Development of a "turn off-on" whole-cell biosensor for sulforaphane detection based on the ultrasensitive activator HrpRS.
Biotechnol Appl Biochem
; 70(2): 798-810, 2023 Apr.
Article
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| MEDLINE
| ID: mdl-36070874
ABSTRACT
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 PhrpL ) 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-µM SFN. Subsequently, a HrpRS-regulated OFF-ON genetic switch was designed by reconstituting a reverse σ70 promoter on the σ54 -PhrpL promoter sequence; this was coupled with dual-color reporter genes to construct a "turn off-on" whole-cell SFN biosensor. The PhrpLB 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 µM (R2 = 0.99429) and 10-100 µM (R2 = 0.99465) and a detection limit of ~0.1 µM.
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Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Proteínas Bacterianas
/
Factores de Transcripción
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Idioma:
En
Revista:
Biotechnol Appl Biochem
Asunto de la revista:
BIOQUIMICA
/
BIOTECNOLOGIA
Año:
2023
Tipo del documento:
Article
País de afiliación:
China