Difference between revisions of "Part:BBa K535001:Design"

Latest revision as of 04:01, 26 September 2011

HydG-> Chlamydomonas reinhardtii

Design Notes

Some codons of the original Chalmidomonas' sequence had been changed for synonimous ones according to the Codon adaptation index (CAI) procedure in order to optimize its expression and to optimize 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 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.

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. We avoided sequences with restriction sites.

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

Unwanted restriction sites had been changed for synonimous codons:

• 4 sites PstI comprising the codons CTG CAG were changed: The codon CTG was replaced for the codon CTC.
• One BglII comprising the codons GAG ATC was changed. The codon GAG was replaced for the codon GAA.

Now this sequence has 7 restriction sites, 2 in the Preffix (EcoRI and XbaI), other 2 in the suffix (SpeI and PstI) and 3 SalI sites, however this sites were not taken into account for the design of our constructions.

This sequence was synthesized.

Source

This gene is in a putative operon next to HydEF and the [Fe] hydrogenase structural gene in the Chalydomonas’ genome. HydEF and HydG are co-expressed anaerobically with the hydrogenase genes.

References

• Yvain Nicolet, Lydie Martin, Cécile Tron, Juan C. Fontecilla-Camps (2010) A glycyl free radical as the precursor in the synthesis of carbon monoxide and cyanide by the [FeFe]-hydrogenase maturase HydG. FEBS Letters 584, 4197–4202.

• 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.

• 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.