I have one more experiment to do for our toxin/antitoxin manuscript. I need to make sure that survival into and recovery from stationary phase is normal in the antitoxin-knockout mutant. This strain overexpresses the toxin gene and cannot inactivate the resulting toxin protein. We already know that it produces a normal-looking growth curve using the Bioscreen; one of the lines in the graph below is for the antitoxin knockout), but this analysis is based on changes in culture turbidity and does not consider whether some of the cells contributing to turbidity might be dead. This isn't a concern for rapidly growing cultures, but is for cells that have ceased growing. So I need to complement the Bioscreen result with growth curves made by diluting cultures and plating the cells, to measure viable 'colony-forming-units' rather than just turbidity.
I would normally set up four cultures (wildtype, toxin knockout, antitoxin knockout, double knockout), but there's a complication. The double knockout antitoxin mutant only exists in a 'marked' version (with a spectinomycin cassette inserted in place of the missing genes) and the antitoxin knockout only exists in an 'unmarked' version (no spectinomycinR cassette). If this cassette influences growth or survival this difference could cause anomalous results. The toxin knockout exists in both forms, so I'll include both of them in the analysis.
First step is to restreak all the strains from the freezer stock. I did this last week but foolishly let the cells die on the plates rather than restreaking them.
nice
ReplyDelete