Yesterday I looked at changes in the numbers of viable cells (cfu, colony-forming units) after cells of different genotypes (wildtype, purH, purR) had been transferred to the starvation medium MIV. I also did this after transfer of wildtype cells to the defined medium cMMB without inosine.
The experiment had a couple of problems. I'd wanted to compare survival of sxy- cells, but the only vials of frozen cells we have date from before the big freezer meltdown of 2004, and the culture I started from one vial remained resolutely non-turbid all day. However I streaked it out and some colonies grew up overnight, and I've now confirmed that they are indeed kanamycin-resistant (the knockout is an insertion of miniTn10kan). So I can test them tomorrow. I also had intended to use RM- rather than cMMB-, but my new stock of the 10x base for MMB smelled very nasty after autoclaving, and had a large precipitate, so I suspected that the glutathione I'd included had broken down (though the published instructions say to autoclave it). So I threw it out and made fresh without glutathione, but this has also thrown a big precipitate. Now I wonder if the problem is that this time I carefully adjusted its pH up before autoclaving, which the instructions don't say to do. The solution started to become a bit cloudy when the pH got above 7.0, so I adjusted it down a bit, to 6.8, before autoclaving. I'll try again tomorrow.
Anyway, both the mutant strains survived in MIV just as well as wildtype, with the cfu increasing 2-3-fold in 60 minutes. I left the cells incubating overnight and plated them again this morning, so we'll see if there's any differences in longer-term survival. The wildtype cells transferred to cMMB didn't divide at all, nor did they become even a tiny bit competent.
Now that I know roughly what to expect, I can do a more detailed test, and include the sxy knockout mutant.
Welcome to the 4th Reich part 1.
16 hours ago in Angry by Choice