Hey everyone,
I am working on S. cerevisiae SK1 cells to study meiosis. I have transformed my cell cycle protein tagged with FRB into SK1. Checked on WB. I am trying to establish anchor-away system for my protein, the experimental strain (complete anchor-away system, including tor1 and fpr1 mutations) doesn't grow on rapamycin and shows big cell size as expected. But, for my control strain (carrying FRB-tagged protein, tor1 and fpr1 mutations), they don't grow on rapamycin!! And the cells are a bit big in size (cell cycle defect phenotype)!
I am thinking that the tag hinders the activity of my protein, which is essential, in the presence of rapamycin. But I don't understand how they became sensitive to rapamycin without FKBP12!!
Could they be temperature sensitive? I am checking this right now. Any other possibilities/suggestion?
Appreciate your input!
Update:
They are not temperature-sensitive. They grow normally in 37 degrees!
Hello,
Your problem does not seem to be easy to solve/overcome, whichever if the FRB tag compromises the function of your gene of interest or not.
Does it really need to be anchoring away?
Can't you use auxin-induced degron( AID) system?
There are couple of options (N-terminal or C-terminal, full length AID, 1x mini AID, and 3x mini AID) and they seem to have different leakiness or negative impact on the gene function.
The system will require similar degree of modifications to your host cells.
But perhaps you can test first if adding the AID tag affects the function of your gene of interest, if the gene is essential.
--
Shin
Thank you for your reply Shin.
I forgot to add that SK1 cells with My protein-FRB grow normally in YPD (without Rapamycin). So the mysterious phenotype appear when I add Rapamycin.
Not necessarily. Anchor-away technique is used successfully in my lab for many other proteins. Perhaps, tagging the FRB to the other side of my protein (N-terminus) would give me a different output! Let's see.
And thank you, I will include AID system in my plan as well.
Best,
Mohammad
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