FGE‐sulfatase‐based bacterial biosensor with single copy evolved sensing cassette for arsenic detection

BACKGROUND Arsenic (As) is classified as a Group 1 human carcinogen, and the maximum arsenic contamination level allowed in drinking water is10 μg L−1. In this study, a genetic‐based whole‐cell biosensor for arsenic detection was constructed and engineered for rapid and more efficient arsenic detect...

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Published in:Journal of chemical technology and biotechnology (1986) 2020-04, Vol.95 (4), p.1173-1179
Main Authors: Sangkaew, Watsawan, Sallabhan, Ratiboot, Ritcharoon, Benjarat, Mongkolsuk, Skorn, Loprasert, Suvit
Format: Article
Language:English
Subjects:
Arsenic
Biosensors
Carcinogens
Chromosomes
Contamination
Cysteine
directed evolution
Disruption
Drinking water
E coli
Efflux
error‐prone PCR
Fluorescence
formylglycine generating enzyme
Performance enhancement
Polymerase chain reaction
Reporter gene
repressor
Residues
Sulfate
toxic metals
Water pollution
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Summary:BACKGROUND Arsenic (As) is classified as a Group 1 human carcinogen, and the maximum arsenic contamination level allowed in drinking water is10 μg L−1. In this study, a genetic‐based whole‐cell biosensor for arsenic detection was constructed and engineered for rapid and more efficient arsenic detection. RESULTS The promoter and repressor responsive to arsenic (ArsBR) were newly isolated from Bacillus cereus ATCC14579. The reporter gene was atsBA carrying formylglycine‐generating enzyme (FGE) and sulfatase, which provided a fluorescent product after reacting with 4‐methylumbelliferyl sulfate (4‐MUS). To improve the performance of the arsenic biosensor, firstly, more efficient Escherichia coli host strains were constructed, including the disruption of the arsenic efflux pump, multidrug efflux pump and arsenical‐resistant genes; secondly, a repressor (ArsBR) was engineered by an error‐prone polymerase chain reaction (PCR) technique, aiming to lower the detection limit; lastly, a single copy biosensor cassette in an E. coli chromosome was constructed. CONCLUSION The stable and sensitive biosensor was able to detect As(III) at 5 μg L−1 and As(V) at 50 μg L−1 in 3.5 h with a very low fluorescent background. Moreover, the roles of all seven cysteine residues of the repressor were also investigated. Interestingly, the six cysteine residues Cys 28, 34, 36, 89, 90 and 100 are functionally involved in arsenic sensing, but not Cys 101. © 2019 Society of Chemical Industry
ISSN:0268-2575
1097-4660
DOI:10.1002/jctb.6302