promoter function - (Feb/24/2009 )
i have transformed a vector in yeast. this vector has two expression cassettes, one is antibiotic for screening transformants and another is for expression of gene of interest. both of them have the same promoter. i have the transformants which is resistant to the antibiotic and also i checked the transformation by PCR. but the expression of the gene wasn't examined. if the promoter worked in the antibiotic cassette, if it should work in another cassette? thanks a lot.
why nobody explain something, please.
assuming everything went all right, it should work.
however, this being biology, it is no certainty that the gene will be expressed well or expressed in a functional form. Things such as codon usage of the gene of interest, unexpected post translation modifications, protein toxicity, aggregate formation can cause problems.
Can the activity of the protein being expressed be directly observed?
additionally, the gene might need some stimuli to be active. and maybe instead of protein activity (in case the product is not functional as suggested by perneseblue) you could investigate mRNA levels of this gene by rt-pcr.