We think that the previous attempts at inducing E. coli's ppdD were unsuccessful because the researchers didn't know that this gene has a CRP-S promoter that requires Sxy, and didn't know what we know about regulation of sxy expression and of competence genes in H. influenzae.
We know that competence is induced by an abrupt nutritional downshift, so I'll try to induce E. coli sxy the same way. The previous researchers tested nutritionally-limiting conditions, but only in steady state (log phase) culture. So I'll grow E. coli in rich medium (LB or sBHI) and transfer it to either MIV or the simple salts used in various E. coli defined media. I'll also test the effect on ppdD of abruptly cutting off the culture's oxygen supply, and of the onset of stationary phase.
Because we have evidence that in H. influenzae sxy induction depends on depletion of nucleotide pools, I'll try various genetic tricks that may cause E. coli to suddenly run out of nucleotides for DNA or RNA synthesis. The most straightforward is culturing purine auxotrophs in rich medium, and then abruptly transferring them to a defined medium lacking purines.
cAMP induces sxy transcription in H. influenzae, but I don't think the E. coli sxy gene has any obvious CRP site. However I'll probably add cAMP to the various test media, both because we know the CRP-S promoters need it and just in case sxy transcription does too.
Friday Fabulous Flower - Palm flowers
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