FUS3/Cdc28 --------------------> Phospholocator
- How to make Fus3 constitutively active?
- Look for papers where this has been done
- Mutate residues in Ste7 or something?
In the next few days:
- Improve SUMO gel.
- Get better resolution
- Quantify amount of protein in each band?
- Pour plates.
- Grow yeast!
- Get pGEV construct out of e. coli (miniprep).
- Transform pGEV in to yeast so we can have linear expression of the phospholocator controlled by beta-estradiol.
- Order antibodies.
- Figure out protocols for using antibodies.
- IP has low yield... how to avoid this?
- Could run a gel with Fus3- and Fus3+ on it to locate Fus3 band
- Getting a Fus3- strain: use a Fus3/Leucine/Fus3 knockout plasmid
- How to locate amount of active Cdc28:
- Run 2 gels at the same time (one with anti-Cdc28 and one with anti-Phosphotyrosine)
- Do IP (probably more conventional)
- Can you us the beta-estradiol system on a Gal4+ strain of yeast? How important is it to have a Gal4 knockout, if you are growing on glucose anyway?
- Do we have beta-estradiol lying around?
- We're using an integrating plasmid. Is this the best choice? (Apparently yes.)
Once we get supplies (antibodies - listed File:Fus3cdc28.doc), here are the things we can measure:
- Amount of phospholocator in cell.
- Relative amounts of phospholocator in nucleus vs. in cytoplasm.
- Amount of Fus3 in cell.
- Amount of ACTIVE Fus3 in cell.
- Amount of Cdc28 in cell.
- Amount of ACTIVE Cdc28 in cell.
What to do First?
- First I need to see how the antibodies work. So I should try to isolate total Fus3 and active Fus3 and measure their levels in asynchronous cells just to get a baseline.
- Do same with total Cdc28 and active Cdc28.
- This means doing... Western Blots! (But not sumo gels. That's only for gel-shift.)
- Should test the linear induction system. Should set up 2 arrays of cells. One of them will eventually be arrested with nocodazole, the other with alpha factor. Each array will recieve a gradient of beta-estradiol at the outset - so some plates will get none, some very little, and some quite a lot. (Note: I have no idea what constitutes "a lot".) Under the microscope, I should see 1 array of cells with varying degrees of cytoplasmic brightness, and one array with varying degrees of nuclear brightness.
- Figure out a way to influence levels of Fus3 and Cdc28 in the cell. Very important.
- Can use the ATP analog to do this. Add varying amounts of analog; could perhaps create a graph. X axis is [analog] and Y axis is the gain.
- Or you could add different amounts of alpha factor to try and influence Fus3. Not as direct, though. HAS THIS BEEN DONE? Find out.
- Longer term: Find out how changing the levels of Fus3, Cdc28, and the phospholocator affects the system. For example, does lowering Fus3 have no effect until a certain point? Then Fus3 is probably in excess. Can you lower the phospholocator to a very low level with no change in brightness? Then perhaps you could increase the gain of the system by adding more Fus3.
- How is this helpful? You could use the information about the inner workings of the system to optimize it to get a better gain.