Time course results and prospects

Well, my execution of the time course experiment was near-flawless, but the results leave a bit to be desired.  Once again the colony counts were erratic, and some of the results are inconsistent with results of previous 'replicates'.  (Is replicate the right word here?).

I do have a new hypothesis to explain the colony-count problems: too short of an 'expression time' for the antibiotic resistance allele.  When cells recombine an allele coding for an antibiotic-resistant version of a protein, they don't instantly become resistant -- some time is needed for the new allele to be transcribed and translated into protein, and full resistance may take an hour or more.  However this matters more for some antibiotics and some forms of resistance than for others.  

With kanamycin and H. influenzae, experiments I did when I first set up my lab showed that cells could be spread on kanamycin agar plates right away (15 minutes for DNA uptake, 5 minutes for DNaseI, maybe 5 minutes for dilutions and plating), and every cell that had the new allele could form a colony.  When I started working with A. pleuropneumoniae I was told to use a very high concentration of kanamycin to prevent sensitive cells growing on the kanamycin plates, and found that cells did need an hour for expression of the allele before being able to form colonies on plates.  But I tested lower concentrations and found that sensitive cells couldn't grow at a much lower concentration.  In writing up that test I speculated that maybe they wouldn't need expression time to grow on this concentration.  But I seem to have then just gone on to do subsequent experiments without expression time without ever really testing whether it was needed.  So maybe my erratic results and low colony counts on kanamycin plates are because many cells that had acquired the resistance allele didn't have time to become phenotypically resistant before they encountered the kanamycin.

To resolve this, I can thaw out and transform some frozen competent A. pleuropneumoniae cells and test their need for expression time.  But I now wonder if these cells, having been starved to induce competence, might actually need less expression time than the cells growing in rich medium, because the antibiotic causes most of its killing when cells are actively replicating their DNA.  This would be consistent with my results, because the plating problems did mainly happen with growing cells. That's an interesting issue in its own right, and but would entail doing a more complicated test.  If the tests showed that expression time was the problem (at least a big part of the problem), I'd redo the time course.

The issue may be moot, because we may now leave the time course out of the manuscript.  One of the authors from the other research group (the leader of the group) strongly feels that we need to do more experiments before submitting this for publication, even as a 'Note' to a fairly minor journal.  But of course he wouldn't be the one doing the experiments, and the rest of us think we should just send it in and see what the reviewers think.  I and the postdoc can also think of desirable experiments, but our lists don't overlap with his.  So we'll try to persuade him by sending him our list and arguing that we can't possibly do all of these experiments, so let's wait and see which ones the reviewers might want us to do, rather than trying to read their notoriously unpredictable minds.