The postdoc and former RA generated some great RNAseq data, which I'll write about in another post. But we have some money that needs to be spent on sequencing in the next couple of months, so we need to decide which additional RNA seq runs we should do. And then I'm going to grow the cultures and prep the RNAs.
We have data sets showing how RNA levels change after transfer to competence-inducing MIV medium for several Haemophilus influenzae strains: wildtype (2 expts), sxy- (2 expts), HI0659- (1 (antitoxin?, 1 expt) and HI0660- (toxin?, 1 expt). For each we have samples at t=0, t=10, t=30 and t=100 minutes. (The figure shows a comparison between wildtype and sxy- at t=0 and t=10; the red circles are CRP-regulated genes and the blue ones are competence genes.) We need to do at least one replicate of the HI0659 and HI0660 cultures. If we also did another replicate of everything, that would be a full 24 sample run (one lane?) for the sequencer, and enough data that we could do proper statistical analyses.
But I also want to get RNAseq data for strains with other mutations, especially the hypercompetence-causing mutation murE749 in exponential growth. This would be a single condition, replicated once or twice, so 2 or 3 samples total. I might be able to squeeze this in with the run described above; depending on what other experiments we plan to do with these strains, two replicates of some might be enough. Or it might be better to do a second run since we have the funds, doing a more comprehensive analysis of other conditions and other mutants too.
One condition I'd like to examine is 'late-log' growth, where wildtype cells develop moderate levels of competence. I want to see if these levels are comparable to those in the HI0659 antitoxin mutant, which shows no competence at all although it appears to have (compared to wildtype cells) only a slight decrease in competence gene expression at t=10, no decrease at t=30, and a moderate decrease at t=100. It's possible that the toxin acts only by decreasing mRNA levels of other competence genes, but the disproportion between its absolute competence defect and modest RNA defect makes me wonder if it also does something else.
We have three other hypercompetence mutants with mutations in murE - I don't know if it would be worth doing one sample of each of these in exponential growth. We also have a hypercompetent mutant of unknown genotype - RNAseq might find the mutation as well as show us the RNA changes. Might it be worth testing a crp mutant or cya mutant, to confirm our understanding of cAMP/CRP regulation? Or the purR or purH mutant, or cells whose MIV-induced competence development has been blocked by adding AMP? Or cells whose DNA replication has been blocked by hydroxyurea (depletes dNTP pools). Or the hfq mutant, which has 10-fold lower competence.
Welcome to the 4th Reich part 1.
16 hours ago in Angry by Choice