(Sorry for the infrequent posts. Teaching biology to 400 first-year students, and the associated administrative crap, has been sucking all the oxygen out of my brain.)
Once classes end (only a couple more weeks...) I'm planning research in two directions. One is the preliminary work for the laser tweezers measurements of DNA uptake. I'll post about that later. The other is searching for conditions that induce expression of the E. coli sxy gene.
In Haemophilus influenzae expression of sxy induces the genes of the CRP-S operon, and these in turn enable the cell to take up DNA. E. coli has most of the same CRP-S genes, and it has a sxy homolog. The grad student (we're down to one; the other one finished) has shown that artificial expression of E. coli sxy does induce expression of its CRP-S genes, but nobody has found conditions that naturally induce these genes. Such conditions would, we expect, be conditions that induce expression of sxy. Understanding how sxy is regulated, in both E. coli and H. influenzae, is key to understanding the evolutionary function(s) of the CRP-S regulon.
So my plan is to subject E. coli to the same kinds of conditions that induce sxy in H. influenzae. To tell when sxy has been induced, I can either use a fusion of the E. coli lacZ gene to the sxy gene, or use a fusion of the lacZ gene to the ppdD gene, which is one of the CRP-S genes that we know to be strongly induced by Sxy. Then I can easily detectg expression of lacZ, either in broth by incubation with the lactose analog ONPG (turns yellow), or on plates with the lactose analog X-GAL (turns colonies blue). The grad student has emailed the French researchers who studied ppdD induction, asking if they will send us their ppdD fusion.
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