Device

Part:BBa_K1465105

Designed by: iGEM-Team Bielefeld 2014   Group: iGEM14_Bielefeld-CeBiTec   (2014-10-07)

Fumarate reductase (Fum) under control of the T7 promoter

Fumarate reductase (Fum) under control of the T7 promoter

Usage and Biology

Fumarate reductase is part of the anaerobic fumarate respiration in bacteria. It catalyzes the reaction from fumarate into succinate using fumarate as a final electron acceptor in anaerobic fumarate respiration. The related enzyme in aerobic respiration is the succinate dehydrogenase, which catalyses the reaction from succinate to fumarate. The electrons are transferred from succinate to FAD+ producing fumarate and FADH2. The succinate dehydrogenase is also a membrane enzyme and it is part of the citric acid cycle. They both belong to the respiration complex II. Naturally there is no activity of both enzymes at the same time in E. coli cells. (Iverson et al., 1999)
In our project we used the fumarate reductase in combination with an extracellular mediator as electron donor to transfer electrons into bacterial cells. The reduced mediator crosses the outer membrane of E. coli through the outer membrane porine OprF (BBa_K1172507). Mediators adsorb at the inner membrane and transfer electrons to the fumarate reductase. After that the reduced fumarate reductase transfer electrons to fumarate producing succinate. This process has been shown for the fumarate reductase in Actinobacillus succinogenes by Park et al..
As published by Park et al., 1999 the fumarate reductase from A. succinogenes shows high activity accepting electrical reduced neutral red as an electron donor and so stimulates the metabolism in a electrobiochemical reactor system. We planed to compare activity of fumarate reductase Fum of A. succinogenes with fumarate reductase Frd of E. coli (a href="https://parts.igem.org/Part:BBa_K1465102">BBa_K1465102) with regard to activity accepting neutral red or bromphenol blue as alternative electron acceptors in our electrobiochemical reactor system.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 765
    Illegal XhoI site found at 2572
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2911
    Illegal AgeI site found at 3072
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2988
    Illegal BsaI.rc site found at 1151


Results

The fumarate reductase Fum from A. succinogenes just as Frd from E. coli (BBa_K1465102) is a membrane associated enzyme, which consists of four subunits. Because there was no full genomic DNA available from A. succinogenes, we ordered the Fum gene via custom gene synthesis at gBlocks® Gene Fragments. Before ordering DNA we ensured for optimal codon usage adapted to E. coli by jcat online optimization tool. Furthermore we checked the sequence for illegal restriction sites (EcoRI, PstI, NotI, XbaI) and eliminated them if required.
Because the sequence is too large for synthesis in one part with gBlocks® system, we divided the sequence of fum into two different parts: fumA and fumBCD (also called fumB). FumA includes the coding sequence of the fumarate reductase (Fum) subunit A (BBa_K1465103). However fumB consists of the fumarate reductase subunits B, C and D (BBa_K1465104). The fumarate reductase subunits were designed with their own ribosome binding site respectively. Two different subunits of the fumarate reductase Fum from A. succinogenes had to be cloned into the shipping vector pSB1C3. Several problems occurred during the cloning of the ordered fumA and fumB into pSB1C3 via gibson assembly.
Cloning of fum was not successful for a long time. Unfortunately we could not realize the ligation of fumA and fumB creating an active fumarate reductase (Fum). So there are no characterizations of fumarate reductase (Fum) available.

References

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