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Bio-electric interface
Background
Part of our project was focusing on designing, constructing and testing a user-friendly bio-electric interface. Our idea was to create a standardised and regulated electron export system that would work in Escherichia coli as well as other microbes. In consequence that would allow for an output measurable by simple electronic methods, making the whole process easy and reliable. The bio-electric interface would connect biological and electronic systems and allow integration of biological devices with computers via generation of electrons. The final goal was to achieve a non-toxic, portable and user-friendly device. We've decided to use Shewanella oneidensis genes for molecular machinery and we used half fuel cells to measure the change in potential over time, reflecting the export of electrons outside of the cell.
Shewanella oneidensis is one of the organisms known to produce electricity. It gives it an evolutionary advantage over its competitors by allowing S. oneidensis to utilise multitude of extracellular electron acceptors. Even though S. oneidensis electron export system is a complicated, multi-component molecular machinery, Jensen et al [1] demonstrated that mtrCAB gene cluster is sufficient to engineer an efficient extracellular electron transport system. Therefore we've decided to research and model the system of extracellular electron transport in S. oneidensis and an engineered strain of E. coli. We have identified MtrCAB, cymA and napC to be elements with the most potential to influence efficiency of electron export and ccm cluster to be responsible for cytochrome maturation [1]. Following this conclusion we have modelled the system and attempted to test the selected genes in E coli.
In both organisms, electrons are being generated by TCA cycle and need to be transported to a terminal electron acceptor. The first step is generation of intracellular quinol pool which serve as electron shuttles, transporting electrons in the inner membrane of the cell [2,3]. The electrons are then transferred to appropriate transmembrane proteins: napC in E. coli (napC is a tetraheme cytochrome c quinol dehydrogenase, involved in nitrate reduction pathway [2]) and cymA in S. oneidensis (tetraheme cytochrome c quinol dehydrogenase, related to napC [3]). The transmembrane proteins then interact with MtrA (S. oneidensis decaheme cytochrome c) which is free floating in the periplasm. Reduced MtrA transfers electrons to MtrC (S. ondeidensis outer membrane decaheme cytochrome c); the MtrA and MtrC interaction is facilitated by MtrB (S. oneidensis outer membrane beta barrel porin). This way, electrons are transported across the membrane and MtrC can interact with a multitude of electron acceptors. S. oneidensis can also utilise nanowires and mediators to further improve efficiency of the process [4].
Figure 1: Engineering MtrCAB electron export system in E. coli. a) electron export system in S. oneidensis b) plasmids used by Jensen et al. for E coli transformation. C) electron export system in engineered E coli strains (transformed with mtrA or mtrCAB) Jensen H M et al. PNAS 2010;107:19213-19218
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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