Reforming Genetic Character Of Lactobacillus Lactobacillus Plantarum ZJ316 For Improving Bacteriqcin Production

(1) Quorum sensing system (QS) is the cell concentration decisive network system, which regulate many complex metabolic processes within the cell. In 1970, scientists first revealed QS system of Vibrio fischeri,and then Gram-positive bacteria’s and Gram-negative bacteria’s QS system have been described. In recent years, with the development of QS system research, people began to use the QS system to the service of humanity.(2) Lactobacillus plantarum ZJ316 has good antibacterial activity, the bacteriocin production is regulated by QS, but we found that QS system of Lactobacillus plantarum ZJ316 is sort of response regulator.So we want recombinant plnC/plnD gene into Lactobacillus bacteria ZJ316 for the study of response regulator PlnC/PlnD, and hope that through the transformation of QS system, improve its bacteriocin production, get a high yield of bacteriocin strain.(3) Extract Lactobacillus plantarum genomes, having good antibacterial effect strains,and PCR, determine the plnC, plnD gene, and obtain the target band, by enzyme cutting and connecting plasmid PNZ8048, successfully constructed recombinant plasmid plnC-PNZ8048 and plnD-PNZ8048, and electroporation of E.coli DH5.(4) Through Lactobacillus plantarum ZJ316 electroporation conditions explored, we found that the cells were cultured at 600nm of 1.1-1.2, add 10 g/L lysozyme, then cultured at 37 ℃ for 1h cells. And using glycerol/sucrose solution as the electroporation buffer processing transformation efficiency was the highest, and we successfully obtained plnC-PNZ8048-ZJ316, plnD-PNZ8048-ZJ316 gene engineering bacteria. After obtaining the gene engineering bacteria, we determine the growth curve and pH, and compared with the wild bacteria,the growth of gene engineering bacteria has no evidently distinguish. Through the determination of growth curve, we can induce gene expression of gene engineering bacteria easily.(5) Through the orthogonal experiment, the optimal induction conditions:in add 15 ng/mL of chloramphenicol in MRS liquid culture, and culture at a temperature of 30 ℃, at 600 nm of 0.4-0.5 add 2 ng/ml Nisin induced by 16 h,resultly we found in the 27K Da near the band. Through further antibacterial experiment found that, compare with the wild bacteria, the inhibitory effect of plnC-PNZ8048-ZJ316 on Staphylococcus citreus is no significant difference, but plnD-PNZ8048-ZJ316 has distinct reduced. As response regulator, PlnC, PlnD can adjust the bacteriocin production. PlnC was not on the bacteriocin produced activation was obvious, while PlnD has demonstrated its certain inhibition. Although genetic engineering, we can study function of PlnC/PlnD, but genetic engineering still has a certain defects, the expression product have the excess amino acid sequence, which may influence the objective function of proteins.(6) Although we initially identified PlnC, PlnD function, but because of the defect of gene engineering, PlnC does not appear in the anticipated results.At the same time, we also did not obtain a higher antibacterial activity Lactobacillus plantarum. The antibacterial activity of Lactobacillus plantarum 9# also has good antibacterial activity, so we hope that through Genome shuffle technology, transform Lactobacillus plantarum ZJ316.Genome shuffle technology is the biological technology,which can change the microbial properties efficiently, it has advantage that other technology have not. It can overcome interspecific sterility, the springoff can inherit the parents’excellent characters.Through genome shuffling of Lactobacillus plantarum ZJ316 and Lactobacillus plantarum 9#, we want to obtain a good antibacterial activity Lactobacillus plantarum.(7) Through Genome shuffle technology, we successfully obtain the fusion.And then through electron microscope, we found the fusion cell morphology than the wild strain has lengthened, and SDS-PAGE analysis, find the fusion protein has both two protein bands of the parents,it can prove successful shuffling of two strains.Then antibacterial experimen results, compared with two of parents, the antibacterial effect of the fusion are significantly improved.