Novel Detection Technology Of D-Gal And Its Related Small Molecules By Using GalR To Protect DNA Sequence From Cleavage

Objective: Small molecules of metabolites in organisms can be used as important indicators for disease diagnosis and prognosis evaluation.Accurate quantification of small molecules of metabolites can provide important clues for doctors’ diagnosis and treatment.The detection of some metabolites is limited by instruments,equipment,detection reagents and the professional quality of operators,and cannot be widely used in practice.Therefore,it is necessary to develop fast,accurate and simple methods for small molecular metabolites detection.Transcription factors(TFs)regulate gene expression by responding to the concentration of metabolites in cells.Inspired by this trait,a biosensor can be constructed using transcription factor to detect small metabolites in vitro.The purpose of this thesis is to construct a new type of biosensor based on Galactose repressor(GalR)to detect D-galactose(D-Gal),and further expand the application range of this biosensor combined with disaccharide hydrolysis to successfully detect lactose and melibiose.Finally,the detection target of this biosensor can be further extended to achieve the detection purpose of screening transcription factor inhibitors.Methods: 1.A novel biosensor for the detection of D-galactose was developed by using the restriction endonuclease(RE)combined with the GalR,which named as repressor-RE.(1)Isolation and purification of target protein GalR(2)Feasibility analysis and optimize the condition of D-Gal detect by repressor-RE(3)Characterizing the D-Gal assay by repressor-RE2.Lactase is used to hydrolysis lactose to produce galactose,and lactose is detected by combining with repressor-RE.(1)Hydrolysis of lactose to produce galactose(2)Analysis the feasibility of repressor-RE to detect D-Lac(3)Characterizing the D-Lac assay by repressor-RE3.Screening of galactose repressor inhibitors with repressor-RE.(1)Analysis the feasibility of repressor-RE to screen inhibitors of galactose repressor(2)Characterizing the galactose repressor inhibitors screening assay by repressor-REResults: repressor-RE biosensor was successfully constructed in vitro,and the conditions of nucleic acid concentration,GalR concentration,restriction endonuclease concentration and enzyme digestion time in the reaction system were optimized.Under the optimal conditions,the minimum limit of detection for D-Gal is 4.82 μM.And this method can effectively distinguish galactose from other similar monosaccharides.When using this method to detect galactose in actual samples,the detection recovery can reach 95.2-103.4% under the premise that the concentration of galactose is 0.1 m M.When combined with lactase to detect D-Lac,the minimum detection limit of this method for D-Lac can reach 11.10 μM.The biosensor can effectively distinguish lactose from other types of disaccharides.When using this method to detect the lactose content in milk,the consistency between repressor-RE and the commercially available kit can reach 95.75%.The results of screening galactose repressor inhibitors using this biosensor showed that Isopropylβ-D-thiogalactoside,D-Glucose and L-Fucose could all produce inhibitory effects,but Isopropyl β-D-thiogalactoside had the strongest inhibitory effect.Conclusion: Combining Galactose repressor with restriction endonuclease,we have constructed a new biosensor effectively.It can detect small metabolites such as D-Gal,D-Lac and inhibitors of GalR in biological samples.