Research About Gene Regulation Line Response To Threonine

Synthetic biology is a hot spot in the current study,of which the gene line related research is an important part of synthetic biology.This paper applied biological function components in order to realize the gene line response to threonine,and to construct a series of stains response to threonine.First,using glycine degradation promoter,strain response to glycine is constructed.Second,to examine the glycine degradation promoter of threonine response.Then,use of threonine operon leader peptide region,strains response to threonine are constructed.Then,application of sRNA jamming technology,deter expression system is constructed.Finally,the research about threonine operon leader peptide region is combined with sRNA interference,and the strains response to threonine are constructed.The main results are as follows:First of all,upon the basis of the construction of strain response to glycine,it is found that the strain is also response to threonine.Therefore,the research on if gcv promoter of glycine degradation operon is also response to threonine is done.Then,using isothermal titer heat meter monitor the interaction between gcvR transcriptional repressor and threonine,and draw the conclusion: there is weak binding reaction between gcvR and threonine,the phenomenon that the strain response to glycine is also response to threonine should be due to the one step conversion of threonine to glycine.Second,upon the basis of knowing of THR operon leading regulatory mechanism,the strains response to threonine are constructed by coupling of the leading region with red fluorescent reporter gene.Additionally,by point mutation transformation of thr L,the strains of which thr L region codes four threonines and ten threonines are constructed.And results show that as the accumulation of threonine,the expression intensity of reporter gene in the three strains are all on the decline and the linear response range is roughly at 0-3.2 mM,the response concentration of different mutants is in different degrees of deviation.Then,according the design principle of sRNA,sRNA for red fluorescent gene is designed and built in plasmids pACYC184,pET28 a,p LX07 with red fluorescent gene.The three plasmids represent respectively low,medium,high copys.And results show that under the low copys expression system,sRNA with promoter Pr represses the expression of red fluorescent gene by about 50%.Under the medium and high copys expression system,sRNA with promoter Pr represses the expression of red fluorescent gene by about 75%.Finally,thr L region coding eight threonines is coupled with sRNA and built in plasmids pET28 a and pLX07,which is expected to realize the regulation of transciptional quantity of sRNA gene by different levels of threonine and then influence the expression of red fluorescent gene.Results show that under the medium copys expression system,the strain can response for threonine at 0-10 mM while response for threonine at 10-80 mM under the high copys expression system.However,the expression of red fluorescent gene in the two expression systems are both negatively related the concentration of threonine,which is not consistent with expectation.In spite of this,to establish a controllable regulation strategy of gene expression is still worth continuing to explore.This new idea can offer some quick and convenient method for transformation of metabolic engineering.