So last post we said we'd go into accessing the genetics of peppers; our goal is to find the sequence of the capsaicin synthase, so we can create some kind of interference in our peppers to prevent it from expressing.
So we can hop over to pubmed and search "capsaicin synthase" under "Nucleotide" and we get seven or eight hits, all 981 bp.
Unfortunately, we've been reading too much recent literature on the subject and this raises red flags: according to an RNA-sequencing profiling project of Lin, Lei, et al, ( 2012)
1: There is no complete sequence of the pepper genome.
2: Within the more limited sequencing projects that have occurred, no one is quite sure what the capsaicin synthase gene is.
Back to the genes we found on pubmed:
They mostly stem from a paper published in PNAS in 2006, which was retracted in 2008. The paper is fairly exhaustive: they did a column fractionation of placental enzymes, then tested each fraction for capsaicin synthase activity, by putting the enzyme mixture in a solution of vanilllylamine and 8- methyl-nonenoic acid, and testing the amount of capsaicin that resulted on HPLC.
They then purified the enzymes on a column containing bound vanillylamine, in the hopes that the affinity CS has for vanillylamine would purify it from other enzymes.
Now that they have a fairly pure product, they sequence it, starting with its amino acids, and go to the point of taking their genomic sequence, putting it into an E.coli vector, and making it heterologously. However, although they give a number for the activity of the heterologous csy1, there are no figures. One would think that you would want to blast that piece of proof right up front where everyone can see it.
The retraction states that the genetic sequence shows homology to a protein kinase found later, by a M. Rapolu. Since the homology is for a small part of this kinase (981 bp for CS of > 3000 bp for the kinase), this suggests that their gene was wrong, although chances are good that they did purify some kind of synthase, before the sequencing part.
This means that there probably isn't any juice to this protein. It also suggests that they never inserted the gene in E. coli, or didn't get any activity, or something in the media / E. coli spontaneously creates capsaicin, which is why there are no controls.
But that is somewhat rude. If biotechnology was as easy as taking one beaker and pouring it into another, we wouldn't have to write this blog. Messing with genes is hard. Why is it so hard?
- Most genes are very hard to see, and when you purify them or do PCR on them, they mutate, sometimes in a way that makes them inactive.
- Although it is easy enough to align genes with other genes that you may have some idea of their function, actually figuring out the function of any particular gene, in the context of the thousands of other genes, can be a combinatorially hard problem.
With that cautionary note, next time we will discuss the candidates for capsaicin synthase, and how we might go about testing them.
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