Development of a Multiplexed Cadmium Biosensor for the Detection of Heavy Metals in the Environment

Continuous environmental monitoring is necessary for rapid identification of dangerous contaminants present in our ecosystems. Heavy metals are of particular concern with anthropogenic activities resulting in an increase in levels present in soil and water.  Current methods for detection of heavy metals are reliant on the transport of samples to off-site facilities, resulting in a time delay between sample collection and analysis. This delay can result in accidental consumption of contaminated substances, consequently leading to cellular accumulation and health related problems, such as cancer. Engineering bacteria to act as on-site microbial biosensors is one approach which provides the ability to rapidly detect heavy metals in the environment.

We are developing and testing the abilities of multiplexed cadmium responsive biosensors. The biosensing construct consists of an operator/promoter sequence regulated by the transcriptional regulator, CadR. CadR binds to the operator sequence, inhibiting transcription of downstream genes. Typically, these genes are responsible for cadmium uptake, reduction and export. The transcriptional regulator, cadR, together with the cognate operator/promoter was cloned upstream of a promoterless gfp. The biosensing construct was sub-cloned into a broad-host range vector, along with a constitutively expressed rfp. In this system an uptake of cadmium chloride into the bacterial cell results in GFP expression which increases over time, allowing qualitative and quantitative analyses.

The biosensing construct was transferred into commonly used laboratory bacteria; Escherichia coli, Shewanella oneidensis and Pseudomonas aeruginosa, alongside wild-type Enterobacter spp.  originally isolated from cadmium contaminated soil. Fluorescence microscopy and cadmium slope agar plates were used as an initial assessment of each biosensors functionality. The limit of detection of the microbial biosensors in response to cadmium chloride was found to range between 0.005-0.5 µg ml^-1with the response time ranging from 5 minutes to 24 hours. These results highlight the microbial biosensors effectiveness at detecting cadmium chloride at levels deemed dangerous to human health by the World Health Organisation. The specificity of the microbial biosensors was also tested and it was found that exposure to alternate heavy metals, particularly mercury chloride, results in a detectable output. This revealed that the biosensing construct can produce a multiplexed response to multiple inputs. The cadmium biosensors are currently being applied for the detection of cadmium in environmental samples including ground and ocean water, along with soil. Alongside these assays the facultative anaerobe biosensors are currently being tested under anaerobic conditions, opening the potential for the incorporation of alternate outputs.

Future research on the capabilities of the multiplexed cadmium biosensors will add knowledge to how bacteria can be applied as long-term environmental monitoring devices. This PhD project has been financially supported by the Human Protection and Performance Program of the Defence Science Institute, Australia.