Part:BBa_K1465101

Cytochrome c from Geobacter sulfurreducens

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 352
Illegal SapI.rc site found at 433

Usage and Biology

Although the exact mechanism of electron transfer by cytochromes is not clarified up to now there are a few basic approaches that indicate which ones could have an important function. Those periplasmatic cytochrome are essential for electron uptake and function as an intermediate electron acceptor. Therefore we decided to work with a key-type cytochrome c, called PccH of Geobacter sulfurreducens because it has been shown that biofilms of this organism are capable of accepting electrons from an electrode. Furthermore it has been demonstrated that the mechanism of electron delivery and electron uptake varies in view of involved proteins. In this case transcriptome analysis by microarray revealed that the corresponding gene ( GSU 3274) of PccH is far more abundant in current-consuming cells using an electrode as electron donor in order to reduce fumarate than the genes known to be essential for electron delivery. Additionally deletion of GSU 3274 inhibits the electron uptake completely and complementation of it afterwards can fully restore electron consumption. Based on this we aim to express PccH (BBa_K1465101) in E. coli to bring the electrons into the cell by reducing the membrane-bound fumarate reductase.(Strycharz et al., 2011; Dantas et al., 2013)
Figure 1 shows the general principle of the electron flow.

Figure 1: The electron flow mediated by redox active molecules.

Therefore the periplasmatic cytochrome leaves the periplasm through the porins OprF (BBa_K1172507) in the outer membrane, gets reduced at the cathode and enters the periplasm again. There the fumarate reductase (BBa_K1465102) which is located in the inner phospholipid membrane oxidized the cytochrome again and is thereby the connective element which transfers the electrons into the cell. These supplied electrons increase the succinate production and together with the succinate dehydrogenase the metabolic activity of the cells should be enhanced.
One of the challenges going along with expressing periplasmatic cytochromes is the correct localization of the them in the periplasm. It is documented that PccH has its own signal peptide for localization and therefore we initially want to use this signal peptide(Dantas et al., 2013). Furthermore we consider to use E. coli specific signal peptides afterwards if necessary.
Another challenge is the correct folding of PccH because there are specific cytochrome c maturation (ccm) genes needed. Those genes also exist in E. coli but are normally just expressed under anaerobic conditions (Jensen et al., 2010). In view of module two and three and of the characterization of PccH completely anaerobic conditions are not possible and can not be guaranteed. This is why we have to bring the ccmA-H genes on a plasmid in E. coli to express it under aerobic conditions. Furthermore we bring it under the control of a constitutive anderson promoter by an appropriate design of primer.
The expression strength of both, PccH and ccmA-H, are another challenge because they have to be balanced correctly (Jensen et al., 2010). Therefore we want to express ccmA-H constitutivley whereas the promoter of PccH should be changed to test the optimal ratio between both.

References

Stycharz, S. M., Glaven, R. H., Coppi, M. V., Gannon, S. M., Perpetua, L. A., Liu, A., Nevin, K. P. &amp. Lovley, D. R. (2011):Gene expression and deletion analysis of mechanisms for electron transfer from electrodes to Geobacter sulfurreducens. In: Bioelectrochemistry 80, pp. 142 - 150.
Dantas, J. M., Tomaz, D. M., Morgado, L. & Salgueiro, C. A.(2013): Functional charcterization of PccH, a key cytochrome for electron transfer from electrodes to the bacteium Geobacter sulfurreducens. In: FEBS Letters 587, pp. 2662 - 2668.
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. In: PNAS 107, pp. 19213–19218.

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