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Recombination-Based DNA Assembly and Mutagenesis Methods for Metabolic Engineering

  • Xiquan Liang
  • Lansha Peng
  • Billyana Tsvetanova
  • Ke Li
  • Jian-Ping Yang
  • Tony Ho
  • Josh Shirley
  • Liewei Xu
  • Jason Potter
  • Wieslaw Kudlicki
  • Todd Peterson
  • Federico KatzenEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 834)

Abstract

In recent years there has been a growing interest in the precise and concerted assembly of multiple DNA fragments of diverse sizes, including chromosomes, and the fine tuning of gene expression levels and protein activity. Commercial DNA assembly solutions have not been conceived to support the cloning of very large or very small genetic elements or a combination of both. Here we summarize a series of protocols that allow the seamless, simultaneous, flexible, and highly efficient assembly of DNA elements of a wide range of sizes (up to hundred thousand base pairs). The protocols harness the power of homologous recombination and are performed either in vitro or within the living cells. The DNA fragments may or may not share homology at their ends. An efficient site-directed mutagenesis protocol enhanced by homologous recombination is also described.

Key words

Recombineering Recombinational Yeast Mutation Double-strand break repair Synthetic biology 

Notes

Disclosure

Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

References

  1. 1.
    Raymond C. K., Pownder T. A. and Sexson S. L. (1999) General method for plasmid construction using homologous recombination. Biotechniques 26, 134–8, 140–1Google Scholar
  2. 2.
    Willer D. O., Yao X. D., Mann M. J. and Evans D. H. (2000) In vitro concatemer formation catalyzed by vaccinia virus DNA polymerase. Virology 278, 562–9PubMedCrossRefGoogle Scholar
  3. 3.
    Hamilton M. D., Nuara A. A., Gammon D. B., Buller R. M. and Evans D. H. (2007) Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase. Nucleic Acids Res 35, 143–51PubMedCrossRefGoogle Scholar
  4. 4.
    Zhu B., Cai G., Hall E. O. and Freeman G. J. (2007) In-fusion assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. Biotechniques 43, 354–9PubMedCrossRefGoogle Scholar
  5. 5.
    Gibson D. G., Benders G. A., Andrews-Pfannkoch C., Denisova E. A., Baden-Tillson H., Zaveri J., et al. (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319, 1215–20PubMedCrossRefGoogle Scholar
  6. 6.
    Gibson D. G., Young L., Chuang R. Y., Venter J. C., Hutchison C. A., 3rd and Smith H. O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6, 343–5PubMedCrossRefGoogle Scholar
  7. 7.
    Cheo D. L., Titus S. A., Byrd D. R., Hartley J. L., Temple G. F. and Brasch M. A. (2004) Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: functional analysis of multi-segment expression clones. Genome Res 14, 2111–20PubMedCrossRefGoogle Scholar
  8. 8.
    Hartley J. L., Temple G. F. and Brasch M. A. (2000) DNA cloning using in vitro site-specific recombination. Genome Res 10, 1788–95PubMedCrossRefGoogle Scholar
  9. 9.
    Bubeck P., Winkler M. and Bautsch W. (1993) Rapid cloning by homologous recombination in vivo. Nucleic Acids Res 21, 3601–2PubMedCrossRefGoogle Scholar
  10. 10.
    Zhang Y., Buchholz F., Muyrers J. P. and Stewart A. F. (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20, 123–8PubMedCrossRefGoogle 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, 6640–5PubMedCrossRefGoogle Scholar
  12. 12.
    Liu Q., Li M. Z., Leibham D., Cortez D. and Elledge S. J. (1998) The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes. Curr Biol 8, 1300–9PubMedCrossRefGoogle Scholar
  13. 13.
    Lebedenko E. N., Birikh K. R., Plutalov O. V. and Berlin Yu A. (1991) Method of artificial DNA splicing by directed ligation (SDL). Nucleic Acids Res 19, 6757–61PubMedCrossRefGoogle Scholar
  14. 14.
    Aslanidis C. and de Jong P. J. (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18, 6069–74PubMedCrossRefGoogle Scholar
  15. 15.
    Li M. Z. and Elledge S. J. (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4, 251–6PubMedCrossRefGoogle Scholar
  16. 16.
    Gao X., Yo P., Keith A., Ragan T. J. and Harris T. K. (2003) Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-fidelity assembly of longer gene sequences. Nucleic Acids Res 31, e143PubMedCrossRefGoogle Scholar
  17. 17.
    Orr-Weaver T. L., Szostak J. W. and Rothstein R. J. (1981) Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci USA 78, 6354–8PubMedCrossRefGoogle Scholar
  18. 18.
    Hinnen A., Hicks J. B. and Fink G. R. (1978) Transformation of yeast. Proc Natl Acad Sci USA 75, 1929–33PubMedCrossRefGoogle Scholar
  19. 19.
