Smith MC (2015) Phage-encoded serine integrases and other large serine recombinases. Microbiol Spectr 3(4) doi: 10.1128/microbiolspec.MDNA3-0059-2014
  6. 6.
    Kim AI, Ghosh P, Aaron MA, Bibb LA, Jain S, Hatfull GF (2003) Mycobacteriophage Bxb1 integrates into the Mycobacterium smegmatis groEL1 gene. Mol Microbiol 50(2):463–473CrossRefPubMedGoogle Scholar
  7. 7.
    Thorpe HM, Smith MC (1998) In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proc Natl Acad Sci U S A 95(10):5505–5510CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ghosh P, Wasil LR, Hatfull GF (2006) Control of phage Bxb1 excision by a novel recombination directionality factor. PLoS Biol 4(6):e186CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Khaleel T, Younger E, McEwan AR, Varghese AS, Smith MC (2011) A phage protein that binds ϕC31 integrase to switch its directionality. Mol Microbiol 80(6):1450–1463CrossRefPubMedGoogle Scholar
  10. 10.
    Colloms SD, Merrick CA, Olorunniji FJ, Stark WM, Smith MC, Osbourn A, Keasling JD, Rosser SJ (2014) Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination. Nucleic Acids Res 42(4):e23CrossRefPubMedGoogle Scholar
  11. 11.
    Rowley PA, Smith MC, Younger E, Smith MC (2008) A motif in the C-terminal domain of phiC31 integrase controls the directionality of recombination. Nucleic Acids Res 36(12):3879–3891CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Olorunniji FJ, Rosser S, Smith MC, Colloms SD, Stark WM (Submitted for Publication) Serine integrases fused to their recombination directionality factors promote efficient reverse-direction DNA site-specific recombination. Nucleic Acids ResGoogle Scholar
  13. 13.
    Pace CN, Vajdos F, Fee L, Grimsley G, Gray T (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci 4(11):2411–2423CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Femi J. Olorunniji
    • 1
  • Christine Merrick
    • 2
  • Susan J. Rosser
    • 2
  • Margaret C. M. Smith
    • 3
  • W. Marshall Stark
    • 1
  • Sean D. Colloms
    • 1
    Email author
  1. 1.Institute of Molecular, Cell and Systems Biology, University of GlasgowGlasgowScotland
  2. 2.SynthSys—Synthetic and Systems Biology, School of Biological Sciences, University of EdinburghEdinburghScotland
  3. 3.Department of BiologyUniversity of YorkYorkUK

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