Team:Edinburgh/Project/Bioelectric-Interface/Microbial-Half-Fuel-Cells

From 2012.igem.org

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Bio-electric interface
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Bio-electric Interface:
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Microbial half fuel cells
Microbial half fuel cells
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Procedure
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Methods
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- Fuel cells were constructed using carbon weave electrodes and reference electrodes provided by Matthew Knighton from Dr Bruce Ward’s lab.
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The half fuel cells were constructed using the following components provided by Matthew Knighton from Dr Bruce Ward’s lab: <br />
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- 250 ml or 500 ml glass bottle <br />
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- A standard plastic cap with two holes drilled for electrodes <br />
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- carbon weave electrode fixed to the cap with silicone sealant <br />
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- reference electrode "red rod" REF201 available for sale from Radiometer analytical <br />
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- Following the assembly, bottles were autoclaved (reference electrodes were instead sterilised with alcohol as they are temperature sensitive). Under sterile conditions, reference electrodes were dipped in alcohol, inserted into the cap of the bottles and sealed with silicon sealant. The half fuel cells were then filled with media, inoculated with bacteria and sealed with parafilm in order to ensure anaerobic growth. The bacteria were left to grow at room temperature. (Figure 1)
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- Fuel cells were assembled by inserting bottle cap with attached carbon weave electrode into 500 or 250 ml standard glass bottles. The electrodes were attached to the caps using silicone sealant. Bottles were then autoclaved. In sterile conditions, reference electrodes were dipped in alcohol, inserted into the cap of the bottles and sealed with silicon sealant. Half fuel cells were then filled with media and inoculated with bacteria and sealed with parafilm in order to ensure anaerobic growth. They were then grown in room temperature
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- Media used: standard LB or M9 (<a href="http://openwetware.org/wiki/M9_medium/minimal">minimal growth medium</a>) supplemented with 1% lactose, 0,4% glycerol or 0,4% sodium acetate.
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- media used: standard LB, M9 (<a href="http://openwetware.org/wiki/M9_medium/minimal">minimal growth medium</a>) supplemented with 0,4% glycerol or sodium acetate.
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- Measurements were obtained using a digital multimeter.
- Measurements were obtained using a digital multimeter.
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- We have examined the behaviour of <i>S. oneidensis</i> and <i>E. coli</i> in different media using half fuel cells. We managed to obtain results using the following media: LB, M9 with glycerol and M9 with sodium acetate. The results are summarised in the figure below. We also performed a measurement for <i>Citrobacter freundii</i> to see whether it differs from other bacteria.<br /><br />
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- We have examined the behaviour of <i>S. oneidensis</i> and <i>E. coli</i> in different media using half fuel cells. We managed to obtain results using the following media: LB, M9 with glycerol and M9 with sodium acetate. The results are summarised in figures 2 and 3 below. We also performed a measurement for <i>Citrobacter freundii</i> to see whether it differs from other bacteria.<br /><br />
<img id="fig01" src="https://static.igem.org/mediawiki/2012/e/e4/Bio-el-interface-fig09.JPG"><br />
<img id="fig01" src="https://static.igem.org/mediawiki/2012/e/e4/Bio-el-interface-fig09.JPG"><br />
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Figure 1: Microbial half fuel cells with <i>S. oneidensis</i> and <i>E. coli</i>
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<b>Figure 1:</b> Our microbial half fuel cells with <i>S. oneidensis</i> and <i>E. coli</i>
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<img id="fig02" src="https://static.igem.org/mediawiki/2012/9/91/Bioelec1.jpg"><br />
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Figure 2: Half fuel cells experiments 1 and 2, using LB medium for growth of <i>S. oneidensis</i> and <i>E. coli</i>. Experiment 1 (left) was performed using 500 ml of medium while experiment 2 (right) was performed using 250 ml of medium.
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<b>Figure 2:</b> Half fuel cells experiments 1 and 2, using LB medium for growth of <i>S. oneidensis</i> and <i>E. coli</i>. Experiment 1 (left) was performed using 500 ml of medium while experiment 2 (right) was performed using 250 ml of medium.
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<img id="fig03" src="https://static.igem.org/mediawiki/2012/c/cc/Bioelec2.jpg"><br />
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<img id="fig03" src="https://static.igem.org/mediawiki/2012/4/4a/Bio-el-interface-fig11.JPG"><br />
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<b>Figure 3:</b> Half fuel cell experiments 3 and 4, using M9 medium for growth of <i>S. oneidensis</i>, <i>E. coli</i> and <i>Citrobacter freundii</i>. Experiment 3 (left) was performed using 250 ml of medium M9 with 0,4% glycerol while experiment 4 (right) was performed using 250 ml of medium M9 with 0,4% sodium acetate. In experiment 4, <i>C. freundii</i> was also tested.