    Larionov V., Kouprina N., Eldarov M., Perkins E., Porter G. and Resnick M. A. (1994) Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks. Yeast 10, 93–104PubMedCrossRefGoogle Scholar
  20. 20.
    Ma H., Kunes S., Schatz P. J. and Botstein D. (1987) Plasmid construction by homologous recombination in yeast. Gene 58, 201–16PubMedCrossRefGoogle Scholar
  21. 21.
    Marykwas D. L. and Passmore S. E. (1995) Mapping by multifragment cloning in vivo. Proc Natl Acad Sci USA 92, 11701–5PubMedCrossRefGoogle Scholar
  22. 22.
    Ebersole T., Okamoto Y., Noskov V. N., Kouprina N., Kim J. H., Leem S. H., et al. (2005) Rapid generation of long synthetic tandem repeats and its application for analysis in human artificial chromosome formation. Nucleic Acids Res 33, e130PubMedCrossRefGoogle Scholar
  23. 23.
    Larionov V., Kouprina N., Graves J., Chen X. N., Korenberg J. R. and Resnick M. A. (1996) Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination. Proc Natl Acad Sci USA 93, 491–6PubMedCrossRefGoogle Scholar
  24. 24.
    Gibson D. G. (2009) Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides. Nucleic Acids Res 37, 6984–90PubMedCrossRefGoogle Scholar
  25. 25.
    Lartigue C., Vashee S., Algire M. A., Chuang R. Y., Benders G. A., Ma L., et al. (2009) Creating bacterial strains from genomes that have been cloned and engineered in yeast. Science 325, 1693–6PubMedCrossRefGoogle Scholar
  26. 26.
    Gibson D. G., Benders G. A., Axelrod K. C., Zaveri J., Algire M. A., Moodie M., et al. (2008) One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome. Proc Natl Acad Sci USA 105, 20404–9PubMedCrossRefGoogle Scholar
  27. 27.
    Gibson D. G., Glass J. I., Lartigue C., Noskov V. N., Chuang R. Y., Algire M. A., et al. (2010) Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 52–6PubMedCrossRefGoogle Scholar
  28. 28.
    DeMarini D. J., Creasy C. L., Lu Q., Mao J., Sheardown S. A., Sathe G. M., et al. (2001) Oligonucleotide-mediated, PCR-independent cloning by homologous recombination. Biotechniques 30, 520–3PubMedGoogle Scholar
  29. 29.
    Raymond C. K., Sims E. H. and Olson M. V. (2002) Linker-mediated recombinational subcloning of large DNA fragments using yeast. Genome Res 12, 190–7PubMedCrossRefGoogle Scholar
  30. 30.
    Vidal M., Brachmann R. K., Fattaey A., Harlow E. and Boeke J. D. (1996) Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions. Proc Natl Acad Sci USA 93, 10315–20PubMedCrossRefGoogle Scholar
  31. 31.
    Chevray P. M. and Nathans D. (1992) Protein interaction cloning in yeast: identification of mammalian proteins that react with the leucine zipper of Jun. Proc Natl Acad Sci USA 89, 5789–93PubMedCrossRefGoogle Scholar
  32. 32.
    Struhl K., Stinchcomb D. T., Scherer S. and Davis R. W. (1979) High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci USA 76, 1035–9PubMedCrossRefGoogle Scholar
  33. 33.
    Clarke L. and Carbon J. (1980) Isolation of a yeast centromere and construction of functional small circular chromosomes. Nature 287, 504–9PubMedCrossRefGoogle Scholar
  34. 34.
    Murray J. A. (1987) Bending the rules: the 2-mu plasmid of yeast. Mol Microbiol 1, 1–4PubMedCrossRefGoogle Scholar
  35. 35.
    Kline B. C. (1985) A review of mini-F plasmid maintenance. Plasmid 14, 1–16PubMedCrossRefGoogle Scholar
  36. 36.
    Boeke J. D., LaCroute F. and Fink G. R. (1984) A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197, 345–6PubMedCrossRefGoogle Scholar
  37. 37.
    Hutchison C. A., 3rd, Phillips S., Edgell M. H., Gillam S., Jahnke P. and Smith M. (1978) Mutagenesis at a specific position in a DNA sequence. J Biol Chem 253, 6551–60PubMedGoogle Scholar
  38. 38.
    Stemmer W. P. and Morris S. K. (1992) Enzymatic inverse PCR: a restriction site independent, single-fragment method for high-efficiency, site-directed mutagenesis. Biotechniques 13, 214–20PubMedGoogle Scholar
  39. 39.
    Kunkel T. A. (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82, 488–92PubMedCrossRefGoogle Scholar
  40. 40.
    Hemsley A., Arnheim N., Toney M. D., Cortopassi G. and Galas D. J. (1989) A simple method for site-directed mutagenesis using the polymerase chain reaction. Nucleic Acids Res 17, 6545–51PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Xiquan Liang
    • 1
  • Lansha Peng
    • 1
  • Billyana Tsvetanova
    • 1
  • Ke Li
    • 1
  • Jian-Ping Yang
    • 1
  • Tony Ho
    • 1
  • Josh Shirley
    • 1
  • Liewei Xu
    • 1
  • Jason Potter
    • 1
  • Wieslaw Kudlicki
    • 1
  • Todd Peterson
    • 1
  • Federico Katzen
    • 1
    Email author
  1. 1.Life Technologies CorporationCarlsbadUSA

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