The RA found a stock of our original purR::kan knockout strain (made 10 years ago) and showed by PCR that it had the expected disrupted purR gene. So I've been checking its competence phenotype.
The original phenotyping (done 10 years ago by the grad student who made the mutant) found that the purR mutant strain grew normally and developed normal competence in the starvation medium MIV and had a slight reduction (maybe 7-fold) in how competent it became at high density in rich medium (I'll use the term "late log" for this growth condition even though the cells are not in log phase). I expected this to be reliable information because the grad student was very careful, and I've now confirmed it, finding normal growth, normal MIV competence and about a 10-fold reduction in rich medium. At present the 10-fold reduction in late-log competence is the only way this mutant differs from wildtype. That's too bad, as it doesn't give us much to work with genetically.
We do have another mutant with a stronger late-log competence defect. This is the strain that I thought was purR::kan; I made it a few years ago in an attempt to recreate the original mutant from its DNA, as I thought the mutant cells were all dead (freezer meltdown 5 years ago). I transformed wildtype cells with DNA from the purR mutant, and assumed that the KanR cells I got had the knockout. But the RA found that this strain's purR gene was the same size as the wildtype one, so I set it aside. (I'll go back and put in links to the blog posts about this strain.)
But now I suspect that this strain may indeed have a purR defect, because it had a stronger version of the late-log competence defect (~100-fold). So I think we need to find out how this strain differs from wildtype and from the purR::kan knockout. The RA suggests sequencing its wildtype-sized purR PCR fragment to see if this carries any mutations, and using inverse PCR to sequence outward from its kan cassette to find out where it is in the genome. I hope that these analyses will show differences that give us some insight into what causes the phenotype.
Welcome to the 4th Reich part 1.
16 hours ago in Angry by Choice