Lissa plan

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FUS3/Cdc28 --------------------> Phospholocator

Update:

  1. How to make Fus3 constitutively active?
    1. Look for papers where this has been done
    2. Mutate residues in Ste7 or something?


In the next few days:

  1. Improve SUMO gel.
    1. Get better resolution
    2. Quantify amount of protein in each band?
  2. Pour plates.
  3. Grow yeast!
  4. Get pGEV construct out of e. coli (miniprep).
  5. Transform pGEV in to yeast so we can have linear expression of the phospholocator controlled by beta-estradiol.
  6. Order antibodies.
  7. Figure out protocols for using antibodies.
    1. IP has low yield... how to avoid this?
    2. Could run a gel with Fus3- and Fus3+ on it to locate Fus3 band
      1. Getting a Fus3- strain: use a Fus3/Leucine/Fus3 knockout plasmid
    3. How to locate amount of active Cdc28:
      1. Run 2 gels at the same time (one with anti-Cdc28 and one with anti-Phosphotyrosine)
      2. 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 Image:Fus3cdc28.doc), 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.
    1. 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.
    2. 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.
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