Construction And Directed Modification Of Glycine Riboswitch Sensor Of Clostridium Pasteurianum

Riboswitch is an RNA functional element located in the untranslated region at the 5'end of a gene.It can specifically bind metabolites without the participation of other proteins or transcription factors,thereby achieving efficient and dynamic regulation of gene expression.However,the range of natural riboswitch response substrate concentrations is often limited,and the response is normally one-directional,which greatly limits the wide application of riboswitches as biosensor in metabolic engineering and synthetic biology.Owing to their special properties of riboswitches,they have great scientific significance and practical application for dynamic regulation of metabolic pathways,especially synthesis pathways.Glycine riboswitch is the only activation(ON)riboswitch among more than 20 natural riboswitches currently known.It is located in the upstream non-coding region of the gcvTHP operon that encodes gens for the glycine cleavage enzyme system,and contains two concatenated fitness regions that bind glycine.Clostridium pasteurianum has a high value for industrial applications.It is also of scientific interests for metabolic engineering and synthetic biology.The purpose of this study was to develop glycine riboswitches derived from C.pasteurianum and to determine the glycine concentration range that regulates gene expression.Furthermore,directional modifications of the glycine riboswitch of C.pasteurianum was carried out,and the mechanism of the dynamic regulation of gene expression by the riboswitch was elucidated.Specifically,Escherichia coli W3110 was employed as a host to construct a fusion vector of glycine riboswitch and galactosidase gene(lacZ)gene and red fluorescent protein gene(mRFP),respectively.By measuring the enzyme activity and fluorescence intensity of galactosidase,the effective concentration of the native glycine riboswitch in response to glycine was determined to range from 10 mM to 60 mM.Subsequently,it was determined that aptamer-2 is more important than aptamer-1 in regulating gene expression on account of the red fluorescent protein reporter vector.On this basis,a bidirectional screening platform for riboswitch was established to further transform the natural ON riboswitch into a repressed OFF riboswitch.A series of glycine riboswitches that can respond to different concentrations of glycerin was obtained.Overall,this thesis obtained a number of glycine riboswitches as useful tools for metabolic engineering and synthetic biology of pathways related to glycine metabolism.