The RA has finished making our purH knockout mutant. The PurH protein catalyzes the last common step in de novo synthesis of purine nucleotides, so this mutant should be entirely dependent on purine salvage for growth.
It grows fine on agar plates, and yesterday I tested its growth and competnece development in broth. The colonies are still too small to count so I won't have numbers until tomorrow, but I can tell that its growth rate and levels of competence are not dramatically different from wildtype.
Today I'm going to make up some defined medium with and without inosine as a source of purines for salvage. I predict that wild type cells (and the purR mutant) will be able to grow without inosine but that the purH mutant will not.
On Monday we should get the sequences of our various purR mutants, and then we'll be able to decide how to proceed with them.
Later: Curses! The defined medium recipe calls for glutathione (a tripeptide). I thought we would have some in the lab from the last time I made this medium (years ago), but I can't find any. And it's expensive (~$160 for 500 mg)! We've already modified the medium recipe by adding casamino acids, so I'll try just replacing the glutathione with extra casamino acids.
Next day: Damn! Neither the purH mutant nor the wildtype control grew in the defined medium, with or without inosine. Maybe they really need the glutathione, or maybe I left something out. I don't want to spend all that money getting some glutathione, so perhaps I'll try the older defined medium (Herriott et al. 1970), or maybe I can scrounge a bit of RPMI tissue culture medium from a colleague and try the new-fangled medium (Coleman et al. 2003).
1 day ago in Variety of Life