Difference between revisions of "Part:BBa K535005"

Latest revision as of 03:39, 26 September 2011

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