Coding

Part:BBa_K535000:Design

Designed by: iGEM11_UNAM-Genomics_ Mexico   Group: iGEM11_UNAM-Genomics_Mexico   (2011-08-24)

HydEF -> C. reinhardtii


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 2774
    Illegal NotI site found at 3080
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 127
    Illegal NgoMIV site found at 148
    Illegal NgoMIV site found at 258
    Illegal NgoMIV site found at 931
    Illegal NgoMIV site found at 1051
    Illegal NgoMIV site found at 1114
    Illegal NgoMIV site found at 1528
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 334
    Illegal BsaI.rc site found at 754
    Illegal BsaI.rc site found at 961
    Illegal BsaI.rc site found at 2764
    Illegal BsaI.rc site found at 3121


Design Notes

Codons of the original Chalmidomonas' sequence were changed for synonimous codons according to the Codon adaptation index (CAI) procedure in order to optimize its expression and Rhizobium etli’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 sequences according to the CAI procedure we first obtained relative adaptiveness (w) for each codon (1 being the most frequent codon, 0 a non-existent codon) in R. etli and then we substituted codons in target CDS with all synonymous codons with greatest w.

Some standard restriction sites arose from this optimization. They were removed changing one of the codons spanning these sites for synonimous codons.

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

The constitutive promoter from LacZ has been added to regulate the expression of HydEF.

This sequence was synthesized.

Source

This sequence originaly comes from the genome of the unicelular photosynthetic green alga Chlamydomonas reinhardtii, but as specified in "Design Notes" it has been modified in some parts.

References

• Sofia, H. J., Chen, G., Hetzler, B. G., Reyes-Spindola, J. F. & Miller, N. E. Miller. (2001). Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods. Nucleic Acids Res. 29, 1097–1106

• 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

• Paul M. Sharp, Wen-Hsiung Li (1986). Codon usage in regulatory genes in Escherichia coli does not reflect selection for 'rare' codons. Nucleic Acids Res. 14, 7737-7749.