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Help: My Gal Promoter Doesn’t Work

Dear Aunt Yersinia,

I work with a yeast vector where my gene is under a Gal promoter. My boss told me to grow yeast in medium with sucrose, and then add galactose to induce the promoter. I did it, but my protein is not induced; the Western blot is empty. I sequenced a part of the vector and found out that the promoter and the gene are correct. What can I do?

Anni

 

Dear Anni,

The Gal1 promoter from fission yeast is used in many vectors for regulated gene expression. The working of the promoter is caused by the fact that for yeast, nothing beats glucose as a source of carbon and energy. As a result, the Gal promoter from the operon of galactose catabolism can be in three states: repressed, derepressed and induced.

If you grow yeast in glucose, the promoter is repressed. If you grow it on a neutral sugar such as sucrose, the promoter is derepressed – you may have a few copies of your protein per cell. And finally, in the presence of galactose, the promoter is induced, producing thousands of copies of your protein.

At least, this is the theory. In practice, even the purest sucrose source often contains traces of glucose, which is enough to repress the Gal promoter, and repression is not cancelled by galactose.

I find that cells grown in raffinose – at least in my experience – never have this problem. Admittedly, raffinose is more expensive, but if your experiment doesn’t work, you will also spend money and time trying to solve the problem.

Bonus information. Some people transfer yeast from sucrose or raffinose into medium containing pure galactose (2%), which is expensive.  Plus the cells slow down due to the sudden carbon source change and because galactose is an awkward carbon source to grow on.

This sudden change is unnecessary. Just as traces of glucose repress the promoter, even small amounts of galactose will induce it. So you can try using 0.2% galactose in your medium, or if you want to be on the safe side, 1% of your sugar and 1% of galactose.

Also, you didn’t say why you want to express your gene, but bear in mind that in some cases the Gal promoter, with its massive overexpression, may be not ideal: too much protein may be detrimental for the cell. If you require a tighter controlled promoter, you may want to use the TetO regulated promoter.

Good luck with your Gal, gal.

Sincerely yours,

Aunt Yersinia

1 Comment

  1. Uche Ogbede on May 29, 2019 at 3:02 am

    This is a very great explanation; Thanks Yersinia

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