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Tytuł:
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Creation of Golden Gate constructs for gene doctoring.
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Autorzy:
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Thomson NM; Quadram Institute Bioscience, Norwich Research Park, Norwich, Norfolk, NR4 7UQ, UK.
Zhang C; Quadram Institute Bioscience, Norwich Research Park, Norwich, Norfolk, NR4 7UQ, UK.; National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China.; Guangdong Key Laboratory for Veterinary Drug Development and Safety evaluation, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China.
Trampari E; Quadram Institute Bioscience, Norwich Research Park, Norwich, Norfolk, NR4 7UQ, UK.
Pallen MJ; Quadram Institute Bioscience, Norwich Research Park, Norwich, Norfolk, NR4 7UQ, UK. .; School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, Norfolk, NR4 7TU, UK. .; School of Veterinary Medicine, University of Surrey, Daphne Jackson Road, Guildford, Surrey, GU2 7AL, UK. .
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Źródło:
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BMC biotechnology [BMC Biotechnol] 2020 Oct 07; Vol. 20 (1), pp. 54. Date of Electronic Publication: 2020 Oct 07.
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Typ publikacji:
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Journal Article; Research Support, Non-U.S. Gov't
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Język:
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English
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Imprint Name(s):
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Original Publication: [London] : BioMed Central, 2001-
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MeSH Terms:
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Genetic Engineering*
Mutagenesis*
Bacteria ; Chromosomes ; DNA ; Enterobacteriaceae ; Escherichia coli/genetics ; Gene Deletion ; Gene Editing ; Genetic Vectors ; Mutagenesis, Insertional ; Plasmids ; Whole Genome Sequencing
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References:
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Grant Information:
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BB/R012504/1 United Kingdom BB_ Biotechnology and Biological Sciences Research Council; BBS/E/F/000PR10351 United Kingdom BB_ Biotechnology and Biological Sciences Research Council
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Contributed Indexing:
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Keywords: Chromosome; Deletion; Enterobacteria; Gene doctoring; Golden Gate assembly; Insertion; Mutagenesis; Recombineering
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Substance Nomenclature:
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9007-49-2 (DNA)
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Entry Date(s):
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Date Created: 20201008 Date Completed: 20210907 Latest Revision: 20210907
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Update Code:
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20240105
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PubMed Central ID:
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PMC7542709
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DOI:
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10.1186/s12896-020-00648-5
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PMID:
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33028286
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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.
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