Construction Of CRISPR/Cas9 Gene Editing Vector And Study On Electrotransformation Conditions Of Lactobacillus Paraplantarum

Lactobacillus paraplantarum is a kind of lactic acid bacteria with many probiotic characteristics.With the development of gene editing technology,people are not limited to the basic physiological properties of Lactobacillus paraplantarum.The research on its genetic manipulation is a hot topic in current research.Studies have shown that CRISPR/Cas technology can be used for gene editing of lactic acid bacteria.Due to the complex and dense cell wall structure of Lactobacillus paraplantarum,the transfer of foreign plasmids becomes difficult,limiting the study of their genetic manipulation.Therefore,this study will start from the construction of CRISPR/Cas gene editing vector and explore the transformation conditions.Based on plasmids pLCNICK and pLP-gba,the promoter fragment of Cas9 and the terminator fragment of erythromycin resistance gene were amplified from plasmid pLCNICK,and the CDS region fragment of LP404142 fusion gene was amplified from plasmid pLP-gba,were cloned into linear pUC19 vector by Easy Geno rapid recombination cloning method,and named as plasmid A.Then,the long fragment containing kanamycin resistance gene-gRNA gene and the erythromycin resistance gene fragment containing promoter and the terminator were amplified from plasmid pLCNICK,were cloned into linear pUC19 vector by Easy Geno rapid recombination cloning method,and named as plasmid B.Finally,the promoter-LP404142-terminator fragment of plasmid A,kanamycin-gRNA-erythromycin gene fragment of plasmid B and cas9 gene fragment of plasmid pLCNICK,were fused into a plasmid and named plasmid C.Plasmid C was identified by double enzymes digestion and sequencing,and could be used for gene editing of Lactobacillus paraplantarum.In this study,the plasmid pMG36 e was used to explore the electrotransformation conditions of Lactobacillus paraplantarum.The results of single factor experiment and orthogonal experiment showed that the suitable conditions for preparing competent cells were that Lactobacillus paraplantarum were cultured in MRS medium containing 2% glycine(and containing 0.3 mol/L sucrose)for 6 hours.The suitable conditions for electrotransformation were as follows: added 15 ?g plasmid pMG36 e to competent cells then pulsed under specificconditions(voltage 1.6 kV,capacitance 25 ?F and resistance 400 ?),then cultured in the recovery medium for 2 hours.The transformation efficiency was 145.50 CFU/?g DNA under the optimal conditions.The plasmid C constructed by us will provide convenience for the study of gene function of Lactobacillus paraplantarum,and our transformation method can also provide reference for the transformation of other Gram-positive bacteria.