Creation of Golden Gate constructs for gene doctoring

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

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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,sdg 3 - good health and well-being ,/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: 22 Oct 2022 07:21
DOI: 10.1186/s12896-020-00648-5

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