Creation of Golden Gate constructs for gene doctoring

BMC Biotechnology

Nicholas M. Thomson¹  Eleftheria Trampari¹  Chuanzhen Zhang²  Mark J. Pallen³ 

[1] Quadram Institute Bioscience, Norwich Research Park, NR4 7UQ, Norwich, Norfolk, UK;Quadram Institute Bioscience, Norwich Research Park, NR4 7UQ, Norwich, Norfolk, UK;National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, College of Veterinary Medicine, South China Agricultural University, 510642, Guangzhou, China;Guangdong Key Laboratory for Veterinary Drug Development and Safety evaluation, College of Veterinary Medicine, South China Agricultural University, 510642, Guangzhou, China;Quadram Institute Bioscience, Norwich Research Park, NR4 7UQ, Norwich, Norfolk, UK;School of Biological Sciences, University of East Anglia, Norwich Research Park, NR4 7TU, Norwich, Norfolk, UK;School of Veterinary Medicine, University of Surrey, Daphne Jackson Road, GU2 7AL, Guildford, Surrey, UK;

关键词: Gene doctoring;    Recombineering;    Golden Gate assembly;    Mutagenesis;    Enterobacteria;    Chromosome;    Insertion;    Deletion;   

DOI  :  10.1186/s12896-020-00648-5

来源: Springer

【 摘 要 】

BackgroundGene 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.ResultsWe 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.ConclusionsOur 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.

【 授权许可】

CC BY