The Enterococcus Faecalis CRISPR/Cas System Function Exploration Study In Escherichia Coli

Enterococcus faecalis is the normal intestinal microflora of people and many other animals. However,is widespread in the majority of clinical isolates have pathogenicity islands (PAIs), and there are very large differences in healthy intestinal strains. The research shows that most of the virulence genes and the resistance genes may be carried by the mobile DNA elements, how to suppress the horizontal transfer of these mobile elements has been becoming the primary task. Clustered, regularly interspaced short palindromic repeat (CRISPR loci) are long-term produced in bacterial evolution and adaptation process, as a self-protection mechanism to against invasion of exogenous gene. Mainly using the CRISPR/Cas immune system inhibition of the exogenous DNA for the source of the bacteriophage and plasmid, and forcing the pathogenic genes and the resistance genes transfer failure, thereby protecting the integrity of the host genome.This study investigated the functional integrity of the CRISPR/cas genes in E. coli, namely the inhibitory effect of exogenous DNA. Through specific PCR amplification, found in the42clinical strains preserved in our laboratory, the E.faecalis of11.90%strains containing the complete CRISPR/cas gene. After sequencing analysis showed that this fragment total7919bp, wherein four adjacent cas genes with these others corresponding sequences homology of99.4%, and was found of the13spacers in the repeat region, and the direct repeat sequence having neck ring structure, and L sequences relative to the reference sequence of only a mutation, indicating that its relatively conservative within species. Application the recombinant plasmid pCRISPRl transformed E.coli competent cells with our fabrication, with the recombinant plasmid pET28-S1transforming recombinant bacteria E.coli cells, and found that the presence of the CRISPR/cas gene restrictions the plasmid pET28-S1entry of the bacteria, which indicates that the CRISPR/cas gene in E. coli can be suppressed the DNA transfer of the exogenous plasmid. By the phage Ph.D.-7TM infecting the recombinant E.coli (pCRISPR1-ER2738), and employ mitomycin C and uv-induced to screening may be the formation of anti-phage strains. After sequencing sequence show that the new DR-S sequence not found in the CRISPR loci of the recombinant ER2738strain.