The five double mutant strains I constructed appear to be correct. They should have the purR knockout combined with each of our five sxy hypercompetence mutations. I know they have the knockout because they're resistant to kanamycin (an insertion of the kanR gene is what knocks out purR). And I know they still have the sxy mutations, because I tested their competence under conditions that only slightly induce competence: the sxy+ purR- control had a transformation frequency of only 2 x 10^-7, but the sxy mutant purR- strains had transformation frequencies 100-500 times higher.
Now I'm waiting for the grad student's results of testing whether these mutants are now insensitive to the transformation-inhibiting effects of nucleotides. He finished the experiments last night, but won't know the results until the cells have had enough time to grow into colonies.
I'm hopeful, because my results already suggest our hypothesis is correct. As part of constructing the double mutants, I had measured the transformation frequencies of wildtype and sxy mutant cells (all purR+) under similar slightly-inducing conditions. The culture medium wasn't deliberately supplemented with nucleotides, but it's a 'rich' medium and we think that the nucleotides it naturally contains are part of the reason it allows only weak induction of competence. In this medium the sxy (purR+) mutants were only 4-10-fold more competent than the control sxy+ cells. Adding the purR knockout has made them 100-500 times more competent, suggesting that PurR does normally repress competence in this rich medium.
1 day ago in Variety of Life