Development of a 2-Nitrobenzoate-Sensing Bioreporter Based on an Inducible Gene Cluster

Abstract

Based on the sole information of structural genes of the 2-nitrobenzoate (2NBA) utilizing catabolic gene cluster (onbX1X2FCAR1EHJIGDBX3), 2NBA-sensing bioreporters were constructed by incorporating egfp into the onb gene cluster of Cupriavidus sp. strain ST-14. Incorporation of reporter gene in proximal to the hypothesized promoter region in conjunction with the disruption of the gene encoding inducer-metabolizing enzyme was turned out to be advantageous in reporter gene expression at low inducer concentration. The bioreporter strain was capable of expressing EGFP from the very 1st hour of induction and could detect 2NBA at (sub) nanomolar level exhibiting a strict specificity toward 2NBA, displaying no response to EGFP expression from its meta- and para-isomers as well as from a number of structurally related compounds. The present study is a successful demonstration of the development of a 2NBA-sensing bioreporter with respect to ease of construction, inducer specificity, and sensitivity, without prior knowledge of the associated inducer-responsive promoter-regulator elements. The present approach can be used as a model for the development of bioreporters for other environmental pollutants.

Engineering a Rhamnose-Inducible System to Enhance the Extracellular Electron Transfer Ability of Shewanella Genus for Improved Cr(VI) Reduction

“…Simply by varying induction time or inducer concentrations, inducible promoters can change the expression kinetics of the genes of interest. To date, inducible expression systems are widely utilized in genetically encoded biosensors for detection of environmental pollutants and as a biocatalyst for the degradation of pollutants. − In biosensor systems, environment pollutants activate the associated inducible promoters to obtain controlled gene expression of the reporter. , Meanwhile, biocatalysts bearing the catabolic operons involved in pollutant degradation under the control of inducible promoters hold a promise for environmental remediation . An important reason behind such a choice is that the unregulated expression of catabolic enzymes for hazardous contaminants often retards the growth of the engineered strains.…”

“…16−18 In biosensor systems, environment pollutants activate the associated inducible promoters to obtain controlled gene expression of the reporter. 16,17 Meanwhile, biocatalysts bearing the catabolic operons involved in pollutant degradation under the control of inducible promoters hold a promise for environmental remediation. 19 An important reason behind such a choice is that the unregulated expression of catabolic enzymes for hazardous contaminants often retards the growth of the engineered strains.…”

Evaluation of distinct molecular architectures and coordinated regulation of the catabolic pathways of oestrogenic dioctyl phthalate isomers in Gordonia sp.

“…Tables S1 and S2 give primer sequences and information of strains and plasmids used in this study, respectively. The esterase deletion mutant strain was constructed by electroporating the recombinant pCM184 plasmid into electrocompetent cells of the wild-type strain GONU [34,35]. Allelic exchange took place via double recombination of homologous UP and DOWN sites to insert the kanamycin resistance gene cassette from the recombinant pCM184 plasmid into the chromosome of strain GONU by disrupting the esterase gene (Fig.…”