Composite

Part:BBa_K535005

Designed by: iGEM11_UNAM-Genomics_ Mexico   Group: iGEM11_UNAM-Genomics_Mexico   (2011-09-25)
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HydA1 - FeOx construction (Hydrogenase-ferredoxin fussion)

The hydrogenase HydA1 requires electrons to reduce protons and form hydrogen gas molecules. The ferredoxin is the protein that mediates the electron transfer between an electron transfer-donnor and a hydrogenase.

The ferredoxin and the hydrogenase need to physically interact for the circuit to function, actually their interaction surface has been extensively modeled in silico, with evidence that this interaction has a strong electrostatic component.

The putative ferredoxin-binding region is on the N-terminal domain of the hydrogenase, which includes all of the F-clusters that transfer electrons from the surface to the active-site H-cluster.

Experiments had been done in order to improve hydrogen production through physically linking the hydrogenase and ferredoxin to increase the chance of binding and electron transfer between the desired partners. In vivo (in E. coli) activity of the fusion proteins when coexpressed with the PFOR from Desulfovibrio africanus depended on linker length as well as overall configuration.

The highest improvement was seen when the ferredoxin is fused to the hydrogenase N-terminus with a flexible glycine/serine-rich linker of 14 aminoacid long which led to a 3-5 fold increase in hydrogen output from the scaffolded circuit.

We want to express this hydrogenase-ferredoxin translational fusion from Clostridium acetobutylicum ATCC 824 in Rhizobium etli CFN42 with a Desulfovibrio africanus PFOR (Pyruvate Ferredoxin Oxido-Reductase, part PFOR) to make the entire circuit. This construction is regulated by a nifH promoter from Rhizobium etli CFN42 which promotes transcription under the conditions that favor nitrogen fixation which includes a microanaerobic environment, necessary for HydA correct function.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 793
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1321
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 327
    Illegal NgoMIV site found at 1581
    Illegal NgoMIV site found at 1728
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2203
    Illegal BsaI.rc site found at 1446


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