| H2S and sulfane sulfur are widely present in nature and in living cells.Sulfane sulfur refers to the different forms of zero-valent sulfur inside cells,including polysulfide and persulfide.The understanding and application of sulfane sulfur have intensified its investigatuon.Along with the widespread application of sulfane sulfur,there are negative impacts on ecological environment,agricultural production and human health due to overproduction and massive emission of zero-valent sulfur and its by-products H2S.Therefore,reliable detection of sulfane sulfur and H2S in the environment by convenient and fast detection methods is necessary.However,currently available detection methods usually require toxic organic chemical reagents,expensive detection equipment or materials such as fluorescent probes with complex synthesis processes,which are not very conducive to inexpensive and efficient application in detection environments.Therefore,the use of ecofriendly,inexpensive and simple whole-cell biosensors as a novel method for sulfane sulfur detection will have good application and economic values.In this thesis,we drew on the recently reported natural sulfane sulfur-sensing mechanism in bacteria to initially construct nine biosensor genetic circuits by using the sulfane sulfursensing transcriptional repressors and their controlled natural promoters from nine bacteria as the signal sensing module elements and regulation module elements,respectively,and the reporter protein mKate is the signal output module.Then,the potential dynamic range of the whole-cell biosensor and its performance in response to sulfane sulfur were tested by using E.coli BL21(DE3)as the host cell.Subsequently,through the optimization of plasmid copy numbers and ribosome binding sites,we identified the final element combination of AtBigRbased dual plasmid gene circuit and named it AtBigR-0.01.AtBigR is the BigR repressor from Agrobacterium tumefaciens.After testing,the E.coli AtBigR-0.01 whole-cell biosensor has a very low basal leakage level and high signal output capability with a dynamic range of 300～400-fold.In addition,the sensor is very sensitive to sulfane sulfur’s induction with detection limits as low as 5 μM elemental sulfur and a detection range of 5～400 μM.Further,its response is specific to sulfane sulfur.Thus,it’s a whole-cell biosensor with excellent signal sensing capability.We investigated the application potential of the AtBigR-0.01 gene circuit for four different application purposes.First,AtBigR-0.01 can adapt to different E.coli strains,such as BW25113,Mach1-T1,MG1655 and Nissle 1917 with excellent performance of low basallevel leakage,but the drawback is that the maximum Huorescence intensity of the sensor is reduced,which lows the dynamic range to about 100-fold.The results suggest that AtBigR0.01 has broad application potentials in protein engineering,genetic engineering,biomanufacturing detection and medical diagnostic monitoring.Second,by comparing the induction effect of AtBigR-0.01 with araC-PBAD,LacI-Plac,lacI-Ptrc that commonly used bacterial inducible expression systems in E.coli MC11655 and E.coli BL21(DE3).we found that AtBigR-0.01 and araC-PBAD have similar protein expression efficiency and strict regulatory ability,indicating that AtBigR-0.01 could also be a novel and rigorous inducible expression system.Third,the performance of AtBigR-0.01 in M9 basal medium was examined to determine its potential application for the production detection of industrial products related to sulfane sulfur,and the results showed that the maximum fluorescence expression level of the biosensor was reduced,but still maintained its low detection limit and high sensitivity.Fourth,we constructed a three-plasmid system "AtBigR-0.01-CpSQR"that responds to H2S using sulfide:quinone oxidoreductase(SQR)as the signal transformation element.In E.coli,the system maintained the detection limit of 5 μM and very low basal leakage of the original system and kept a high dynamic range.But the shortcoming is that the system is susceptible to being interfered with by sulfane sulfur if it is present when detecting H2S.As a result,we concluded that AtBigR-0.01-CpSQR has potential as a whole-cell sensor for H2S detection,but further system simplification and performance optimization are needed to improve its performance in terms of specificity and dynamic range.In summary,the research work in this thesis reprogrammed and redesigned the genetic circuit based on the natural regulatory mechanism of sensing sulfane sulfur present in bacteria,and optimized its performance through strategies,such as promoter engineering and protein expression level adjustments.For the first time,we have developed a sulfane sulfur-specific whole-cell biosensor with low basal leakage,high dynamic range and high sensitivity,and explored its possible potential applications in protein engineering,genetic engineering,medical diagnostics,industrial production and environmental monitoring.Furthermore,we constructed the a H2S whole-cell biosensor based on the sulfane sulfur whole-cell biosensor.Our research provides a new theoretical basis and research tools for the study of whole-cell biosensors and sulfane sulfur metabolism. |
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