Study On The Relationship Between Rocg Gene And Nitrogen Metabolism Of Bacillus Subtilis

The rocG gene of Bacillus subtilis encode a catabolic glutamate dehydrogenase which catalyze the chemical reaction of L-glutamate + H2O + NAD=2-ketoglutarate + NH3 + NADH.Interconversion of 2-ketoglutarate and glutamate are a major link between cardon and nitrogen metabolism in all living organism.We intend to researching the relationship between rocG gene and nitrogen metabolism in Bacillus subtilisWe construct the vectors pUC18-2 and pUC18-C which contain the Pxyl and Pspac promotor.The purpose is to replace the promoter in front of rocG gene by the inducible promoter. And we insert the Spc gene in the vector,using the spectinomycin as the resistance maker.Finally,the vectors pUC18-2 and pUC18-C are integrated into the genome of wild strains of B.subtilis 168 by the method of Spizizen, which are named 168-P and 168-E,in order to research the relationship between rocG gene and the nitrogen metabolism of the wild strain B.subtilis 168.Physiological characteristics of the transformants 168-P and 168-E are studied by the way of fermentation,including growth curve,glucose consumption rate and NH₄⁺ consumption rate. the relationship between rocG gene and nitrogen metabolism are researched under different inducer level.We measure the growth curve in M9 culture medium,xylose induction effection is not obvious due to inhibition by glucose; the effect by different concentration of IPTG induction is not obvious for rocG under low concentration of ammonia nitrogen is not a major metabolic pathway, Determined growth curve in LB medium, cell growth increases with the increasing of different xylose concentration; different IPTG inducer concentration induce significant differences,biomass also increase gradually with the increasing IPTG inducer concentration. Using M9 medium for determination the glucose consumption rate of transformants,glucose consumption rate decrease with increasing xylose concentration; For the IPTG induction, in the logarithmic growth phase, When the 2.00mMof IPTG concentration, the glucose consumption rate of consumption is the fastest,while glucose consumption rate is relatively the slowest at the concentration of 0.00mM IPTG.Using M9 medium to measure NH₄⁺ consumption rate, NH₄⁺consumption curve are not significantly different on different xylose inducer concentration; NH₄⁺consumption curve was no significant difference although with different IPTG concentration in low nitrogen conditions of M9 medium.