My transformations worked. The crp knockout cells transformed with the ppd::lacZ or hofM::lacZ fusion plasmids gave me about 200 AmpR KanR colonies each, which should all contain the appropriate plasmid because the no-DNA control gave no colonies. The sxy knockout cells must have not been very competent, as they gave only 1 and 2 transformants with the two plasmids, but the no-DNA control gave none so these too are probably right.
I've streaked all the sxy knockout transformants and 3 of each of the crp knockout transformants. Before I go home (if the new colonies are big enough to see), I'll inoculate them into LB+Amp+Kan, so that tomorrow I can (1) do plasmid minipreps to check that they have the right plasmids, and (2) do beta-galactosidase assays to measure promoter activity in the mutant backgrounds.
I realized this morning that the proper comparison for these strains should not be with the plasmid-carrying strains I already tested but with plasmid-carrying strains that have the same genetic background as the knockouts. We do have the parental strain of the knockouts, so I've streaked it out and tomorrow will make it competent and transform it with the plasmids.
I was growing cells and pouring plates and making P1 lysates today, in preparation for various carefully done transductions, but discovered that the LB broth I was using for today's cultures is contaminated. So I'll need to start this over with fresh medium - probably not for a few days, as two manuscripts are urgently in need of attention. Both need to be completed within the week, as the senior author is about to leave for two months of ecological R&R in Belize. One is the sxy manuscript - data is still being generated and we have four reviewers' comments to address. The other is the manuscript describing what Sxy does at CRP-S promoters. Again data is still being generated, but it's otherwise almost ready to submit.
19 hours ago in Variety of Life