Coding

Part:BBa_K535003:Design

Designed by: iGEM11_UNAM-Genomics_ Mexico   Group: iGEM11_UNAM-Genomics_Mexico   (2011-09-25)
Revision as of 03:36, 26 September 2011 by Alecanda (Talk | contribs) (→‎Design Notes)

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FeOx -> Clostridium acetobutylicums ferredoxin


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 102


Design Notes

Some codons of the original Clostridium acetobutylicum FeOx 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 for all synonymous codons with greatest w.

In our final construction (part BBa_K535005) we coupled this sequence with the N-terminus hydA gene (part HydA) using a flexible glycine/serine-rich linker of 14 aminoacid long in order to make the electron transfer more efficient. At the end we added a poly-His region so we can make an immuno-assay to verify that the whole construction is being exported to the periplasm, this tag is flanked by two AatII restriction sites so we can split it out if needed. We also added a double TAA terminator. The whole construction is regulated by the NifH promoter region (part NifH promoter) so it will be transcribed under microaerobic conditions.

This part was synthesized.

Source

This sequence was copied from the ferredoxin gene encoded in C. acetobutylicum’s genome.

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.