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Thomson, Nicholas M., Zhang, Chuanzhen, Trampari, Eleftheria and Pallen, Mark J. 
ORCID: https://orcid.org/0000-0003-1807-3657
(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,sdg 3 - good health and well-being ,/dk/atira/pure/subjectarea/asjc/1300/1305 |
| Faculty \ School: | Faculty of Science > School of Biological Sciences |
| Related URLs: | |
| Depositing User: | LivePure Connector |
| Date Deposited: | 24 Oct 2020 00:20 |
| Last Modified: | 22 Oct 2022 07:21 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/77425 |
| DOI: | 10.1186/s12896-020-00648-5 |
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