Yesterday I did a big time-course experiment with the ppdA::lacZ fusion strain, to get a more detailed picture of the results in my last post. The information is now in my notebook: 50+ samples, each with sampling time and OD600 (cell density at that time) and volume assayed and assay time and OD420 (ONPG hydrolyzed by beta-galactosidase), but I haven't yet entered it into Excel and analyzed it.
The next important experiment is to demonstrate that the changes in expression are dependent on both CRP and Sxy. I will do this by doing simplified time-course analyses using cells carrying the ppdA fusion plasmid and a knockout of either crp or sxy. I grew up both these strains yesterday and hope to have time to test them this afternoon. If the changes are indeed due to increased activity of the ppdA CRP-S promoter, they shouldn't happen in the knockout backgrounds. I won't do this experiment until after I have analyzed yesterday's data, so I'll know which parts of the time course are most informative.
I didn't have time yesterday to repeat the transfer-to-starvation-medium analysis; I'll wait till I have the knockout results before doing this. That way I'll be sure that the expression changesI'm seeing are indeed due to the cause I want to investagate (changes in activity of CRP-S promoters).
1 day ago in Variety of Life