We convinced our A. pleuropneumoniae collaborators that the manuscript should be submitted in its current form after I do another replicate of the time course (and with some genome-analysis data presented as a table rather than just described in a sentence in the text). So now I first need to do a test of expression-time requirements for starved and growing cells, which should be relatively simple.
Quick plan: start with frozen starved cells, thaw cells, resuspend half in fresh starvation medium (to get rid of the glycerol they're frozen in) and half in a larger volume of rich medium. Add DNA to the first cells, incubate 15 minutes, add DNase I, incubate 5 minutes, add an equal volume of rich medium, continue incubating. At intervals (0, 10, 30, 60 minutes) take samples, dilute and plate on plain and kanamycin plates. Incubate the rich-medium cells until they're moderately dense (OD about 1.0), then add DNA and DNase I and sample as above. Total of 8 samples, needing about 32 plain plates and about 50 kanamycin plates. This time include a no-DNA control.
Then, do another time course like the one I did last week.