CUP1 promoter and Copper toxicity to S. cerevisae
 

Dear Yeast Netters,
There is a question regarding CUP1 promoter:
I placed a gene of interest under the CUP1 promoter. When the cupric
sulphate is above 100 uM, I start to see the dark cell sediment on the
tube bottom. I assume this is due to the copper toxicity. When the
inducer goes up to 500 uM, I saw improved protein production, but the
cell growth was inhibited a lot, and i saw debris by Flow cytometry.
However, I checked with a paper ( Hottiger, T., Furst, P., Pohlig, G. &
Heim, J. Physiological Characterization of the Yeast Metallothionein
(Cup1) Promoter, and Consequences of Overexpressing Its Transcriptional
Activator, Ace1. Yeast 10, 283-296 (1994)), the amount of cupric
sulphate was much lower than the toxicity limit (2mM).

Would you please share with me your experience on CUP1 promoter? Is
there any substitute to replace CUP1 promoter if the toxicity is a
really problem?

Thanks a lot!

Jingjing

Re: CUP1 promoter and Copper toxicity to S. cerevisae
 

I've typically done CUP1 inductions at 250 uM maximum - that's the concentration of Cu²⁺ our lab tends to use. I've not noticed ill effects on the yeast at this concentration but it might be because I haven't looked hard enough.

Side note: the CUP1 promoter is always on at a moderate level of expression even without the presence of Cu²⁺.

Re: CUP1 promoter and Copper toxicity to S. cerevisae
 

Didn't know that, thank you.

Re: CUP1 promoter and Copper toxicity to S. cerevisae
 

I think it's an important point that most people tend to ignore - promoters are never truly "off", and basal transcription rates vary greatly. The CUP1 promoter without Cu²⁺ just has a basal rate that is significantly higher than the GAL1 promoter in dextrose.

That leads me to a side note, that the GAL1 promoter in dextrose, even an attenuated promoter, expresses three essential proteins I've worked with at high enough levels to allow proliferation, albeit very slow, of yeast :frusty:. This can complicate genetic analyses, and should be something that we all are aware of.

Re: CUP1 promoter and Copper toxicity to S. cerevisae
 

thank u sir............
i have need complete research article
on toxicity........