Detection Of Biohazardous Materials In Water Upon The Charateristics Of Fluorescent Sensor Frex

In recent years,as an effective mean of monitoring biological toxicity in water environment,luminescent bacteria get more and more attention.It has a negative correlation between toxicant concentration and luminous intensity,thus it is reasonable to judge the acute toxicity by the luminous intensity of luminescent bacteria.Strong fluorescence can be aroused due to Frex combined with NADH,so the fluorescence intensity vary because of the change of NADH level in the bacteria,thus we can use microplate reader to detect the effect on microbial metabolism,5o as to achieve the goal of real-time monitoring of toxicant.The plasmid pRSETb-Frex was transformed into E.coli BL21(DE3)pLysS with genetic engineering technology,it is proved that fluorescent protein Frex was expressed in the bacteria correctly through westerm-blot.Purified Frex can be through affinity chromatography column with the concentration of 300mM imidazole.The fluorescence intensity of Frex was studied with a certain concentration of substrate NADH,NAD+,NADP+,ADP as time goes by.At the same time,we have proved that the Frex is specific to NADH,and draw a curve of the functional relations between different concentration of NADH and fluorescence intensity of Frex as time goes by.The result indicated that Frex is sensitive to NADH,they can produce strong fiurescence after combination.We studied the effect of expression level and fluorescent activity of the fusion protein Frex by condition,such as inducing temperature,inducing time and the final concentration of inductor IPTG The recombinant fluorescent bacteria was applied to the initial detection of toxic substances in water environment.HgCl2?3.5-Dichlorophenol?Potassium dichromate?Zinc sulfate heptahydrate four international standard substances of biological toxicity test were chosed to conduct the biological toxicity test.The result suggest that most of the substances can cause a decrease of fluorescence of bacteria.This kind of method has the property of fast-response and high sensitivity respectively.Meanwhile,we optimized the condition of biological toxicity test,provided the foundation for subsequent application,expand the scope of biological toxicity test in other aspects.