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Figure 2. Escherichia coli cadmium and mercury biosensors on slope agar plates containing increasing concentrations of mercuric ions (Hg 2+ ) and cadmium ions (Cd 2+ ). Increasing concentration of heavy metals induce expression of gfp in the biosensors. Development of Microbial Biosensors using Electroactive Microbes for Detection of Hazardous Analytes in the Environment Sanja Aracic 1 , Gülay Mann 2 and Ashley E. Franks 1 1 Department of Microbiology, La Trobe University, Bundoora, Victoria, Australia 2 Land Division, Defence Science and Technology Organisation, Port Melbourne, Victoria, Australia MERCURY BIOSENSOR CONSTRUCT Rapid and reliable detection of a wide range of hazardous substances in the environment is required to mitigate ecological harm. Analytes of these hazardous substances can enter and accumulate in the food chain potentially causing harm to humans. Current detection methods are not always practical as they are time-consuming, costly and require off-site testing. The ability to genetically manipulate regulatory elements to detect analytes and produce a measurable signal has resulted in the utilisation of laboratory microorganisms as biosensors 1 . In order for whole cell biosensors to be feasible for the detection of contaminants in the environment, a wider range of microbial chassis with integrated output systems is required. FUTURE BIOSENSOR CONSTRUCT REFERENCES 1. Bereza-Malcolm, L., Mann, G. & Franks, A. E. (2014). Environmental sensing of heavy metals through whole cell microbial biosensors: A synthetic biology approach. ACS Synth Biol. 2. Aracic, S., Semenec, L. & Franks, A. E. (2014). Investigating microbial activities of electrode-associated microorganisms in real-time. Front Microbiol 5, 663. 3. Semenec L. & Franks A. E. (2014). The microbiology of microbial electrolysis cells. Microbiol Aust 35, 201-206. ACKNOWLEDGMENTS This project is funded by the Office of Naval Research Global (Award no. N626909-13-1- N259), the ARC (Award no. LP140100459), the Defence Science Institute (Synthetic Biology Initiative) and the Defence Science and Technology Organisation. Constitutively expressed gfp Empty vector Cadmium biosensor Mercury biosensor INTRODUCTION FUTURE DIRECTIONS Input: hazardous analytes Output: fluorescent or electrochemical signal Microbial chassis: Escherichia coli Pseudomonas aeruginosa Shewanella oneidensis Geobacter sulfurreducens [Hg 2+ ] [Cd 2+ ] Figure 1. Mercury-inducible gfp biobrick. The gfp gene is under a mercury-inducible promoter whose transcription is regulated by the transcriptional regulator, MerR. MerR controls the expression of gfp (from the mercury-inducible promoter, P mer ) in response to the concentration of mercuric ions (Hg 2+ ) in the environment. Broad-host range plasmid Absence of Hg 2+ Presence of Hg 2+ merR gfp P merR Broad-host range plasmid merR gfp P merR P mer 0 1000 2000 3000 4000 5000 0 1 5 RFU Hg 2+ [μg ml -1 ] Broad-host range plasmid merR omc P merR Broad-host range plasmid merR omc P merR P mer Lanes: 1. Ladder 2. Empty vector 3. Constitutively expressed gfp 4. Uninduced mercury biosensor 5. Induced (1 μg ml -1 Hg 2+ ) mercury biosensor 6. Induced (5 μg ml -1 Hg 2+ ) mercury biosensor kDa 30 20 1 2 3 4 5 6 Absence of Hg 2+ Presence of Hg 2+ SDS-PAGE (1 h at 200 V) Empty vector Constitutively expressed gfp Mercury biosensor 0 1000 2000 3000 4000 0 1 5 RFU Hg 2+ [μg ml -1 ] Pseudomonas mercury biosensor 0 50 100 150 200 0 1 5 RFU Hg 2+ [μg ml -1 ] Shewanella mercury biosensor Figure 4. SDS-PAGE of total soluble protein extracted from E. coli mercury biosensor expressing GFP (27 kDa) following induction with Hg 2+ for 4 h. Aim: To utilise electroactive microorganisms (Pseudomonas, Shewanella and Geobacter) as whole-cell biosensors which will generate electrochemical outputs in response to various hazardous analytes. Figure 5. Fluorescence assay of P. aeruginosa and S. oneidensis mercury biosensor induced with 1 and 5 μg ml -1 of Hg 2+ for 4 h. The expression of gfp in the mercury biosensor is induced in the presence of Hg 2+ . Figure 6. Genes encoding electroactive cytochromes have been cloned downstream of heavy metal-inducible promoters and their cognate transcriptional regulators. These redox-active proteins have specific electrochemical signals that can be detected using cyclic voltammetry 2 . Pseudomonas, Shewanella and Geobacter can interact directly with electrode surfaces and have the potential to be integrated into electronic devices 3 . The developed biosensors for heavy metals will be incorporated into existing field-deployable microbial fuel cells designed for environmental monitoring. Figure 3. Fluorescence assay of E. coli mercury biosensor induced with 1 and 5 μg ml -1 of Hg 2+ for 4 h. The expression of gfp in the mercury biosensor is induced in the presence of Hg 2+ .