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Figure 3: Half fuel cells experiments 3 and 4, using M9 medium for growth of <i>S. oneidensis</i>, <i>E. coli</i> and <i>Citrobacter freundii</i>. Experiment 3 (left) was performed using 250 ml of medium M9 with 0,4% glycerol while experiment 4 (right) was performed using 250 ml of medium M9 with 0,4% sodium acetate. In experiment 4, <i>C. freundii</i> was tested.
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Discussion and conclusions
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For the fuel cell experiment we have obtained a series of interesting results. In our half fuel cells, <i>E. coli</i> seems to exhibit properties similar to <i>S. oneidensis. E. coli</i> generates potential which closely relates to <i>S. oneidensis</i> outputs and the results repeat throughout multiple media, except for the final experiment using M9 with sodium acetate, which limited the growth of <i>E. coli</i> altogether as well as limiting the electrogenicity of other bacteria. It seems that electrogenicity can be linked to the growth of cultures, at least in the minimal media. This shows a great potential for using microbial half fuel cells in combination with different promoters and selectable markers. We are intending to further test this idea by using cells with arsenic promoter linked to sucrose hydrolase gene. In such a system, detection of arsenic would induce expression of sucrose hydrolase, necessary for growth in media containing sucrose as sole carbon source. In consequence such a system could be used as a reliable bio-detector generating data which would be easy to obtain and link to a computer system. With potential for automation and miniaturisation this system offers a potential advancement in the field of biosensors.
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We are also intending to proceed in testing our BioBricks napC and MtrA. After linking them to a promoter we would like to test their influence of potential generation. Overall the system we have constructed gives repeatable results with <i>E. coli</i> and with further test we hope to create a system capable of providing reliable data which can be coupled with a variety of promoters and genes.
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Growth-based biosensor
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We have designed and tested a growth-based arsenic biosensor with a direct electric output. In order to test the principle of this device, we have transformed <i>E. coli</i> JM109 with Edinburgh 2006's <a href="http://partsregistry.org/Part:BBa_J33203">BBa_J33203</a> BioBrick (arsenic promoter with <i>arsR</i> repressor linked to <i>lacZ'</i> gene responsible for lactose degradation). We have then prepared 3 half-fuel cells with lactose medium (M9 with trace elements and thiamine + 1% lactose): <br />
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1) BBa_J33203 transformants  in medium with sodium arsenate (100 parts per billion concentration)<br />
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2) BBa_J33203 transformants in medium without sodium arsenate <br />
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3) control, wild type <i>E. coli</i> in medium with sodium arsenate (100 parts per billion concentration)<br /><br />
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<img src= "https://static.igem.org/mediawiki/2012/2/2e/Biosensor_final.jpg" width="700"> <br />
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<b>Figure 4</b>: Growth-based arsenic biosensor: change in voltage over time using <i>E. coli</i> transformed with BBa_J33203 + lacZ' and WT <i>E. coli</i> as control<br /><br />
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The growth of cells and associated change in voltage was much slower compared to our previous experiments. This can probably be attributed to the lower temperature of incubation as the fuel cells were incubated at room temperature. Despite the slower growth rate, the results we have obtained are encouraging. BBa_J33203 transformed cells in the presence of arsenate show a faster drop in voltage compared to other samples. This is especially important compared to the BBa_J33203 cells in the medium without arsenate. These results show promising prospect for growth-based biosensors. With more sophisticated measurement methods it would be possible to connect our system to a computer which would allow for automated and quantitative analysis of the data, allowing for simple and automated contamination detection. <br />
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Current results are encouraging but background growth is still present in the media and therefore further experiments are necessary to optimise the growth parameters. One possible improvement includes the addition of the <i>cscA</i> BioBrick that we have designed this year. Using sucrose instead of lactose may reduce background growth and allow for tighter control of the system.
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Acknowledgements
Acknowledgements
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We would like to thank Dr Bruce Ward and Matthew Knighton for their help with the fuel cells and for lending us their lab equipment.
We would like to thank Dr Bruce Ward and Matthew Knighton for their help with the fuel cells and for lending us their lab equipment.
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<a href="https://2012.igem.org/Team:Edinburgh/Project/Bioelectric-Interface/Bio-electric-Interface-BioBricks-Cloning"><span class="intense-emphasis">&lt;&lt;Prev</span></a><span style="color:white;">__</span>3/4</span></a><span style="color:white;">__</span><a href="https://2012.igem.org/Team:Edinburgh/Project/Bioelectric-Interface/Discussion"><span class="intense-emphasis">Next&gt;&gt;</span></a></span>
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Latest revision as of 00:37, 27 October 2012

