Most of the B. subtilis culture and transformation problems have been solved. What appeared to be growth of the trp- and met-requiring strain Y on minimal-glucose agar with no tryptophan or methionine turned out to be tiny crystals (of CaPO4?) that were seeded in the agar by the act of streaking over it. I now realize that I shouldn't have added CaCl2 to the agar at all.
The only problem remaining is that strain Y can grow moderately well on plates without added methionine, because the defined medium recipe says to add a small amount of 'casamino acids' to the agar. Casamin acids is the mix of amino acids obtained by breaking down the milk protein casein with acid; this destroys the tryptophan but the mixture is about 1% methionine by weight. The genotype of strain Y shows that it should only need methionine and tryptophan, and strain W shouldn't need any amino acid at all. My first test of medium without any casamino acids had no growth of either strain, but maybe I messed up. I've now tested using half and one sixth as much casamino acids as the recipe specifies (cells grow fine), and now I'm testing one tenth as much and none again.
I put strain Y through the B. subtilis competence ritual: Grow )shaking vigorously) in the minimal glucose medium plus casamino acids and a bit of yeast extract until growth has been slowing for 90 minutes, then dilute tenfold into more of the same medium with extra magnesium and calcium and shake for another hour, then mix cells with DNA of strain W. I definitely got transformants to Trp+, but the frequency was quite low. (I couldn't tell about transformation to Met+ because of the background growth due to the casamino acids.) The low frequency could be because the cells weren't very competent or because the strain W DNA was very old (it's been in the fridge for somewhere between 10 and 20 years ).
So now I'm going back into the lab to inoculate a culture of strain W so I can make some fresh DNA tomorrow.
1 day ago in Variety of Life