Research On Construction Of Mutant Library Of SPT15 By Error-prone PCR

How to safely and effectively breed the fermentation micrograms for the fermentation quantity is a scientific problem, especially to strains of gentility black unclear. In order to get fine properties of microbes,this study researched on T yeast which added in soy sauce brewing . Firstly, the SPT15 gene mutant library was constructed, and then connected with the plasmid. Then the mutant plasmid library of YEplac195-P-T-SPT15 transformed into Uracil auxotroph strains, by screening the advantageous strains were got. At last, the goal of high efficient breeding was achieved.In this study, with the template of SPT15, the conditions of PCR reaction including the temperature, Mg2+concentration and different ratios of dATP, dTTP, dCTP and dGTP were used. The results showed electrophoresis bandings were very bright when the annealing temperature was 42°C-58?,when the concentration of Mg2+ at 2mM-2.7mM, when changing amount of dATP ,dTTP and dGTP in PCR system. A large number of error-prone PCR products were collected , which indicated the SPT15 gene mutant library was successfully constructed.In this paper, the promoter and terminator were amplified by PCR technique, and the plasmid YEplac195-P-T was constructed with the vector YEplac195. The mutant gene of SPT15 gene and YEplac195-P-T were digested with double enzymes, and the SPT15 gene mutation plasmid library was obtained through the ligation reaction. This mutant plasmid library of YEplac195-P-T-SPT15 transformed into Saccharomyces cerevisiae W303, by screening in solid uracil auxotrophs of different salt concentration salt tolerant strains was got, and was verified through the dilution experiment. The plasmids in the salt tolerant strains were extracted again and the mutation position was analyzed after sequencing of PCR products. The better salt tolerance of the mutant gene was got. Afterwards, the above plasmids were transformed into uracil deficient T yeast WM2 constructed by previous study,and were verified by dilution experiments. The engineering strains containing different plasmids were added into the soy sauce fermentation juice. The results showed that the contents of amino nitrogen in soy sauce fermented by engineering strains all were higher than that in the control group.