Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: iGEM11_UNAM-Genomics_ Mexico
Date created: 2011-09-24 11:00:00
Date modified: 2015-05-08 01:12:36
HydA1 - FeOx construction (Hydrogenase-ferredoxin fussion)
| Types | DnaRegion |
| Roles | engineered_region Composite |
| Sequences | BBa_K535005_sequence (Version 1) |
Description
The hydrogenase HydA1 requires electrons to reduce protons and form hydrogen gas molecules. The ferredoxin is the protein that mediates the electron transfer between the 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 express this hydrogenase-ferredoxintranslational fusion from Clostridium acetobutylicum ATCC 824 in Rhizobium etli CFN42 with a Desulfovibrio africanus PFOR (Pyruvate Ferredoxin Oxidoreductase) 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.
Notes
Some codons of the original Clostridium acetobutylicum ATCC 824 HydA1 and FeOx sequences have been changed for synonimous ones according to the Codon Adaptation Index (CAI) procedure in order to optimize its expression and to optimize Rhizobium etli CFN42 fitness as well.The CAI indicates how similar the Codon Usage (CU) in a coding sequence (CDS) is to that of highly/constitutively expressed genes. It is not a cause of high gene expression, but it is necessary to optimize resource usage. To optimize a sequence according to the CAI procedure we first obtained relative adaptiveness (w) for each codon (1.- most frequent codon. 0.- non-existent codon) in R. etli and then we substitute codons in target CDS with all synonymous codons with greatest w.
The fusion between HydA1 and FeOx is mediated by a 14aa long glycine/serine rich linker. The whole construction transcription is mediated by a Rhizobium etli CFN42 nifH promoter which is active under the nitrogen fixation conditions which include low oxygen concentration.
We added at the end a double TAA terminator.
A NifH promoter from Rhizobium etli CFN42 has been added to regulate the expression of this construction because we want it to be expressed under the same conditions that the genes related to nitrogen fixation are expressed.
Unwanted restriction sites had been changed for synonimous codons.
A 6 aminoacid long polyhistidine tag has been added at the C-terminus. This tag is flanked by two HindIII restriction sites.
Source
Both genes are part of Clostridium acetobutylicum ATCC 824 chromosome, some modifications to the natural sequence were made, for more details check out the ???design notes??? section. Ferredoxin NCBI accession number NP_346944; Hydrogenase NCBI accession number AAB03723.| Access | Instance | Definition |
| public public public | BBa_K535004 BBa_K535002 BBa_K535003 | BBa_K535004 BBa_K535002 BBa_K535003 |
| Sequence Annotation | Location | Component / Role(s) |
| BBa_K535004 BBa_K535002 BBa_K535003 | 1,203 210,2093 2102,2347 | BBa_K535004 BBa_K535002 BBa_K535003 |