Bio-electric Interface:

Microbial half fuel cells

Methods


The half fuel cells were constructed using the following components provided by Matthew Knighton from Dr Bruce Ward’s lab:
- 250 ml or 500 ml glass bottle
- A standard plastic cap with two holes drilled for electrodes
- carbon weave electrode fixed to the cap with silicone sealant
- reference electrode "red rod" REF201 available for sale from Radiometer analytical

- Following the assembly, bottles were autoclaved (reference electrodes were instead sterilised with alcohol as they are temperature sensitive). Under sterile conditions, reference electrodes were dipped in alcohol, inserted into the cap of the bottles and sealed with silicon sealant. The half fuel cells were then filled with media, inoculated with bacteria and sealed with parafilm in order to ensure anaerobic growth. The bacteria were left to grow at room temperature. (Figure 1)

- Media used: standard LB or M9 (minimal growth medium) supplemented with 1% lactose, 0,4% glycerol or 0,4% sodium acetate.

- Measurements were obtained using a digital multimeter.

Results

- We have examined the behaviour of S. oneidensis and E. coli in different media using half fuel cells. We managed to obtain results using the following media: LB, M9 with glycerol and M9 with sodium acetate. The results are summarised in figures 2 and 3 below. We also performed a measurement for Citrobacter freundii to see whether it differs from other bacteria.


Figure 1: Our microbial half fuel cells with S. oneidensis and E. coli


Figure 2: Half fuel cells experiments 1 and 2, using LB medium for growth of S. oneidensis and E. coli. Experiment 1 (left) was performed using 500 ml of medium while experiment 2 (right) was performed using 250 ml of medium.


Figure 3: Half fuel cell experiments 3 and 4, using M9 medium for growth of S. oneidensis, E. coli and Citrobacter freundii. Experiment 3 (left) was performed using 250 ml of medium M9 with 0,4% glycerol while experiment 4 (right) was performed using 250 ml of medium M9 with 0,4% sodium acetate. In experiment 4, C. freundii was also tested.

Growth-based biosensor

We have designed and tested a growth-based arsenic biosensor with a direct electric output. In order to test the principle of this device, we have transformed E. coli JM109 with Edinburgh 2006's BBa_J33203 BioBrick (arsenic promoter with arsR repressor linked to lacZ' gene responsible for lactose degradation). We have then prepared 3 half-fuel cells with lactose medium (M9 with trace elements and thiamine + 1% lactose):
1) BBa_J33203 transformants in medium with sodium arsenate (100 parts per billion concentration)
2) BBa_J33203 transformants in medium without sodium arsenate
3) control, wild type E. coli in medium with sodium arsenate (100 parts per billion concentration)


Figure 4: Growth-based arsenic biosensor: change in voltage over time using E. coli transformed with BBa_J33203 + lacZ' and WT E. coli as control

The growth of cells and associated change in voltage was much slower compared to our previous experiments. This can probably be attributed to the lower temperature of incubation as the fuel cells were incubated at room temperature. Despite the slower growth rate, the results we have obtained are encouraging. BBa_J33203 transformed cells in the presence of arsenate show a faster drop in voltage compared to other samples. This is especially important compared to the BBa_J33203 cells in the medium without arsenate. These results show promising prospect for growth-based biosensors. With more sophisticated measurement methods it would be possible to connect our system to a computer which would allow for automated and quantitative analysis of the data, allowing for simple and automated contamination detection.
Current results are encouraging but background growth is still present in the media and therefore further experiments are necessary to optimise the growth parameters. One possible improvement includes the addition of the cscA BioBrick that we have designed this year. Using sucrose instead of lactose may reduce background growth and allow for tighter control of the system.

Acknowledgements

We would like to thank Dr Bruce Ward and Matthew Knighton for their help with the fuel cells and for lending us their lab equipment.



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Bibliography (expand)

1. Jensen, H. M., Albers, A. E., Malley, K. R., Londer, Y. Y. , Cohen, B. E., Helms, B. A., Weigele, P., Groves, J. T. & Ajo-Franklin, C. M. (2010). Engineering of a synthetic electron conduit in living cells. PNAS 107, 19213-19218

2. Stewart, V., Lu, Y. & Darwin, A. J. (2002). Periplasmic Nitrate Reductase (NapABC Enzyme) supports Anaerobic Respiration by Escherichia coli K-12. Journal of Bacteriology 184, 1314-1323

3. Marritt, S. J., Lowe, T. G., Bye, J., McMillan, D.G.G., Shi, L., Frederickson, J., Zachara, J., Richardson, D. J., Cheesman, M. R., Jeuken L.J.C. & Butt, J. N. (2012). A functional description of CymA, an electron-transfer hub supporting anaerobic respiratory flexibility in Shewanella. Biochemical Journal 444, 465-474

4. Richter, K., Schicklberger, M., Gescher, J. (2011). Dissimilatory reduction of extracellular electron acceptors in anaerobic respiration. Applied and Environmental Microbiology 78, 913-921

Close bibliography.