synthetic biology approach. ACS Synth Biol. 2. Aracic, S., Semenec, L. & Franks, A. E. (2014). Investigating microbial activities of electrode-associated ...
Development of Microbial Biosensors using Electroactive Microbes for Detection of Hazardous Analytes in the Environment Sanja
1 Aracic ,
Gülay
2 Mann
and Ashley E.
1 Franks
1Department
of Microbiology, La Trobe University, Bundoora, Victoria, Australia 2Land Division, Defence Science and Technology Organisation, Port Melbourne, Victoria, Australia
INTRODUCTION 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 biosensors1. 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.
Input: hazardous analytes Microbial chassis: Escherichia coli Pseudomonas aeruginosa Shewanella oneidensis Geobacter sulfurreducens
Output: fluorescent or electrochemical signal
Aim: To utilise electroactive microorganisms (Pseudomonas, Shewanella and Geobacter) as whole-cell biosensors which will generate electrochemical outputs in response to various hazardous analytes.
MERCURY BIOSENSOR CONSTRUCT Absence of Hg2+
Presence of Hg2+ Broad-host range plasmid
Broad-host range plasmid PmerR
merR
[Cd2+]
[Hg2+] Constitutively expressed gfp Empty vector Cadmium biosensor Mercury biosensor
PmerR
gfp
merR
gfp Pmer
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, Pmer) in response to the concentration of mercuric ions (Hg2+) in the environment.
Figure 2. Escherichia coli cadmium and mercury biosensors on slope agar plates containing increasing concentrations of mercuric ions (Hg2+) and cadmium ions (Cd2+). Increasing concentration of heavy metals induce expression of gfp in the biosensors.
1
5000
RFU
4000
2 3
4
5 6 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
pBB Empty vector
3000
pBBgfp Constitutively expressed gfp
2000
1000
pBBmergfp Mercury biosensor kDa
0 0
1 Hg2+ [µg ml-1]
30
5
ml-1
Figure 3. Fluorescence assay of E. coli mercury biosensor induced with 1 and 5 µg of Hg2+ for 4 h. The expression of gfp in the mercury biosensor is induced in the presence of Hg2+.
Shewanella mercury biosensor
4000
200
3000
150 RFU
RFU
Pseudomonas mercury biosensor
2000
1000
20
SDS-PAGE (1 h at 200 V)
Figure 4. SDS-PAGE of total soluble protein extracted from E. coli mercury biosensor expressing GFP (27 kDa) following induction with Hg2+ for 4 h.
FUTURE BIOSENSOR CONSTRUCT Absence of Hg2+
100
Broad-host range plasmid
Presence of Hg2+ Broad-host range plasmid
50 merR
0
PmerR
omc
merR
PmerR Pmer
omc
0 0
1
Hg2+ [µg
ml-1]
5
0
1
Hg2+ [µg
ml-1]
5
Figure 5. Fluorescence assay of P. aeruginosa and S. oneidensis mercury biosensor induced with 1 and 5 µg ml-1 of Hg2+ for 4 h. The expression of gfp in the mercury biosensor is induced in the presence of Hg2+.
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 voltammetry2.
FUTURE DIRECTIONS Pseudomonas, Shewanella and Geobacter can interact directly with electrode surfaces and have the potential to be integrated into electronic devices3. The developed biosensors for heavy metals will be incorporated into existing field-deployable microbial fuel cells designed for environmental monitoring.
REFERENCES
ACKNOWLEDGMENTS
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.
This project is funded by the Office of Naval Research Global (Award no. N626909-13-1N259), the ARC (Award no. LP140100459), the Defence Science Institute (Synthetic Biology Initiative) and the Defence Science and Technology Organisation.
