yeast expression (gal1-promotor) - expression in glucose?? (Jun/04/2011 )
I'm trying to express my genes with a gal1-promotor in yeast (the sequences are derived from cds).
I got colonies after transformation of yeast with the vector and i have taken a positive colony-pcr.
I have isolated rna of this yeast cells and made cdna with a kit. the concentrations are good and the rt-pcr works! but that's the problem. i have positive pcr-products of the galactose AND glucose samples. it means, that my gene is also expressed in glucose containing medium. could it be? there is not direct repression by glucose (but the pcr-product is only a little bit lesser than the cell-samples in galactose-medium).
The samples are genomic dna-free, I have checked this with control primers for the vector. there are also no genes in yeast, which was amplified with my primers (I have also checked this with a wildtype-sample).
plz, can somebody help me?
It is possible that gal1 promoter sequence (of vector) is not 100 % homologous to the original gal1 sequence. It seams that about 200 bp of promoter sequence are very important for control of gal genes. Can you check your sequence to the original one (just do a simple blast). If so, promoter is leaky and even a low concentration of rna is enough for rt-pcr.
I was using gene A (keeps cells alive)under constitutive control and gene B (kills cells) under gal1 borrowed from pGilda. Compared to A only the number of colonies was considerably lower in the presence of A + B, on glucose. So there was something going on with pGilda's gal1 promoter. Not to mention cloning and time in e. coli which sometimes introduces some changes.
If there are differences you can get promoter from the yeast and clone it into your vector.