Produce INA Protein On A Large Scale And Expand INA Gene By PCR

Because of the foreground of the ice nucleation active bacteria(INA bacteria), amass of ice nucleation active protein and the ice nucleation gene had been demanded,They were displayed favorable economic trend and social benefit.The main methods:(1)The zymotechnics had been used in shaking experiment,5Lpot,30L pot to consummate the culture medium and improve the yield of INA bacteria.Ultrasonic and chemic extract had been used to get a mass of ice nucleation activeprotein. (2)The technique of PCR had been used primaryly to confirm reactionsystem and expanding condition.INA bacteria Xanthomonas ampelina(TS206) and Erwinia herbicola(A25)wereused in this research. The main researches focus on three fields: (1)Rotate speed wasthe key factor in gaining more biological capacity and less byproduct xanthan gumduring the experiment. The influence degree sequence to bacteria biological producein turn was rotate speed >liquid capacity>sugar consistency in culture medium. In thecondition of the adjusted medium, the viscosity of the fermentation medium had beendeclined greatly and the capability of producing xanthan gum was keeping in alow-level. Although the bacteria quantity had been dropped after the ferment, but thenumerical value of the xanthan gum quantity /bacteria quantity had been descendedrapidly and the difficulty in dealing with the fermentation medium had been reducedalso(.2)The experiment fitting for cosmically extracting ice-nucleation active proteinin Xanthomonas ampelinaTS206 was the combination of the crushing by ultrasonicand extracting by TritonX-100.After the experiment, it was known that ice-nucleationprotein could be extracted 1.2 gram from 10 gram fresh bacteria. So the produce yieldwas about 12%.(3)The experiment method for extracting the whole gene of Erwiniaherbicola in the laboratory had been established. Material composition and optimalDNA expanding condition suitable for 50μl reaction system had been established. Theexperiments was using A25's DNA as the template ,using KOD Dash enzyme as theexpanding enzyme and finally gaining 4.2kb aimed DNA segment, which wasincluded in the size range of the A25's ice-nucleation active gene confirmed by aboardreports.