Construction Of A HrpRS-based “Turn Off-on” Whole-cell Sulforaphane Biosensor

Sulforaphane（SFN）,a defence secondary metabolite with high biochemical reaction activity,is thought to be a major cause of plant insect resistance.As a breakdown product of glucosinolates,plant tissues contain basal levels of SFN,but when cells are damaged or die,the concentration of SFN increases dramatically.Therefore,the detection of changes in SFN concentration provides a reference for the growth and health of plants.In addition,SFN not only has bactericidal effect,but also possesses pharmacological activities such as anti-cancer,antioxidant,anti-inflammatory,anti-aging and so on.At present,the detection of SFN mainly relies on traditional large-scale high-end instruments,which have disadvantages such as expensive equipments and complex sample processing procedures,while the structural instability of SFN easily leads to inaccurate detection results.Therefore,there is an urgent need for low-cost,simple-to-operate,and fast-response biosensing device to specifically response SFN.Based on the coupling of the HrpRS/P_hrpL regulatory system of Gram-negative bacilli with the m RFP1reporter system,a“Turn-off”SFN whole-cell biosensor was constructed for the first time in this study.In the absence of SFN,P_hrpL activates the expression of m RFP1,while in the presence of SFN,SFN can covalently modify Cys209 of Hrp S and inhibit the activity of Hrp S,thereby repressing the expression of m RFP1.However,the“Turn-off”whole-cell biosensor has limitations such as low sensitivity and difficult visualization due to its own working mechanism.To address this problem,this study reversely form the-10and-35 regions of theσ⁷⁰ promoter by targeted mutation on theσ⁵⁴ promoter P_hrpL sequence to design a gene switch（OFF-ON type）with dual-direction signal output.Coupled it with a dual-colour reporter system to successfully constructed an SFN-responsive“Turn off-on”whole-cell biosensor.when SFN is present,the red fluorescence is suppressed and the green fluorescence is enhanced.The research contents are as follows:（1）A“Turn-off”whole-cell biosensor response to SFN was successfully constructed based on the HrpRS/P_hrpL regulatory system.The fluorescence inhibition efficiency of the screened p114（30R-30S）strain reached 91.7%in the presence of 100μM SFN.（2）Through engineering transformation,the screened p115（30R-32S）strain had a 3-folds increase in fluorescence inhibition efficiency in the presence of 10μM SFN,and exhibited good dose-response in the range of 0.1-7.38μM（R²=0.99449）and 7.38-100μM（R²=0.90412）with a detection line of ca.0.1μM and a specific response to SFN.（3）A logic toggle genetic switch that regulates signal output switching by HrpRS was designed by targeted mutation of theσ⁵⁴-type promoter P_hrpL,and the feasibility of designing a gene switch based on promoter remodeling was verified.（4）The P_hrpLB strain exhibited a 11.9-fold increased in GFP and a 85.8%reduced in m RFP1 compared with the system without analyte,realizing the switching of signal output from“Turn-off”to“Turn-on”switch.And the strain showed a good dose-response,linearity and rapid response time.The limit of detection was ca.0.1μM.The above results show that a robust and highly sensitive SFN whole-cell biosensor can be successfully constructed based on the ultrasensitive activator HrpRS,and the gene switch designed on this basis can be modularly applied to other sensing platforms or anabolic dynamic regulatory networks by controlling the presence or absence of HrpRS to promote the development of synthetic biology.