L.paracasei Hd1.7 Prcr Mutagenesis And Pmg36e On Bacteria Transformation

Lactobacillus paracasei HD1.7 was isolated from Chinese sauerkraut juice in 2003. It had been proved that the strain can produce non-acid antimicrobial substance. It was a kind of peptide which had broad spectrum of inhibition. At the same time, it was found that the process of this peptide production showed the feature of quorum-sensing ,just like the others in Gram-positive bacteria. Jiro Nakayama et al.(2003)got a series of genes (prcA,prcK,prcR and prcB) which was defined the putitive quorum-sensing components. They predicted that quorum-sensing system was related with the production of antibacterial peptide, and regulated by PrcR. Just like that, the similar condition was supposed in L.paracasei HD1.7 here. A lot of data in this study would supported the idea and this work will be very significant because the data in this field was few.First of all, according to the data of NCBI, the presence of prcR in L.paracasei HD1.7 was examined by PCR, using the primers designed by ourselves. Then, the information of prcR was revealed and forecasted by bioinformatics method.Secondly, the suicide plasmid, pUC18-prcR-tet, was constructioned by inserting a tetracycline-resistant gene into prcR, whose single site was breakaged by enzymatic digestion of MunI. It was the preparation to getting the mutant strain of prcR deletion by gene knock-out technique.Electrotransformation technique was introducted because it had high effect of transformation and suited all kinds of bacteria. Meanwhile, this technology was influenced by many factors, such as the method of competent cell preparation. So the optimal conditions were researched here. When the recipient cell was treated by 1% of glycine and washed by electrotransformation buffer of AEB1, as well as the 9 kV/cm of field strength and one time of pulse with the A600 value of 0.78, the best result could been got.Eventually, the pUC18-prcR-tet plasmid was imported to the L.paracasei HD1.7. And the mutant strain wad screened by cultureing with tetracycline. PCR would be done to exmain the result.