Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

By Carola Engler, Ramona Gruetzner, R. Kandzia, S. Marillonnet. PLoS ONE, 2009

1,021 citations recorded by the source corpus.

We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call ‘Golden Gate shuffling’, is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.

Subfields: Biotechnology, Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/91308e99646dcbb93bb1aabb6d58d037793a9d24

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Citations in the corpus

Crystal structure of Cpf1 in complex with guide RNA and target DNA (2016, methodology): “Generation of the AsCpf1 Mutants The human codon-optimized AsCpf1 mutants were cloned using the Golden Gate strategy (Engler et al., 2009).”
Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system (2012, methodology): “This cloning method, called Golden Gate cloning, is based on the use of type IIS restriction enzymes combined with restriction-ligation.(8,9) Extremely high cloning efficiency is obtained using a simple one-pot incubation of multiple undigested entry constructs and a destination vector in the presen”
Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting (2011, methodology): “We opted for Golden Gate cloning, a recently developed method of assembling multiple DNA fragments in an ordered fashion in a single reaction (20,21).”
A Modular Cloning System for Standardized Assembly of Multigene Constructs (2011, methodology): “This cloning system is based on the Golden Gate cloning technology, a method that allows highly efficient directional assembly of multiple DNA fragments in a single reaction [14].”
BglBricks: A flexible standard for biological part assembly (2010, methodology): “More recently, there have been reports of a single-reaction DNA shuffling method based on type IIs restriction enzymes, known as Golden Gate Shuffling [28], as well as a single-reaction isothermal method for the assembly of multiple overlapping DNA molecules [29].”
A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering (2012, methodology): “We have further improved the TALE assembly system with a few optimizations, including maximizing the dissimilarity of ligation adaptors to minimize misligations and combining separate digest and ligation steps into single Golden Gat”
Genome Engineering of Crops with Designer Nucleases (2012, methodology): “…TALENs or dTALEs. h ey include Golden Gate assembly methods that allow seamless construction of TALE repeat arrays (Cermak et al., 2011; Engler et al., 2009; Sanjana et al., 2012; Zhang et al., 2011) as well as ligation-based systems (Reyon et al., 2012; Sander et al., 2011a). h ese…”