I want to synthesize several genes with the promoter and terminator, I have chosen the enzymes I want to use and everything is ready to go. But when I requested the company to synthesize it, they told me the promoter have the same sequence of 2 enzymes I want to use. My question is: May I change the promoter sequence in one or two bp (which would be the best for me) or should I change the enzyme? The organism is Saccharomyces cerevisiae and the promoter ir GPD (Glyceraldehyde 3 phosphate promoter)
It depends, if restriction site is not in any functional element of the promoter, you could easily change couple of bases. You could find description of fine GPD promoter structure in N of papers, and imply from there. Still, easiest way is to adjust your scheme and use two new enzymes.
Agreed, if you don't yet know the binding sites for the cognate transcription factors then changing any base is probably a bad idea. When I have this internal site problem, I normally look for an alternative restriction enzyme with a compatible sticky end so that I can still clone into the same vector, even if this destroys the cloning site itself.
You can choose another restrciton enzyme for your sequence from dcaps finder program (http://helix.wustl.edu/dcaps/dcaps.html) then try the enzymes for your sequence on restriction enzyme program (http://www.restrictionmapper.org/).
Thanks a lot everyone!!! I'll do what you told me to do, won't touch the promoter and change the enzymes. Thanks Muhammed Burak for the sites, they will be very useful!
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