Field of Science

inconclusive fraction-competent results

A few posts ago I described surprising results from an experiment measuring the fractions of the cells in different cultures that were competent.  Here they are again:

And here are the new results:

Conditions were a bit different this time.  First, the KW20 (wildtype) culture had not been induced to maximum competence this time - these cells were approaching stationary phase and are 50-100-times less comepetent.  Second, this time I remembered to give all the cultures 60 minutes in rich medium before plating to allow expression fo the spectinomycin resistance.

Including expression time doesn't appear to have significantly changes the transformation frequencies for SpcR selected alone, but it substantially increased the double transformation frequencies (NovR SpcR), and this reduced the apparent fraction competent to below 1.0.

So this new experiment clarifies why I got the anomalously high FC in the previous experiment.  Unfortunately it doesn't address the reason I wanted to measure FC in the hypercompetent mutants in the first place.  The question arose from the analysis of the ∆HI0659 mutant's growth rates.  In this experiment). I had found that cells carrying the HI0659 mutant grew normally.

I did this experiment because I wanted to find out whether unopposed expression of the HI0660 'toxin' harms cells (killing them or inhibiting their growth).  In the ∆HI0659 cells the HI0660 'toxin' is induced but not opposed by the HI0659 'antitoxin' when competence genes are on.  Because competence genes aren't normally on in growing cells anyway, I had tested growth in two hypercompetent mutant backgrounds, sxy-1 and murE749 - this was normal too.

The fraction competent experiments were intended to test whether most of the cells in the hypercompetent cultures has their competence genes on - if not then we might not see a dramatic growth difference even if the toxin does harm cells.  But I foolishly did them with cells approaching stationary phase, when I should have done them with cells in exponential growth.  I don't need to bother doing this for the sxy-1 mutant, since I already know that only a small fraction of its cells are competent then (I did this experiment years ago), but I should test it for murE749.

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