I'd like to create a plasmid for S. cerevisiae (CEN URA3) that scrambles the codon usage of an essential gene to such an extent that the homologous recombination machinery cannot use it as a template to repair the endogenous locus. I'm having problems generating spontaneous suppressors of a mutant allele that are not merely gene conversions back to wild-type by repair off a centromere plasmid. Obviously the expression level may change but that is something that can be fixed by trying multiple promotors. Any help appreciated!
DNA2.0 has a gene analysis program (Gene Designer) that will show you the alternate codons, but it won't automatically assemble the changes into a new gene. The fastest method may be to simply make all the codon changes manually. For just a single gene, that shouldn't take very long.
You can translate to amino acid sequence and then back-translate using E. coli or human/mammal or some other preferred codon set. Or make up a random codon use table to use. But codon use can have a very profound effect on expression of the protein. For one example, HIV-1 genes expressed in mammalian cells produce some 200-fold less protein, than mammal-optimized codons for the same amino acid sequence. It is not a minor difference.
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