I've been futzing around with phage P1, trying to get a high-titer lysate from a single clear (P1vir) plaque, with no success. Part of the problem is the high frequency of what I guess are revertant phage, giving larger and turbid plaques, and partly it's that I've been trying to squeeze the work in between other stuff (peer-reviewing manuscripts and a proposal, preparing my freshman biology final exam, etc.).
But today's clear, and I'm ready to roll. Last night I innoculated cultures from single colonies of the E. coli wildtype strain W3110 and three mutant strains: ppdD::lacZ, sxy::kan and crp::kan. Last night I poured lawns of W3110 with appropriate dilutions of two of my not-very-satisfactory lysates. When I could the plaques this morning I'll know the titer of these lysates, and can then use one or the other to infect the four E. coli strains. I'll do these infections in broth and maybe also on plates.
Then I'll collect the lysates and titer them all overnight on W3110 lawns. I may also do a test transduction overnight, using the W3110 lysate (before I know its titer) to transduce the lacY gene into the lacY mutant strain C600. This should give me all the information I'll need for tomorrow, when I want to infect the sxy and crp knockout mutant strains with the ppdD fusion lysate, and vice versa, and select for transductants that have both the ppdD fusion and the sxy or crp knockout. Then I can test whether the high 'baseline' expression of lacZ in the ppdD::lacZ fusion is due to high baseline activity of its CRP-S promoter, as such activity should drop dramatically if sxy or crp is knocked out.
I'll need to make some minimal-lactose plates to properly score the result of my test transduction, for which I'll need to make up and sterilize stock solutions of the amino acids threonine and leucine, the vitamin thiamine, and lactose, because C600 is thi- thr- leu- as well as lacY-. (I used this strain a lot in grad school, and its genotype is burned into my brain.) And ideally I should also make up some of the TTC we used to put into minimal plates that makes the colonies turn red and easy to see. Right now I can't remember what TTC stands for, but it will come back to me, and I think we have a big bottle of the stuff somewhere.
A new kind of problem
12 hours ago in RRResearch