Construction Of New Biosensors And Their Biological Applications

Biosensors can accurately meet various biochemical analysis goals because of its excellent capabilities to transfer and transform information,it has developed rapidly in recent years and has penetrated into various fields such as people’s lives,production and scientific research.Two biosensors with different uses have been constructed in this work in order to achieve the immediate regulation of the target gene expression in E.coli and the rapid detection of Salmonella typhimurium.The content of the work is as follows:Gene expression is the core manifestation of all life activities,and gene expression regulation plays a vital role in the orderly regulation of life activities such as growth,differentiation,reproduction,and metabolism of living organisms.At the same time,the redox reaction represented by respiration plays an indispensable role in these life activities.Whether artificial intervention of redox reactions in vivo can regulate the expression of related genes to regulate life activities or diagnose and treat diseases is one of research directions in synthetic biology.In this project,E.coli MG1655 with a clear genetic background was selected as the chassis strain for engineering transformation to realize the purpose of using the redox part of the bacterial as a construction element and finally regulating the expression of the target gene of the engineering bacterium through external intervention.In view of the outstanding contribution of the soxRS regulator in the oxidative activation of E.coli,we used it as a promoter element that can respond to external redox signals,subsequently,the fluorescent protein gene phiLOV was used as the final target gene,whose expression process could be regulated by the application of redox molecules,and the expression result was determined according to the fluorescent signal output,finally,the direct regulation of the target gene expression of E.coli was initially achieved.Next,on the basis of the above results,we used the mechanism of quorum sensing between bacteria to construct a biological cascade sensor of redox reaction cascade quorum sensing,and the control signals are sequentially transmitted to the biological relay and the bio-transformer through the redox reaction,thus basically achieving indirect regulation of target gene expression.This research work to convert redox regulatory signals into biological signals will provide a basis for the wider biological behavior by controlled electronically.Salmonella,a typical foodborne pathogen,always seriously threatens the health and even life of both humans and livestock.However,highly sensitive and fast quantitative methods for its detection are still remaining to be challenged.Herein,we presented an efficient method with dual signal amplification strategies by combining immune hybridization chain reaction(HCR)with surface enhanced Raman scattering(SERS)to sensitively detect Salmonella typhimurium in food.After sample preparation,S.typhimurium were specifically captured by immunomagnetic beads(IMBs),then,the aptamer-biosensor triggered the HCR process and the subsequent SERS detection was performed,and finally a good linear relationship between the bacterial concentration ranging from 10 to 105 CFU/mL and the corresponding SERS intensity was obtained,and the detection limit(LOD)within 3.5 hours is 6 CFU/mL.The proposed method has been successfully applied to capture and detect the S.typhimurium in spiked milk samples,and the results were consistent with those of the traditional plate counting methods.The combination of these two strategies achieves double amplification of the detection signal and significantly improves the detection sensitivity of S.typhimurium,and shows good application potential for the highly sensitive detection of other contaminants in food.