The Development And Application Of A Combinational Signal Amplification System Based On Cascade Activation Of T7 RNA Polymerase Biosensors

Because of its simplicity,sensitivity,fastness,and accuracy,biosensors have shown broad application prospects in the fields of life science research,disease diagnosis,agricultural food safety,and environmental pollution monitoring.For example,a well-performed biosensor that can be successfully applied in monitoring environmental pollutants needs to possess high sensitivity,that is,it can quick identify a trace of pollutants to be tested and release strong signals that can be easily quantified.Under this requirement,techniques such as optimizing the components of sensors or amplifying responding signal need to be developed to compensate the insufficient sensitivity of biosensor.Therefore,here,we designed a signal amplification system that combines the cascade activation of T7 RNA polymerase and proteases to improve the sensitivity of whole-cell biosensors.We also plan to integrate this signal application system with existing pollutant detection biosensors to improve their sensitivity.The main work includes the following:(1)Analyzing and optimizing the T7 lysozyme-mediated T7 RNA polymerase inhibitory system.T7 RNAP could be effectively inhibited by a N-terminal T7 lysozyme through a flexible linker,which resulted in the repression of transcription and expression of report gene under its regulation in E.coli cells.However,a crucial problem existed in the system is the high background caused by the incomplete suppression.Using sf GFP as the report gene,we optimized the length of flexible linker between T7 lysozyme and T7 RNA polymerase,and found that compared to the previously reported linker with 28 aa,32aa-length linker enabled T7 RNA polymerase to be more efficiently inhibited by T7 lysozyme.The results showed that the background fluorescence of sf GFP decreased approximately 91%,88%,and 76% compared to the positive control after induction for 20 h,12 h,and 6 h at 18 ?,30 ?,and 37 ?,respectively.(2)Activating T7 RNA polymerase inhibitory system by human rhinovirus 3C protease.The inhibition of T7 RNA polymerase by T7 lysozyme can be sufficiently removed if the linker between these two enzymes could be effectively cleaved by a highly active and specific protease.Based on this,we placed the substrate sequences of tobacco etch virus(TEV)protease and human rhinovirus(HRV)3C protease within the flexible linker between T7 lysozyme and T7 RNA polymerase,respectively,and evaluated their effect of activating the sf GFP fluorescence in the T7 lysozyme-mediated T7 RNA polymerase inhibitory system.Our results showed that HRV 3C protease performed better than TEV protease,and at the same time,HRV3 C protease exhibited high activity under a wide temperature range.After induction for 20 h,12 h,and 4 h at 18 ?,30 ?,and 37 ? respectively,the sf GFP fluorescence recovery efficiencies reached to 29%,42%,and 43% of the total fluorescence.(3)Constructing the proteases cascade amplification system.In order to further amplify the signal,we also try to establish a protease cascade amplification system,in which the catalytic activity of HRV 3C protease(the first-stage protease)needs to be inhibited first,and then another protease(the second-stage protease)is introduced to activate HRV 3C protease.1)Inhibiting the HRV 3C protease(the first-stage protease).We anchored the Ssr A degradation tag to the C-terminal of HRV 3C protease to facilitate its degradation by Clp X/P proteasome system within E.coli cells.In this way,the T7 lysozyme-mediated T7 RNA polymerase inhibitory system cannot be activated due to the degradation of the first-stage HRV 3C protease,and the sf GFP fluorescence thus maintained to be suppressed.2)Identifying the second-stage protease in the protease cascade amplification system.We first tested the possibility of TEV protease as the second-stage protease to remove the Ssr A tag from the C-terminal of HRV 3C protease for its activation.Our preliminary experimental results showed that this proteases cascade amplification system can restore about 14% of the system fluorescence.Currently,we are testing to use HRV 3C protease mutant or foot-and-mouth disease virus(FDMV)3C protease as the second-stage protease to optimize this system.(4)Integrating with existing naphthalene biosensors.We have tested the combination of a P.putida derived naphthalene biosensor(Nah R-Psal-Reporter)with our developed signal amplification system using TEV protease as the second-stage protease.However,no effective sf GFP fluorescence recovery was obtained so far in the T7 lysozyme-mediated T7 RNA polymerase inhibitory system,probably due to the effectiveness of the naphthalene biosensor in E.coli,insufficient proteolytic activity of TEV protease,or strong degradation effect of Ssr A tag to primary HRV 3Cprotease.The evaluation will continue after a suitable secondary protease was identified.In summary,we designed a combinational signal amplification system based on the cascade activation of the T7 RNA polymerase and proteases,which could not only be combined with the existing biosensors to amplify the responding signal and increase sensitivity of biosensor,but also expected to provide valuable references for other types of in vivo reactions which need signal amplification.