Had my part IIs yesterday and got a lot of good ideas but mostly need to start working on the two projects that I have.
1) Streptavidin evolution with the unnatural
-For this I need to check if IDT will make me a DTB-primer.
-I want to see if some of matt's solubility mutants work in my system. If they do this will mean I might be able to skip the system evolving solubility mutants from my variant pool and focus instead on evolving the DTB-binding variants already!
I am looking for good ways to get a randomly mutated pool and I printed out Matsumura and Ellington's protocol for the same. I know there is also sexual PCR and the MnCl2 PCR, I think I need to go over those again. There is also what looks like a nice reference in it called, "The effect of high-frequency random mutagenesis on in vitro protein evolution: a study on TEM-1 beta lactamase." from J. Mol Bio in 1999. Looks pretty relevant. I think this is the most important task in this project going forward.
-Andy suggests getting some PURE system in here and testing to see how that works in comparison to my stuff. Doable. Lets see.
2)Rationally designed yeast synthetase
- M.Winkler suggested testing to see if glutathione works better than DTT. I need to check this the next time I make a reaction mix, maybe do a couple of reactions to check.
- I need to take out the LacIq portions from all the synthetase plasmids and try to make lysates with them.
- Need to make primers to PCR up the synthetase mutants.
Ok, thats it for today. I'm kinda done. And I need a vacation.
