Coding

Part:BBa_K535002:Design

Designed by: iGEM11_UNAM-Genomics_ Mexico   Group: iGEM11_UNAM-Genomics_Mexico   (2011-09-25)
Revision as of 00:28, 28 September 2011 by Alecanda (Talk | contribs) (→‎Design Notes)

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HydA (hydrogenase I)-> Clostridium acetobutylicum ATCC 824


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 584
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1112
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 118
    Illegal NgoMIV site found at 1372
    Illegal NgoMIV site found at 1519
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1237


Design Notes

Some codons of the original Clostridium acetobutylicum ATCC 824 hydA sequence have been changed for synonimous ones according to the Codon Adaptation Index (CAI) procedure with respect to Rhizobium etli CFN42 codon usage in order to optimize its expression and to optimize R. etli CFN42’s (where we will express this gene) fitness as well.

The Codon adaptation Index 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.

We added a periplasm export tag at the C-terminal domain because we want that the protein works in the periplasm. This tag is flanked by two NdeI restriction sites in the final construction (composite part BBa_K535005) so we can split it out if needed.

The sequence that we use in our project is linked to the FeOx gene (part BBa_K535003), is regulated by a NifH promoter (part BBa_K535004) from Rhizobium etli and at the end it has two TAA stop codons. Please refer to the composite part BBa_K535005 for details.

Unwanted restriction sites had been changed for synonimous codons.

This sequence was synthesized .

Source

This sequence belongs to the Clostridium acetobutylicum ATCC 824 hydA hydrogenase gene which is in the chromosome of this organism. It has been modified, please refer to the “Design Notes” section for more details.

References

  • Matthew C. Posewitz, Paul W. King, Sharon L. Smolinski, Liping Zhang, Michael Seibert, and Maria L. Ghirardi. (2004). Discovery of Two Novel Radical S-Adenosylmethionine Proteins Required for the Assembly of an Active [Fe] Hydrogenase. J. Biol. Chem. 279, 25711-25720
  • Kuchenreuther JM, Grady-Smith CS, Bingham AS, George SJ, Cramer SP, et al. (2010) High-Yield Expression of Heterologous [FeFe] Hydrogenases in Escherichia coli. PLoS ONE 5(11): e15491.
  • Paulette M. Vignais (2007) Hydrogenases and H+-Reduction in Primary Energy Conservation. Results Probl Cell Differ. 45, 223-252.
  • Christina M Agapakis, Daniel C Ducat, Patrick M Boyle, Edwin H Wintermute, Jeffrey C Way, Pamela A Silver (2010) Insulation of a synthetic hydrogen metabolism circuit in bacteria. Journal of Biological Engineering 4:3.
  • Daniel C. Ducat, Gairik Sachdevac, and Pamela A. Silver (2011) Rewiring hydrogenase-dependent redoxcircuits in cyanobacteria. PNAS 108, 3941-3946.