The Construction Of Gene EfaA~+ Enterococcus Transformant And Preliminary Study Of Its Influence On Biofilm Formation

Objectives:Construct pAT28+efaA+His recon and entrococcus efaA- control strain and efaA+ transformant, pave the way for further study of efaA gene's function , do the groundwork for prevention and cure of patients from the infection of entrococcus faecalis.Methods:One:The construction and identification of entrococcus efaA- control strain and efaA+ transformant.1. Choose efaA-/gel-/cyl- entrococcus faecalis as the wild strain by PCR(Saved in our laboratory).2. Introduce pAT28 vector into wild strain as a control by calcium chloride co-precipitation method.3. Construct pAT28+efaA recombinant, transform it into the wild strain S14 as the transformant: Get the hole efaA gene from Genebank, amply efaA gene's openning reading flame (ORF) by PCR, clone it to pAT28 vector, transform it into DH5αEcoli strains by calcium chloride co-precipitation. After X-gal filter, PCR identification, double enzymes disaggregation and DNA sequencing, transform the correct recon into the wild strain as the transformant. Induce efaA gene expression with IPTG, conform by SDS-PAGE and Western blot.Two: Preliminary study of gene efaA's influence on biofilm formation. Detect the biofilm OD values of entrococcus wild strain, pAT28 control strain and efaA+ transformant (about 5×106-10×106 CFU/ml) cultivating in 96-well 3 and 24 h by visible light semi-quantitative method, inquire the influence of efaA gene on biofilm formation by comparing the biofilm formation ability of entrococcus faecalis before and after efaA genetic transformationResults: 1. we can see colonies of the control strain (Transform pAT28 into the wild train) on ELB culture plate containing spectinomycin (final concentration of 500 ug/ml); we can see white colonies of the transformant (Transform pAT28+efaA recombinant into the wild train) on IPTG/X-Gal ELB culture plate containing spectinomycin (final concentration of 500 ug/ml). Induce efaA gene to express by IPTG, analysis by SDS-PAGE and Western Blot,a belt is found near 35 kd; 2. OD595 values of the transformant's biofilm adhering on the bottom of 96-well are thicker than the wild strain's and control strain's both preliminary adherence and biofilm formation.Conclusions: 1. Successfully build entrococcus control strain and transformant; 2. efaA gene is beneficial for the enterococci's preliminary adherence and biofilm formation.