Construction Of Two Novel Metabolic Biosensors Based On Transcription Factors And Their Applications In The Detection Of Galactose And Tryptophan

Objective: Metabolites are small molecular substances produced in the complex metabolic process of organisms.The detection of metabolites in organisms is helpful to reflect the pathophysiological state of organisms.The current methods for the detection of small molecules of metabolites either require expensive instruments and equipment,professional operators,or have characteristics,such as specificity,which faces the challenge of transforming from laboratory to bedside testing.Therefore,it is important to develop new methods for the detection of metabolites with sensitivity,speed,high specificity and low cost.It is reported that one-third of the transcription factors(TFs)in Escherichia coli organisms can respond to metabolites and regulate gene expression.The selectivity and affinity of these naturally evolved recognition proteins in response to metabolites have become ideal recognition elements for developing metabolite detection biosensors.The objective of this study is to use two different types of repressive TF,to develop new metabolite detection biosensors.These biosensors can be applied to perform Point-of-care testing(POCT).We selected two typical transcriptional repressors,galactose repressor(GalR),tryptophan repressor(TrpR),to test the feasibility of novel biosensors for detecting metabolites.Methods: 1.GalR can bind to specific sequence upstream of the promoter to suppress transcription,while D-Gal can relieve this inhibition.We combine the "switching function" of repressor with RPA technology to develop a new biosensor technique that can detect D-Gal,named as repressor-RPA.(1)Constructing recombinant GalR in vitro(2)Constructing recombinant vector containing a GalR recognition sequence(3)Constructing and optimizing the repressor-RPA system(4)Characterizing the D-Gal assay by using the repressor-RPA system2.L-Trp can bind TrpR and enhance its binding affinity to recognition sequence and they form a co-repressor to suppress transcription.We combined the repressor with MB,initially propose a TF-splinting strategy.Based on this,we develop a new biosensorTFSD(transcription factors splinting duplex)to detect L-Trp.(1)Expression and purification of recombinant TrpR in vitro(2)Construction of TFSD biosensor for detecting L-Trp(3)Optimization of TFSD system(4)Characterization of L-Trp assay by TFSD system Results: We realized the detection of D-Gal,and proved the feasibility of biosensor to detect metabolites by using repressor-RPA.In the experiment,recombinant GalR was expressed in vitro and an RPA amplification template was constructed successfully.The method was verified by agarose gel electrophoresis.Then the template concentrations and GalR concentrations were optimized.Under the optimal conditions,the method was characterized.The sensitivity for D-Gal detection of the biosensor was 3.36 ?M,and it can specifically distinguish D-Gal from other types of monosaccharides.We realized the detection of L-Trp,and proved the biosensor based on repressor combined with TF-splinting strategy to detect metabolites successfully.The first step in this study was to acquire the recombinant protein-TrpR in vitro.Next,we verified the feasibility of the method by MB fluorescence spectroscopy,and carried out a series of optimization of the system.Under the optimal conditions,the method was characterized.The detection limit for L-Trp detection of the biosensor was 0.79 ?M,and it can specifically distinguish L-Trp from other types of amino acids and tryptophan analogs.Conclusion: We have successfully constructed two types of new biosensors.They can make fast detection of small metabolites such as D-Gal and L-Trp in biological samples.