Creation of Golden Gate constructs for gene doctoring

Thomson, Nicholas M., Zhang, Chuanzhen, Trampari, Eleftheria and Pallen, Mark J. (2020) Creation of Golden Gate constructs for gene doctoring. BMC Biotechnology, 20 (1). ISSN 1472-6750

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Abstract

Background: Gene doctoring is an efficient recombination-based genetic engineering approach to mutagenesis of the bacterial chromosome that combines the λ-Red recombination system with a suicide donor plasmid that is cleaved in vivo to generate linear DNA fragments suitable for recombination. The use of a suicide donor plasmid makes Gene Doctoring more efficient than other recombineering technologies. However, generation of donor plasmids typically requires multiple cloning and screening steps. Results: We constructed a simplified acceptor plasmid, called pDOC-GG, for the assembly of multiple DNA fragments precisely and simultaneously to form a donor plasmid using Golden Gate assembly. Successful constructs can easily be identified through blue-white screening. We demonstrated proof of principle by inserting a gene for green fluorescent protein into the chromosome of Escherichia coli. We also provided related genetic parts to assist in the construction of mutagenesis cassettes with a tetracycline-selectable marker. Conclusions: Our plasmid greatly simplifies the construction of Gene Doctoring donor plasmids and allows for the assembly of complex, multi-part insertion or deletion cassettes with a free choice of target sites and selection markers. The tools we developed are applicable to gene editing for a wide variety of purposes in Enterobacteriaceae and potentially in other diverse bacterial families.

Item Type: Article
Uncontrolled Keywords: chromosome,deletion,enterobacteria,gene doctoring,golden gate assembly,insertion,mutagenesis,recombineering,biotechnology ,/dk/atira/pure/subjectarea/asjc/1300/1305
Faculty \ School: Faculty of Science > School of Biological Sciences
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Depositing User: LivePure Connector
Date Deposited: 24 Oct 2020 00:20
Last Modified: 17 Nov 2020 01:08
URI: https://ueaeprints.uea.ac.uk/id/eprint/77425
DOI: 10.1186/s12896-020-00648-5

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