The Construction And Optimization Of Arsenic Whole-cell Biosensor

Arsenic(As)is a toxic substance that poses a threat to human health and the environment.Therefore,a quick and easy method is needed to monitor the concentration of arsenic in drinking water sensitively and specifically.In this context,based on the arsenic binding protein Ars R and its specific promoter P_ars,a variety of gene circuit of multi-assembled arsenic biosensor was designed,and the detection limit,sensitivity and specificity of the biosensor were optimised by coupling a positive feedback amplification system.Eight biosensor chassis cells were constructed based on designed gene circuit of the arsenic whole-cell biosensor.The fluorescence detection results showed that only the fluorescence signals expressed by biosensors AM and AE formed a gradient relationship with the concentration of arsenic ions,and the background value of biosensor AM was lower and the sensitivity was higher than that of AE.So the fluorescent gene mcherry is selected as the reporting element.The arsenic biosensor AL chassis cells were obtained based on the constructed AM gene circuit coupled positive feedback amplifying circuit.When using the As(III)concentration of semi-saturated arsenite,the sensitivity of AL with positive feedback system is 20 times higher than that of AM without positive feedback system,and the minimum detection limit is reduced by 5 times to 0.1?M,lower than the World Health Organization standard for the arsenic level in drinking water.Due to the amplification of the output signal,the positive feedback biosensor AL can provide a detectable signal in a shorter time,and the cell culture time of the chassis can be shortened by 3 to 4hours under the same fluorescence signal intensity.Specific experiments showed that AL showed good specificity to As(III)at the lowest detection limit,and the coupling of the positive feedback circuit reduced the metal ion concentration that can be specifically detected by the biosensor by 10 times and specific signal ratio increased by 1.5 to 7.5times.In addition,in the coexistence of As(III)and Cd(II),the positive feedback biosensor AL and the cadmium biosensor CM can be used to detect As(III)of 0.1?100?M and Cd(II)of 1?100?M.Collectively,the designed positive feedback amplifier biosensor meets the requirements for As(III)detection with high sensitivity and specificity,and has good application value.This work also demonstrates the importance of genetic circuit engineering in designing whole-cell biosensors and the genetic positive feedback amplifier should be taken into account when developing whole-cell biosensors to detect other inducers.