OK, the fusion of the ppdA promoter to lacZ produces 100-fold more beta-gal when Sxy is overexpressed. What are the important things to do with it?
1. Find out how sensitive the ppdA promoter is to Sxy: The pASKAsxy plasmid produces a great deal of Sxy when its promoter is induced with IPTG, but we know that most of this Sxy aggregates into insoluble and probably nonfunctional 'inclusion bodies'. So I could try inducing with decreasing concentrations of IPTG, and measure the effect on beta-gal production. It may be that only very slight induction of Sxy will give a big induction of the ppdA promoter. Ideally the amount of Sxy would be measured too, but we don't (yet) have the antibody to E. coli Sxy that would let us easily make these measurements. The beta-gal vs IPTG assay will be easy. If I find that ppdA induction needs only a tiny bit of IPTG, I'll have more confidence that natural induction will need only modest induction of Sxy expression from its normal promoter.
2. I should also do a time course of induction, so I know how quickly the steps happen (IPTG causes sxy transcription, Sxy protein is made, Sxy causes transcription of ppdA::lacZ, beta-gal is made). I can do this with the standard (1 mM) IPTG concentration I used yesterday. Yesterday I waited 2 hours after adding IPTG before assaying beta-gal; now I'll try 1 min, 2 min, 5 min, 10 min, 30 min, 60 min, 90 min, 120 min. The results will let me optimize the assay conditions I'll use when looking for culture conditions that induce Sxy expression from its normal promoter.
What culture conditions should I test first? My previous finding that the baseline expression of this fusion is independent of Sxy and CRP means that I shouldn't bother looking for conditions that reduce expression, so I won't try adding glucose to the medium to turn off CRP. I will try adding cAMP to the medium to make sure CRP is fully active. I will do a time course, following growth of the culture in LB, looking especially at expression as the cells' growth slows at high cell density. I will transfer the cells from LB to minimal salts (with a small amount of amino acids) to mimic the inducing effect of transferring H. influenzae from rich medium to MIV. Maybe I'll try growing the E. coli in BHI before such a transfer, as BHI is a much richer medium than LB. Perhaps I should get an E. coli mutant that can't synthesize purines (or pyrimidines), to see if transferring it to minimal gives induction.
Other things I might do:
Should I try to get a strain carrying this fusion in the chromosome rather than on a plasmid? This would eliminate concern about possible variations in the number of copies of the plasmid, due to differences in culture conditions. And if the chromosomal insertion was stable I could eliminate the selective antibiotic from the test cultures. This sounds like a job for recombineering - I wonder if we can still get this working.
Should I sequence the ppdD::lacZ fusion and the hofM::lacZ fusion to see if they have mutations that would explain their failure to be induced by Sxy?
How dumb is too dumb? We still don't know!
7 hours ago in The Phytophactor