Creation of Golden Gate constructs for gene doctoring
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RESEARCH ARTICLE
Open Access
Creation of Golden Gate constructs for gene doctoring Nicholas M. Thomson1, Chuanzhen Zhang1,2,3, Eleftheria Trampari1 and Mark J. Pallen1,4,5*
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. Keywords: Gene doctoring, Recombineering, Golden Gate assembly, Mutagenesis, Enterobacteria, Chromosome, Insertion, Deletion
Background Over 10 years ago, Lee et al. [1] developed the gene doctoring approach for efficient mutagenesis of Escherichia coli, targeting the λ-Red recombination system [2] to a mutation cassette flanked by homologous regions and released from a suicide donor plasmid by the I-SceI meganuclease. Delivery of the mutagenesis cassette from a plasmid rather than as a linear DNA fragment protects the DNA from attack by host nucleases and leads to higher-efficiency recombination. Since then, the * Correspondence: [email protected] 1 Quadram Institute Bioscience, Norwich Research Park, Norwich, Norfolk NR4 7UQ, UK 4 School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, Norfolk NR4 7TU, UK Full list of author information is available at the end of the article
gene doctoring technique has been refined [3] and used in other bacterial species, including Salmonella enterica [4], Pseudomonas putida [5], and Klebsiella pneumoniae [6]. However, construction of donor plasmids suitable for gene doctoring typically relies on multiple, sequential steps. Golden Gate assembly [7, 8] provides a method for one-step combinatorial DNA assembly which, we hoped, could simplify construction of mutagenesis cassettes for gene doctoring. We therefore set about creating a plasmid into which multipl
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