The strain and plasmids we've been waiting for arrived on Wednesday (thank you to the Pugsley lab), so now I can get started on my attempts to induce sxy expression in E. coli. And we do already have an E. coli sxy knockout from the Japanese group, so solving the recombineering problems isn't critical (though I should get to work on that anyway).
What to do first? The initial tests will use a reporter strain carrying a fusion of the ppdD gene to lacZ. The person who sent the strain says it should be pale blue on X-gal plates, indicating weak baseline expression of lacZ. I should first characterize its lacZ expression more precisely by growing it under defined conditions and measuring the amount of beta-galactosidase (the lacZ product) with the substrate ONPG. This is a simple classic assay, done in every introductory molecular biology lab. I'll grow cells in rich medium and minimal medium, doing the assay at various cell densities.
I need to find out whether this baseline expression is independent of the transcriptional regulators Sxy and CRP/cAMP, which we know activate the ppdD promoter. This requires transferring the ppdD::lacZ fusion into strains that carry knockouts of either sxy or crp (or cya, which encodes the adenylate cyclase that makes cAMP), or transferring the sxy and crp knockouts into the ppdD::lacZ strain. We have both these knockouts, but I don't yet have the P1 lysate I'll need to do the transfers. And I need to find out the antibiotic resistances associated with each of these knockouts, so I can plan the selections. And I need to get all the strains from the grad student who's been working with them.
I do have what I need to test whether baseline expression of the ppdD::lacZ fusion is affected by cAMP levels. The grad student tells me cAMP is normally high in the rich medium LB, presumably because LB lacks glucose, causing the phosphotransferase system to activate adenylate cyclase to make lots of cAMP. If this is correct, simply adding glucose to LB should reduce cAMP levels. So should growing cells in a minimal medium with glucose as the carbon source. If the baseline lacZ expression from the ppdD promoter is due to weak activation of the promoter by CRP + Sxy, these conditions should reduce it, and adding exogenous cAMP should restore it. If it's just constitutive activity of the promoter, these conditions should have no effect.
11 hours ago in The Phytophactor