The development and analyses of several Gram-negative arsenic biosensors using a synthetic biology approach

Abstract

Microbial biosensors hold promise as a method to detect bioavailable heavy metals in the environment. However, applications are limited by use of bacterial species with little environmental relevance. In this study, six arsenic biosensors were developed and comparatively analysed. The genetic element consisted of an arsR transcriptional regulator and its cognate operator/promoter region, upstream of a promoterless gfp gene. This allowed for a quantifiable fluorescence output which is regulated by ArsR, in response to arsenic. The genetic element was cloned into a narrow host range, high-copy number plasmid and broad host range, low-copy number plasmid. Preliminary analyses were performed in Escherichia coli whereby microbial biosensor functionality was not effected by plasmid copy number or the stage of bacterial growth. Following, the broad-host range arsenic biosensor construct was transferred into Pseudomonas aeruginosa, Shewanella oneidensis and wild-type Enterobacter spp. Comparative analyses were used to determine the detection limit, induction time and specificity of the arsenic biosensors. The detection limits and induction times varied between bacterial species however, all arsenic biosensors were specific to arsenite. The variations between the microbial biosensors were hypothesized to be related to the genetic element of the biosensor construct, nutrient availability, native transcriptional machinery and presence of endogenous heavy metal resistance pathways