cDNA Libraries pp 167-181

Part of the Methods in Molecular Biology book series (MIMB, volume 729) | Cite as

Generation of Families of Construct Variants Using Golden Gate Shuffling

Protocol

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.

Key words

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

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Icon Genetics GmbHHalleGermany

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