Lissa plan

FUS3/Cdc28 > Phospholocator
Update:
 * 1) How to make Fus3 constitutively active?
 * 2) Look for papers where this has been done
 * 3) Mutate residues in Ste7 or something?

In the next few days:
 * 1) Improve SUMO gel.
 * 2) Get better resolution
 * 3) Quantify amount of protein in each band?
 * 4) Pour plates.
 * 5) Grow yeast!
 * 6) Get pGEV construct out of e. coli (miniprep).
 * 7) Transform pGEV in to yeast so we can have linear expression of the phospholocator controlled by beta-estradiol.
 * 8) Order antibodies.
 * 9) Figure out protocols for using antibodies.
 * 10) IP has low yield... how to avoid this?
 * 11) Could run a gel with Fus3- and Fus3+ on it to locate Fus3 band
 * 12) Getting a Fus3- strain: use a Fus3/Leucine/Fus3 knockout plasmid
 * 13) How to locate amount of active Cdc28:
 * 14) Run 2 gels at the same time (one with anti-Cdc28 and one with anti-Phosphotyrosine)
 * 15) Do IP (probably more conventional)

Questions:


 * 1) 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?
 * 2) Do we have beta-estradiol lying around?
 * 3) We're using an integrating plasmid. Is this the best choice? (Apparently yes.)

Once we get supplies (antibodies - listed ), here are the things we can measure:


 * 1) Amount of phospholocator in cell.
 * 2) Relative amounts of phospholocator in nucleus vs. in cytoplasm.
 * 3) Amount of Fus3 in cell.
 * 4) Amount of ACTIVE Fus3 in cell.
 * 5) Amount of Cdc28 in cell.
 * 6) Amount of ACTIVE Cdc28 in cell.

What to do First?


 * 1) 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.
 * 2) Do same with total Cdc28 and active Cdc28.
 * 3) This means doing... Western Blots! (But not sumo gels. That's only for gel-shift.)
 * 4) 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.
 * 5) Figure out a way to influence levels of Fus3 and Cdc28 in the cell. Very important.
 * 6) 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.
 * 7) Or you could add different amounts of alpha factor to try and influence Fus3. Not as direct, though.  HAS THIS BEEN DONE? Find out.


 * 1) 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.
 * 2) How is this helpful? You could use the information about the inner workings of the system to optimize it to get a better gain.