Abstract
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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Roberts, R. J. (2005) How restriction enzymes became the workhorses of molecular biology. Proc. Natl. Acad. Sci. USA 102, 5905–5908.
Katzen, F. (2007) Gateway® recombinational cloning: a biological operating system. Expert Opin. Drug Discov. 2, 571–589.
Engler, C., Kandzia, R., and Marillonnet, S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One 3, e347.
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.
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.
Szybalski, W., Kim, S. C., Hasan, N., and Podhajska, A. J. (1991) Class-IIS restriction enzymes – a review. Gene 100, 13–26.
Berlin, Y. A. (1999) DNA splicing by directed ligation (SDL). Curr. Issues Mol. Biol. 1, 21–30.
Lu, Q. (2005) Seamless cloning and gene fusion. Trends Biotechnol. 23, 199–207.
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.
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.
Liu, Z. G., and Schwartz, L. M. (1992) An efficient method for blunt-end ligation of PCR products. Biotechniques 12, 28–30.
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.
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.
Acknowledgments
The authors would like to thank Dr. Stefan Werner for critical reading of this manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Engler, C., Marillonnet, S. (2011). Generation of Families of Construct Variants Using Golden Gate Shuffling. In: Lu, C., Browse, J., Wallis, J. (eds) cDNA Libraries. Methods in Molecular Biology, vol 729. Humana Press. https://doi.org/10.1007/978-1-61779-065-2_11
Download citation
DOI: https://doi.org/10.1007/978-1-61779-065-2_11
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-064-5
Online ISBN: 978-1-61779-065-2
eBook Packages: Springer Protocols