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Bio-electric Interface:
Discussion and conclusions
For the fuel cell experiment we have obtained a series of interesting results. In our half fuel cells, E. coli seemed to exhibit properties similar to S. oneidensis. E. coli generates potential which closely relates to S. oneidensis outputs and the results repeat throughout multiple media, except for the final experiment using M9 with sodium acetate, which limited the growth of E. coli 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 the sucrose hydrolase gene. In such a system, detection of arsenic would induce expression of sucrose hydrolase, necessary for growth in media containing sucrose as the 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 its potential for automation and miniaturisation this system offers a potential advancement in the field of biosensors.
We are also intending to proceed with testing our BioBricked napC and mtrA. After linking them to a promoter we would like to test their influence on potential generation. Overall, the system we have constructed gives repeatable results with E. coli and with further test we hope to create a system capable of providing reliable data which can be coupled to a variety of promoters and genes.
We managed to obtain the napC, cymA, ccm and mtrA genes which are now ready for testing, using haem staining and half fuel cells. The mtrA gene still contains an internal PstI site which has to be mutated out prior to submission. We have linked napC, ccm and cymA to the lac promoter to test these new BioBricks using haem staining and half fuel cells, using our current results as reference. However, it is possible that the transformed cells will require multiple genes to function properly. Ccm genes are responsible for cytochrome maturation, which is necessary for the proper folding of multihaem cytochromes such as NapC, CymA and especially the decahaem cytochrome MtrA.
We had some success in cloning the mtrCAB and S. oneidensis ccm genes which may enhance the efficiency of the system. We intend to clone these genes into the pSB1C3 vector (BBa_K917007), link them to a promoter and test them together in order to assess the efficiency of the system.
The longer products (mtrCAB and ccm genes) seem to be more problematic to clone, with the digestion/ligation step being the limiting factor, despite using several alternative techniques (polyA tailing, fusion PCR).
The complete electron export conduit should be able to reliably export electrons in response to an external stimulus. This system can be used to enhance current biosensor systems.
One possible application would be to link our system to the arsenic promoter and construct a reliable, cheap arsenic biosensor which would generate easy to interpret data that can be stored on a computer.
For the fuel cell experiment we have obtained a series of interesting results. In our half fuel cells, E. coli seemed to exhibit properties similar to S. oneidensis. E. coli generates potential which closely relates to S. oneidensis outputs and the results repeat throughout multiple media, except for the final experiment using M9 with sodium acetate, which limited the growth of E. coli 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. To test this concept we have tested J33203 (arsenic promoter) +lacZ construct in our half fuel cells as a growth-based biosensor. We have obtained encouraging results, where transformed cells show faster voltage change compared to controls, showing a good potential for our system to serve as a reliable bio-detector generating data which would be easy to obtain and link to a computer system. With its potential for automation and miniaturisation this system offers a potential advancement in the field of biosensors. We are intending to further test this idea by using cells with arsenic promoter linked to the sucrose hydrolase gene. In such a system, detection of arsenic would induce expression of sucrose hydrolase, necessary for growth in media containing sucrose as the sole carbon source.
We managed to obtained, BioBricked and submitted the napC, cymA, ccm and mtrA genes. We have tested ccm, cymA and napC using haem staining procedure and obtained positive results. We have mutagenised the internal PstI side in mtrA. We had some success in cloning the mtrCAB and S. oneidensis ccm genes which may enhance the efficiency of the system. We would like to clone these genes into the pSB1C3 vector to create a functional BioBrick (BBa_K917007). However, the longer products (mtrCAB and ccm genes) seem to be more problematic to clone, with the digestion/ligation step being the limiting factor, despite using several alternative techniques (polyA tailing, fusion PCR).
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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
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