The Construction And Optimization Of High Sensitivity Cadmium Whole-cell Biosensor

Due to the prevalence of cadmium pollution and its serious harm,it is very important to establish an efficient and rapid detection method for cadmium ion detection.For this goal,the whole cell biosensors for cadmium detection were constructed in this study.The response time,detection limit,sensitivity and specificity of the biosensors were optimized by adding Lux R amplification module,increasing report elements copy number,adding T7 RNAP amplification module.Firstly,the whole cell biosensors of cadmium ion detection were constructed by using cadmium-responsive transcription factors and red fluorescent protein genes.30 types of sensors were designed and constructed from the aspects of the host bacteria selection,types and sources of cadmium responsive-transcription factors and circuit assembly.The sensors were screened by measuring the fluorescence response of cadmium ion concentration,and then the optimal combination of circuit and host bacteria was determined by comparing the response time,detection limit,sensitivity and specificity of the sensor: KT-5-R in Pseudomonas putida KT2440 has a response time of 8 h and a detection limit of 0.04 ?M(which has reached the detection standard for cadmium in drinking water in China,but didn't meet the WHO standard requirements).The sensitivity of KT-5-R was 309.7(slope of linear response curve)and specificity was 5.9(ratio to fluorescence response of metal ions with the strongest interference).Secondly,the detection sensitivity and detection limit of KT-5-R were optimized by coupling Lux R amplification module,increasing the report element copy number and adding T7 RNAP amplification module.Among the three methods,the sensor p2T7RNAPmut-68 coupled with T7 RNAP amplification module has the best performance,which increases the cell's tolerance to cadmium ions without significantly increasing the background fluorescence value.Compared with KT-5-R,p2T7RNAPmut-68 increased the tolerance of cells to cadmium ions,but didn't increase the background fluorescence value significantly.Compared with KT-5-R,the response time of p2T7RNAPmut-68 was shortened by 4 h and the detection limit was reduced by 4 times,which was better than the WHO standard for cadmium detection in drinking water.And the sensitivity and specificity of p2T7RNAPmut-68 were increased by 1.3 times.The detection limit of the double-copy sensor KT-5-R/double and the triple-copy sensor KT-5-R/triple also reached the WHO standard.Compared with KT-5-R,the detection limit was reduced by 2 times,and the response time was shortened by 4 h and 2 h respectively.The sensitivity of KT-5-R/double didn't change significantly,but the specificity decreased by 2.1 times.However,the sensitivity of KT-5-R/triple decreased by 3.6 times,but there was no significant change in specificity.The p2 cad Rlux R-68 with the Lux R amplification module is less effective,and the detection limit is increased by 25 times compared with KT-5-R.which didn't meet the detection standard of cadmium in drinking water in China.In general,the cadmium ion whole cell biosensors constructed in this study has high sensitivity and specificity,which meets the WHO standard for the detection of cadmium in drinking water and had good application value.This study proved the influence of circuit design,host selection and vector plasmids selection on the performance of the sensor.At the same time,it is proved that coupling T7 RNAP amplifier module and increasing the copy number of report elements were feasible to improve the performance of the biosensor.It also provided design ideas for the construction and optimization of other heavy metal ion whole cell biosensors.