Field of Science

Input DNA fragment sizes and shape of uptake peaks

The grad student has completed an analysis of the size distribution of the DNA fragments in the chromosomal DNA preps used for his uptake experiments.  Now we need to think about how we'll use this information.

He used two DNA preps, one sheared to an average length of about 6 kb (the long-fragment prep) and one sheared to an average length of about 250 bp (the short-fragment prep).  He analyzed both with a Bioanalyzer belonging to a neighbouring lab (thanks neighbours!).  This produced intensity traces for each sample (red line), with size-standard peaks (blue).

The intensity traces reflect the number of base pairs at each position in the gel, not the number of fragments, so the values needed to be normalized to fragment length to get the size distribution.  The purple line is the final distribution of fragment sizes.  We see that most fragments are between about 75 and 300 bp.

Now, how do we use this information to predict the shape of the expected uptake peak around an uptake-promoting sequence (a USS)?

We first need to calculate the probability that the position we're looking at will be on the same fragment as a USS (call this value 'U').

 Now do this for each fragment size and plot it.

This is our expected peak shape, if all that matters is whether a USS is present anywhere on the DNA fragment.  We'll compare this to the average shape of well-isolated uptake peaks in the short-fragment dataset - the PhD student has already made a list of this subset of the peaks.

To do the comparison properly we'll need to take peak height into consideration too.  So we should do separate comparisons for different peak-height classes.  If the prediction nicely overlays the observed peaks we'll conclude that a USS anywhere on the fragment is equally effective.

If the location of the USS on the fragment matters, or its orientation, the peak would have a different shape.  For example, if USSs near the ends of fragments don't promote uptake very well, the observed average peak would be narrower than predicted by fragment sizes.

For another example, if USS in the forward orientation promote uptake well when they're near the left end of the fragment but poorly when they're near the right end, we might see different peak shapes for the two orientations - skewed right for 'forward' USSs and skewed left for reverse' USSs.   (Or is that backwards?)  If we only looked at the combined set of USSs in both orientations we might miss this effect.

Is there any other factor we could investigate using this analysis?  And what about the large-fragment data - should we treat it the same way?

Bicyclomycin ≠ bicyclomycin benzoate

A month ago I wrote a post about a planned experiment using the antibiotic bicyclomycin, to see if it induces H. influenzae cells to develop competence.  At the time I couldn't remember why this was a reasonable question, but a commenter pointed me to this paper, which describes the induction of competence by bicyclomycin in Legionella pneumophila.

Bicyclomycin is expensive, and we're close to broke, but a generous colleague had given us 4 mg of it to use in a trial experiment.  So I put our summer undergraduate to work on the project.  She began by testing H. influenzae's ability to grow in different concentrations of bicyclomycin, since we wanted to use a semi-inhibitory (but not lethal) concentration for our experiment.  We had found a paper that reported the minimum inhibitory concentration (MIC) for H. influenzae was 3.1 µg/ml, so she tested a wide range (up to 20 µg/ml).  But she saw no inhibition of growth at all.

That MIC had been for a clinical strain, not the lab workhorse KW20, so she repeated the test (this time using the neighbour-lab's BioScreen system) for both a clinical strain (86-028NP) and KW20, and for a couple of E. coli strains (the same paper reported MICs for E. coli  strains between 6 and 12 µg/ml), using bicyclomycin concentrations up to 50 µg/ml.  Still no evidence of growth inhibition!

But now I think I've solved the mystery.  Before making up our bicyclomycin stock we searched for solubility info.  We learned that it's reasonably soluble in water, but that there's a related antibiotic called bicyclomycin benzoate that needs to be made up in ethanol.  The colleague who gave us the 4 mg remimded me that she'd sent an email saying to dissolve it in ethanol.  I'd forgotten about this email, but reading it now reminded me of the solubility difference, and when I checked with her I found out that what she'd given us was bicyclomycin benzoate.

The same paper that gave us the H. influenzae MIC for bicyclomycin tested a wide range of derivatives, one of which was bicyclomycin benzoate.  It's MIC for H. influenzae was >100 µg/ml.  No wonder our cells didn't care about the concentrations we tested!

Bicyclomycin is about 10 times more expensive that bicyclomycin benzoate ($280/mg) so I don't think we'll be doing this experiment after all.

One more toxin/antitoxin growth experiment

I have one more experiment to do for our toxin/antitoxin manuscript.  I need to make sure that survival into and recovery from stationary phase is normal in the antitoxin-knockout mutant.  This strain overexpresses the toxin gene and cannot inactivate the resulting toxin protein.  We already know that it produces a normal-looking growth curve using the Bioscreen; one of the lines in the graph below is for the antitoxin knockout), but this analysis is based on changes in culture turbidity and does not consider whether some of the cells contributing to turbidity might be dead.  This isn't a concern for rapidly growing cultures, but is for cells that have ceased growing.  So I need to complement the Bioscreen result with growth curves made by diluting cultures and plating the cells, to measure viable 'colony-forming-units' rather than just turbidity.

I would normally set up four cultures (wildtype, toxin knockout, antitoxin knockout, double knockout), but there's a complication.  The double knockout antitoxin mutant only exists in a 'marked' version (with a spectinomycin cassette inserted in place of the missing genes) and the antitoxin knockout only exists in an 'unmarked' version (no spectinomycinR cassette).  If this cassette influences growth or survival this difference could cause anomalous results.  The toxin knockout exists in both forms, so I'll include both of them in the analysis.

First step is to restreak all the strains from the freezer stock.  I did this last week but foolishly let the cells die on the plates rather than restreaking them.