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).
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in The Biology Files
Not your typical science blog, but an 'open science' research blog. Watch me fumbling my way towards understanding how and why bacteria take up DNA, and getting distracted by other cool questions.
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