cDNA Libraries pp 167-181 | Cite as

Generation of Families of Construct Variants Using Golden Gate Shuffling

  • Carola Engler
  • Sylvestre MarillonnetEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 729)


Current standard cloning methods based on the use of restriction enzymes and ligase are very versatile, but are not well suited for high-throughput cloning projects or for assembly of many DNA fragments from several parental plasmids in a single step. We have previously reported the development of an efficient cloning method based on the use of type IIs restriction enzymes and restriction–ligation. Such method allows seamless assembly of multiple fragments from several parental plasmids with high efficiency, and also allows performing DNA shuffling if fragments prepared from several homologous genes are assembled together in a single restriction–ligation. Such protocol, called Golden Gate shuffling, requires performing the following steps: (1) sequences from several homologous genes are aligned, and recombination sites defined on conserved sequences; (2) modules defined by the position of these recombination sites are amplified by PCR with primers designed to equip them with flanking BsaI sites; (3) the amplified fragments are cloned as intermediate constructs and sequenced; and (4) finally, the intermediate modules are assembled together in a compatible recipient vector in a one-pot restriction–ligation. Depending on the needs of the user, and because of the high cloning efficiency, the resulting constructs can either be screened and analyzed individually, or, if required in larger numbers, directly used in functional screens to detect improved protein variants.

Key words

DNA shuffling High-throughput cloning Restriction–ligation Type IIs restriction enzymes Seamless cloning Modular cloning Directed evolution 



The authors would like to thank Dr. Stefan Werner for critical reading of this manuscript.


  1. 1.
    Roberts, R. J. (2005) How restriction enzymes became the workhorses of molecular biology. Proc. Natl. Acad. Sci. USA 102, 5905–5908.PubMedCrossRefGoogle Scholar
  2. 2.
    Katzen, F. (2007) Gateway® recombinational cloning: a biological operating system. Expert Opin. Drug Discov. 2, 571–589.CrossRefGoogle Scholar
  3. 3.
    Engler, C., Kandzia, R., and Marillonnet, S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One 3, e347.CrossRefGoogle Scholar
  4. 4.
    Engler, C., Gruetzner, R., Kandzia, R., and Marillonnet, S. (2009) Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4, e5553.PubMedCrossRefGoogle Scholar
  5. 5.
    Lebedenko, E. N., Birikh, K. R., Plutalov, O. V., and Berlin, Y. A. (1991) Method of artificial DNA splicing by directed ligation (SDL). Nucleic Acids Res. 19, 6757–6761.PubMedCrossRefGoogle Scholar
  6. 6.
    Szybalski, W., Kim, S. C., Hasan, N., and Podhajska, A. J. (1991) Class-IIS restriction enzymes – a review. Gene 100, 13–26.PubMedCrossRefGoogle Scholar
  7. 7.
    Berlin, Y. A. (1999) DNA splicing by directed ligation (SDL). Curr. Issues Mol. Biol. 1, 21–30.PubMedGoogle Scholar
  8. 8.
    Lu, Q. (2005) Seamless cloning and gene fusion. Trends Biotechnol. 23, 199–207.PubMedCrossRefGoogle Scholar
  9. 9.
    Horton, R. M., Ho, S. N., Pullen, J. K., Hunt, H. D., Cai, Z., and Pease, L. R. (1990) Gene splicing by overlap extension. Biotechniques 8, 528–535.PubMedGoogle Scholar
  10. 10.
    Bolchi, A., Ottonello, S., and Petrucco, S. (2005) A general one-step method for the cloning of PCR products. Biotechnol. Appl. Biochem. 42, 205–209.PubMedCrossRefGoogle Scholar
  11. 11.
    Liu, Z. G., and Schwartz, L. M. (1992) An efficient method for blunt-end ligation of PCR products. Biotechniques 12, 28–30.PubMedGoogle Scholar
  12. 12.
    Kotera, I., and Nagai, T. (2008) A high-throughput and single-tube recombination of crude PCR products using a DNA polymerase inhibitor and type IIS restriction enzyme. J. Biotechnol. 137, 1–7.PubMedCrossRefGoogle Scholar
  13. 13.
    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, 214–220.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Icon Genetics GmbHHalleGermany

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