Development of a “Turn off–on” whole‐cell biosensor for sulforaphane detection based on the ultrasensitive activator HrpRS

Sulforaphane (SFN), a defense secondary metabolite, can be used to predict the health status of plants and also has pharmacological effects, including anticancer, antioxidant, and anti-inflammatory properties. The detection of SFN is therefore of great significance for the prevention and treatment of diseases. In this study, a "turn off" whole-cell biosensor that can rapidly and robustly respond to the presence of SFN was constructed based on the orthogonal genetic components (hrpR, hrpS, and P-hrpL) of Pseudomonas syringae (PS). The final optimized biosensor, p114(30R-30S), was able to inhibit 91.7% of the fluorescence intensity in the presence of 100-mu M SFN. Subsequently, a HrpRS-regulated OFF-ON genetic switch was designed by reconstituting a reverse sigma(70) promoter on the sigma(54). P-hrpL promoter sequence; this was coupled with dual-color reporter genes to construct a "turn off-on" whole-cell SFN biosensor. The P-hrpLB variant increased the expression of green fluorescence a factor of 11.9 and reduced the expression of red fluorescence by 85.8% compared with the system in the absence of SFN. Thus, a robust switching of signal output from "turn off" to "turn on" was realized. In addition, the biosensor showed good linearity in the SFN concentration ranges of 0.1-10 mu M (R-2 = 0.99429) and 10-100 mu M (R-2 = 0.99465) and a detection limit of similar to 0.1 mu M.