As well as growing GFAJ-1 and making DNA I've been doing competence assays on Haemophilus influenzae strains. This is old-fashioned microbiology, and I seem to be the lab wizard at these assays (able to do them faster and more reproducibly than anyone else).
The big task is characterizing the starvation-induced competence responses of a number of 'unmarked' knockout mutants the RA has made. She's closing in on her goal of knocking out every gene associated with competence. She's actually made the deletion mutants of all of them, but she hasn't yet succeeded in removing the antibiotic resistance cassette (the 'marker') from eight of them. (This is necessary to eliminate possible confounding effects on downstream genes.) Here's a summary figure showing all the competence genes in the CRP-S regulon:
She recently gave me a list of 12 mutants to test, preferably three replicates of each. I've now done two replicates of 6 of them (plus control wildtype cells), with the expected result that none could be transformed at all. If these results are concordant with earlier results using the marked mutants I don't think we need a third replicate. The knocked-out genes are pilA (the major type 4 pilin), pilC (pilus assembly protein), comC (pilO homolog, pilus assembly), comE (pilQ homolog; secretin pore), comN (pulG homolog, probable minor pilin), and comP (probable minor pilin).
On Saturday I did the other 6 mutants, but I overreached myself, trying to do them along with another experiment (see below). I could tell at the time that it was hard to keep everything straight, and the results bear this out. Some plates that should have lots of colonies have none (I suspect that these were old plates that I forgot to supplement with fresh hemin), and even some of the replicate plates differ by ten-fold or more. The results for some of the strains are about what I expected, but I really don't trust any of the numbers and I think I need to do the whole lot at least twice more. These knocked out genes are HI0659 and HI0660 (cytoplasmic proteins with no known function), pilF2 (outer membrane, required for pilus assembly), dprA (cytoplasmic, protects DNA from degradation), HI1631 (location unknown; no known function), and comJ (cytoplasmic, no known function). All but comJ are competence-induced genes in the CRP-S regulon.
The other experiment I was doing on Saturday was a time course of competence development by three strains. I'll discuss this in the next post.
End of summer &
12 hours ago in The Phytophactor