Study Of CRISPR In E.coli Gene Editing And GBS Elimination

In recent years,gene editing technology has been greatly developed with the development of CRISPR technology.But in practical applications,there are still many bottlenecks.On the one hand,we try to combine the CRISPR with the Red system to create an efficient,simple and fast gene editing tool for optimizing the gene editing of the Escherichia coli.On the other hand,we utilize different bacteria that can secrete and transfer membrane vesicles(MVs)and CRISPR system to complete the control of Streptococcus agalactiae.In the E.coli gene editing optimization experiment,we introduce pKD46,pCas9,pCRISPR plasmids and recombinant ssDNA or dsDNA into it.A series of experiments on point mutation and knockout of E.coli galK gene verified whether Red recombination technology combined with CRISPR system is more effective for gene editing.Then we constructed a new double plasmid system by transforming the traditional plasmid,and carried out some experiments on point mutation,knockout and insertion of galK and lacZ genes.In the prevention and control of Streptococcus agalactiae,the traditional pCas9 plasmid was transformed,and the new plasmid pCas9-2.0 can be replicated in gram positive bacteria.The asd-deficient E.coli with pCas9-2.0 plasmids was used as a vesicular donor.By co culture with GBS,it is found whether the combination of vesicles and CRISPR is useful to Streptococcus agalactiae.The results of the two experiments were all better.First of all,compared with the traditional MAGE technology,it can greatly improve the efficiency of gene editing.The efficiency of point mutation was raised to more than 90%,and the efficiency of fragment recombination was increased to more than 50%.Second,the operation is simple.Finally,for our dual plasmid system,when the gene editing is completed,we can complete the self elimination of two plasmids,avoiding the impact on subsequent experiments.In the control experiments of Lactococcus lactis,experiments show that bacteria can secrete MVs,and plasmids can enter into GBS from E.coli in the form of MVs.Second,the secretion of MVs can increased by prolonging co culture time or adding Kan antibiotics.Third,it is found that vesicle transfer can also make pCas9-cfb plasmid enter into GBS and kill it.The experimental results show that CRISPR has important use value in the gene editing of microbes and the prevention and control of pathogenic bacteria.