We now join a series of experiments already in progress

In the previous post I described the RNA-seq data that indicates that our antitoxin knockout is unlikely to express functional toxin during log-phase growth, because the promoter it would otherwise repress appears to begin transcription well downstream of the toxin start codon.  The graphic I showed was not exactly the right one, but the evidence is good.)This would explain why the antitoxin knockout grows normally in rich medium, and suggests we should look for a…

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I’m actually going to do an experiment!

Last week I went to Philadelphia, to give a talk and spend a few days working with the former postdoc on the toxin-antitoxin manuscript.  This manuscript has a former Honours student as first author; it's has been languishing for most of the past two years, with a few spurts of progress.Until now we have been thinking that the toxin protein was not broadly toxic to cells, but somehow only prevented them from taking up DNA. …

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Does variation in sequencing coverage help explain apparent variation in recombination?

Preamble:  The grad student and I have been looking at the Illumina sequencing coverage of the DNAs used for our DNA-uptake project, and considering how best to exclude from analysis the bits of the genome that consistently have very low coverage.  We're probably just going to exclude all positions that have fewer than 10 reads in the control 'input' DNA sample.  But thinking about this got me thinking about the extent to which position-specific differences…

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Making sense of RNA-seq comparisons

***Hey, it's RRResearch's 10th blogiversary!***Back to work:I'm working on the toxin-antitoxin manuscript, and trying to use the RNA-seq data to decide which genes have changed expression in which mutants.  This information should help us understand how the toxin acts to prevent DNA uptake, and what else it might affect.The comparisons should be straightforward because the former undergrad/summer student left me with a superb set of analyses and R scripts, including EdgeR and DESeq2 analyses comparing…

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Evidence for a likely sample switch in the RNA-seq dataset (or not)

I've been working on the toxin/antitoxin manuscript, trying to extract all the conclusions from the RNA-seq data trove.  We have two odd results, and I now think they are both best explained by a sample switch in the first set of samples.One odd result that the former post-doc drew my attention to is that, when the antitoxin is deleted, expression of the competence genes appears to be down at the last time point ('M3', 100…

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Expression of DNA uptake genes in rich medium – a puzzle

I've been working on the toxin/antitoxin paper.  Right now I'm going through the RNA-seq data for the antitoxin knockout (again!), looking for hints of how unopposed toxin expression prevents DNA uptake.  The two graphs below show mRNA levels of the mutant compared to wildtype cells at the same stage of competence induction (upper panel, 30 min in MIV; lower panel, 100 min in MIV). The green bars are expression in the mutant (unopposed toxin) and…

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Biofilm assay results

The summer undergrad did the biofilm assay this week. The results are quite clear: Haemophilus influenzae does form what might be biofilms on glass tubes, but this is completely independent of competence gene expression or the ability to make Type 4 pili (T4P).  Thus we won't be able to use biofilm assays to clarify how the toxT toxin prevents DNA uptake.The basic assay was as described in the previous post.  She tested four strains:  the…

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Does H. influenzae need DNA uptake genes to form lab biofilms?

This morning I had another Skype conversation with the (most recent) former post-doc.  We mostly talked about the toxin/antitoxin work.  One question that came up was whether the antitoxin knockout strain was unable to form simple biofilms as well as to take up DNA.The kind of biofilm I mean is a simple film of cells that might stick to the surface of the glass or plastic container the cells are being cultured in.  Formation of…

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One more bicyclomycin try!

The previous Bioscreen experiment failed because, as we suspected, the vial we purchased didn't contain the expected mg of bicyclomycin.  The highest concentration we tested (20 µg/ml) caused only a very slight slowing of growth, so we contacted the supplier and had them send us a new vial.  This contained more visible powder than the previous one had, although still a very tiny amount), and we used it for a new Bioscreen experiment, testing concentrations…

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Once again, with the real bicyclomycin!

Last month I wrote that we were abandoning our plan to test whether the antibiotic bicyclomycin induces competence in Haemophilus influenzae, as it does in Legionella pneumophila, because (i) the free 'bicyclomycin' we'd been given by a colleague turned out to be bicyclomycin benzoate, and (ii) the real bicyclomycin we wanted to test cost hundreds of dollars per milligram.But last week I got the budget statement for our NSERC grant and discovered that we're not…

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