Coding

Part:BBa_K535006:Design

Designed by: iGEM11_UNAM-Genomics_ Mexico   Group: iGEM11_UNAM-Genomics_Mexico   (2011-09-25)

PFOR -> D. africanus Pyruvate-Ferredoxin OxidoReductase


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1833
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 146
    Illegal NgoMIV site found at 359
    Illegal NgoMIV site found at 368
    Illegal NgoMIV site found at 395
    Illegal NgoMIV site found at 1117
    Illegal NgoMIV site found at 1244
    Illegal NgoMIV site found at 1279
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2131
    Illegal BsaI.rc site found at 1007
    Illegal BsaI.rc site found at 2048
    Illegal BsaI.rc site found at 2615


Design Notes

Codons of the original Desulfovibrio africanus sequence were changed for synonimous ones according to the Codon adaptation index (CAI) procedure in order to optimize its expression and Rhizobium etli’s fitness as well (where we will express the gene).

The Codon Adaptation Index indicates how similar the Codon Usage (CU) in a coding sequence (CDS) is to those of highly or 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 being the most frequent codon, 0 being a non-existent codon) in R. etli and then we substituted codons in the target CDS with all synonymous codons with greatest w.

5' UTR and the first seven codons were optimized for Gibbs free energy to avoid formation of secondary and tertiary structures. This was achieved by taking “allowed” synonym codons (as determined by w higher than threshold) and using the combination of the first seven codons that together with the six nucleotide long sequence (generated using RSATools) as a spacer, had the least negative ∆G value which indicates a worse folding, relating to a better translation initiation in mRNA. Some standard restrictions sites arose from this optimization. They were removed by changing the codons spanning these sites for synonimous codons.

Two TAA stop codons had been added at the end of the coding region.

PFOR contains at the beginning a periplasm export tag between two NdeI restriction sites intended for removal, at the end it also has a poly His tag for immuno-assays, it is between two HindIII restriction sites.

This sequence was synthesized.

Source

The coding sequence is part of the Desulfovibrio africanus genome. The 5’ UTR was designed by us to include a RBS and spacer sequences for translational optimization.

References

  • 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.
  • Laetitia Pieulle, Bruno Guigliarelli, Marcel Asso, Francois Dole, Alain Bernadac, E. Claude Hatchikian (1995) Isolation and characterization of the pyruvate-ferredoxin oxidoreductase from the sulfate-reducing bacterium Desulfovibrio africanus. Biochimica et Biophysica Acta. 1250 49-59.
  • van Helden, J. (2003). Regulatory sequence analysis tools. Nucleic Acids Res. 2003 Jul 1;31(13):3593-6
  • Thomas-Chollier, M., Sand, O., Turatsinze, J. V., Janky, R., Defrance, M., Vervisch, E., Brohee, S. & van Helden, J. (2008). RSAT: regulatory sequence analysis tools. Nucleic Acids Res.