![]() ![]() Significant features of pDOC-K are displayed. 1) Three DNA fragments (brown arrows) were designed, based on pDOC-K, to form the backbone of pDOC-GG. The first three stages describe the production of pDOC-GG. We reduced the size of the plasmid backbone, to help avoid problems with transformation and stability of a large plasmid prior to mutagenesis.Ĭonstruction of pDOC-GG and its use to produce Gene Doctoring donor plasmids and perform chromosomal modifications. We removed both multiple cloning sites so that only a minimal 34 bp FRT site will remain in the chromosome following removal of a resistance marker. This prevents interference from off-target cleavage of the plasmid by BsaI during cloning. We removed a BsaI site from the pDOC-K backbone, between the HR2 cloning region and the rrnB T1 terminator. Following cloning and transformation, successful colonies can be identified by blue-white screening based upon loss of the drop-out cassette. We replaced the kanamycin resistance cassette of pDOC-K with a lacZα drop-out cassette flanked by BsaI restriction sites to create a Golden Gate cloning region. To improve compatibility with existing Golden Gate libraries, we changed the antibiotic resistance marker on the donor plasmid from bla (ampicillin resistance) to aph (3′)-Ia (kanamycin resistance). Our plasmid, pDOC-GG, was based on pDOC-K from the original Gene Doctoring toolkit, but incorporated five changes to streamline the workflow from cloning to mutagenesis: We therefore set about creating a plasmid into which multiple genetic elements could be inserted via a single Golden Gate reaction to produce a Gene Doctoring donor plasmid. Golden Gate assembly provides a method for one-step combinatorial DNA assembly which, we hoped, could simplify construction of mutagenesis cassettes for gene doctoring. ![]() ![]() However, construction of donor plasmids suitable for gene doctoring typically relies on multiple, sequential steps. Since then, the gene doctoring technique has been refined and used in other bacterial species, including Salmonella enterica, Pseudomonas putida, and Klebsiella pneumoniae. 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. developed the gene doctoring approach for efficient mutagenesis of Escherichia coli, targeting the λ-Red recombination system to a mutation cassette flanked by homologous regions and released from a suicide donor plasmid by the I-SceI meganuclease. The tools we developed are applicable to gene editing for a wide variety of purposes in Enterobacteriaceae and potentially in other diverse bacterial families. 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. We also provided related genetic parts to assist in the construction of mutagenesis cassettes with a tetracycline-selectable marker. We demonstrated proof of principle by inserting a gene for green fluorescent protein into the chromosome of Escherichia coli. Successful constructs can easily be identified through blue-white screening. 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. However, generation of donor plasmids typically requires multiple cloning and screening steps. The use of a suicide donor plasmid makes Gene Doctoring more efficient than other recombineering technologies. 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. ![]()
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