Coding

Part:BBa_K1172401

Designed by: Sebastian Grenz   Group: iGEM13_Bielefeld-Germany   (2013-08-26)


mtrCAB - gene cluster from S. oneidensis for cytochrome expression

Usage and Biology

Figure 5: Overview on the electron transfer pathway from S. oneidensis MR-1 heterologously expressed in E. coli. The electron are transfered from the NapC protein via the Mtr proteins to the outer cell membrane, where an extracellular electron acceptors come into play.

To enable transfer of electrons from the general metabolism to the outside of the cell, the mtrCAB operon from [http://www.ncbi.nlm.nih.gov/genome/1082?project_id=57949 Shewanella oneidensis MR-1] was heterologously expressed in E. coli. This operon encodes for a minimal set of genes required to build an electron shuttle pathway via different c-type cytochromes. Electrons from the native E. coli protein [http://www.ncbi.nlm.nih.gov/protein/EGT66377.1 NapC] are passed to the periplasmic [http://www.ncbi.nlm.nih.gov/protein/NP_717386.1 MtrA], which transports them to the outer membrane protein [http://www.ncbi.nlm.nih.gov/protein/NP_717385.1 MtrB]. Via the membrane-bound [http://www.ncbi.nlm.nih.gov/protein/NP_717387.1 MtrC] the corresponding electrons can be transferred to extracellular electron acceptors like the anode of a microbial fuel cell.


Part uses

Device Regulatory Part Promoter RBS Activity
BBa_K1172403 BBa_K608006 Anderson 0.33 medium medium
BBa_K1172404 BBa_K608002 Anderson 0.77 strong strong
BBa_K1172405 BBa_K525998 T7 induced strong very strong


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2049
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 235


Results

Genetics

  • The amplification of the mtrCAB cluster from the genome of S. oneidensis and the deletion of illegal restriction sites was accomplished.
  • The gene cluster was succesfully ligated into the shipping vector pSB1C3 forming the BioBrick BBa_K1172401
  • The gene cluster was ligated into three expression vectors with varying promoter strength forming the following devices


Characterisation

  • To prove the expression of the Mtr proteins, E. coli cultures were transformed transformed with BBa_K1172403, BBa_K1172405 (uninduced) and BBa_K1172401 as a control and then cultivated anaerobically. Membrane and periplasmatic fractions of the cells were isolated by [http://2013.igem.org/Team:Bielefeld-Germany/Labjournal/ProtocolsPrograms#Cold_osmotic_shock Cold osmotic shock fractioning.] and analyzed by [http://2013.igem.org/Team:Bielefeld-Germany/Labjournal/ProtocolsPrograms#Sodium_dodecyl_sulfate_polyacrylamide_gel_electrophoresis_.28SDS-PAGE.29 SDS-PAGE]; the results are shown in Figure 5.
    • The membrane fraction should contain [http://www.ncbi.nlm.nih.gov/protein/NP_717385.1 MtrB] and [http://www.ncbi.nlm.nih.gov/protein/NP_717387.1 MtrC] of the Mtr complex, with sizes of 72 kDa and 69 kDa, respectively.
    • The periplasmatic fraction should only contain the [http://www.ncbi.nlm.nih.gov/protein/NP_717386.1 MtrA] protein of the Mtr complex (32 kDa).


Figure 5: Image of a SDS-PAGE with periplasmatic and membrane fractions of colonies transformed with K1172401 as a control, besides K1172403 and K1172405 (undinduced) for analysis. Ladder: PageRuler unstained Protein Ladder (Fermentas)


  • It was not possible to confirm the expression of the Mtr proteins by [http://2013.igem.org/Team:Bielefeld-Germany/Labjournal/ProtocolsPrograms#Sodium_dodecyl_sulfate_polyacrylamide_gel_electrophoresis_.28SDS-PAGE.29 SDS-PAGE] analysis, as no significant differences between control and experimental condition were visible at the relevant heights of the SDS-PAGE.
  • The redox activity of [http://www.ncbi.nlm.nih.gov/protein/NP_717386.1 MtrA] and [http://www.ncbi.nlm.nih.gov/protein/NP_717387.1 MtrC] was probed via absorption spectroscopy and did not show significant indications for a positive result as the expected shift of the Soret peak from 410 nm to 420 nm could not be observed.


Conclusion

  • The electron transfer system from Shewanella oneidensis MR-1 seems to be suitable for the usage in our Microbial Fuel Cell in principle. However, expression regulation, heme-loading, and correct folding as well as localization of the cytochromes are very complex and therefore our team was not able to produce the functional system in the available time.





References

  • Beliaev AS, Saffarini DA (1998)Shewanella putrefaciens mtrB encodes an outer membrane protein required for Fe(III) and Mn(IV) reduction. [http://jb.asm.org/content/180/23/6292.short J Bacteriol 180:6292–6297]
  • Beliaev, A. S., D. A. Saffarini, J. L. McLaughlin, and D. Hunnicutt. 2001. MtrC, an outer membrane decahaem c cytochrome required for metal reduction in Shewanella putrefaciens MR-1. [http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.2001.02257.x/full Mol. Microbiol. 39:722-730]
  • Grove, J., Tanapongpipat, S., Thomas, G., Griffiths, L., Crooke, H., and Cole, J. (1996)Escherichia coliK-12 genes essential for the synthesis of c-type cytochromes and a third nitrate reductase located in the periplasm. [http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.1996.383914.x/abstract Mol. Microbiol. 19, 467−481]
  • Jensen HM, Albers AE, Malley KR, Londer YY, Cohen BE, et al. (2010) Engineering of a synthetic electron conduit in living cells. [http://www.pnas.org/content/107/45/19213.short Proc Natl Acad Sci USA. 10.1073/pnas.1009645107 Proc. Natl Acad. Sci. USA 107, 19213–19218 (2010).]
  • Myers CR, Myers JM (2002) MtrB is required for proper incorporation of the cytochromes OmcA and OmcB into the outer membrane of Shewanella putrefaciens MR-1. [http://aem.asm.org/content/68/11/5585.short Appl Environ Microbiol68:5585–5594]
  • Sanders, C., Turkarslan, S., Lee, D.-W., and Daldal, F. (2010) Cytochromecbiogenesis: the Ccm system. [http://www.sciencedirect.com/science/article/pii/S0966842X10000442 Trends Microbiol. 18, 266−274]
  • Schicklberger, M., Bucking, C., Schuetz, B., Heide, H., and Gescher, J. (2011) Involvement of the Shewanella oneidensis decaheme cytochrome MtrA in the periplasmic stability of the beta-barrel protein MtrB. [http://aem.asm.org/content/77/4/1520.short Appl. Environ. Microbiol. 77, 1520−1523]
  • Thony-Meyer L, Fischer F, Kunzler P, Ritz D, Hennecke H (1995)Escherichia coligenes required for cytochrome c maturation. [http://jb.asm.org/content/177/15/4321.short J Bacteriol 177:4321–4326]
[edit]
Categories
//cds/membrane
//cds/membrane/transporter
Parameters
None