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Hi, Servers to check for rare codons definitely do exist. http://genomes.urv.es/OPTIMIZER/ http://www.doe-mbi.ucla.edu/~sumchan/caltor.html and several others As to comparing Rosetta and Codon+ - your best bet here is just to dig up product literature from the manufacturers. Rosetta uses pRARE: AGG, AGA, AUA, CUA, CCC, GGA Codonplus BL21-CodonPlus(DE3)-RIPL strain argU (AGA, AGG), ileY (AUA) , proL (CCC), leuW (CUA) BL21-CodonPlus-RIL strain argU (AGA, AGG), ileY (AUA), leuW (CUA) BL21-CodonPlus(DE3)-RIL strain argU (AGA, AGG), ileY (AUA), leuW (CUA) BL21-CodonPlus(DE3)-RIL-X strain argU (AGA, AGG), ileY (AUA), leuW (CUA) BL21-CodonPlus-RP strain argU (AGA, AGG), proL (CCC) BL21-CodonPlus(DE3)-RP strain argU (AGA, AGG), proL (CCC) BL21-CodonPlus(DE3)-RP-X strain argU (AGA, AGG), proL (CCC) Artem > Thanks for the reply. > > I've checked my sequence for rare codons; however, what would be useful to > a > pseudo-molecular biologist like me is a web server which will look at your > input DNA sequence and guesstimate the success of expression in E. coli > (i.e., consider codon frequency). Does one exist? Even better, a web > server > that offers the choice of different commercial forms of E. coli (i.e., > Rosetta and Codon+)? I tried Googling this a few days ago and didn't find > anything too useful? > > Cheers! > > On Tue, Feb 24, 2009 at 11:00 AM, <[log in to unmask]> wrote: > >> Hi, >> >> The question you have to ask yourself first is - does my gene actually >> *have* the rare codons that you're trying to avoid? Experience shows >> that >> usually it's not single codons that are a problem but pairs or triplets >> of >> rare ones. If your gene does not have obviously bad codon combinations >> you >> still may derive a benefit from codon optimization and especially from >> mRNA structure shuffling. There are articles out there that attempt to >> present statistically significant evidence and I tend to agree with them >> - >> the practice of optimizing codon usage AND mRNA structure is a good and >> useful one. >> >> From what you describe, you're working with a kinase. It is not at all >> uncommon to have toxicity of kinases for E. coli. Additional issues >> include heterogenous phosphorylation if the kinase is normally active or >> can auto-activate in E.coli (classical example is PKA which can >> phosphorylate itself in up to 30 places given the right conditions). >> >> Toxicity isn't difficult to spot - classical signs include >> microcolonies, >> heterogenous colonies, slow growth, plasmid instability and so on. Even >> with a native gene you would likely notice these symptoms to some >> extent. >> >> Incidentally - toxicity usually equals folding, i.e. for a kinase to be >> toxic at least some of it has to be folded enough to work. This is >> actually *good news* because toxicity can be combated on a different >> level >> than lack of folded expression. For example, co-expression with >> phosphatases tends to work miracles for kinase-based toxicity. >> >> Finally, to answer your question directly - yes, i've seen several cases >> of proteins not expressing even with rare codon tRNA supplemented in >> trans, but expressing well from optimized DNA. Again - optimized for >> codons AND structure, as I've never separated the two processes. >> >> Synthetic DNA is cheap these days. If you can afford it - it's useful to >> try before taking the next step. In this case the obvious next step is >> attempt at expression in insect cells - kinases usually work out really >> well in IC. >> >> Artem >> >