_(cropped).jpg)
Adding zinc doesn't increase transformation frequency (the simplest and most sensitive measure of DNA uptake). The small differences between most these columns aren't significant, given the modest scale of this experiment. High concentrations of zinc are probably a bit toxic: at 1mM zinc decreased transformation more than 2-fold, and at 3mM it decreased cell viability 2-fold. (I missed adding the DNA to the 3mM tube so I don't have this transformation frequency.) Adding the zinc chelator histidine didn't decrease DNA uptake either.
The conclusion: we can probably rule out an important role for zinc or zinc-finger proteins in DNA uptake. Is this an important enough experiment that I should do it over several times, to get data with good error bars? It's an important experiment, but if the possible effect of zinc is too small to have shown up in this experiment it's probably too small to be interesting.
Two (probably relatively trivial) caveats occur to me:
ReplyDelete1. you didn't test the effect of His and Zn together (seems counterintuitive, but the chelated pair might be, for instance, more available to a transport mechanism than either alone); and
2. if the Zn really were important at trace (contaminant) levels, you might have swamped its effects with toxicity by going all the way up to the mM range -- the zinc-kink paper was presumably not in vivo work?
So I think if it were me, just because I'm reluctant to let ugly facts destroy a beautiful theory, I'd do it again with micro- and even nanoM concentrations of Zn, and test Zn/His (1:1 molar ratio) at total concentrations up to maybe 1mM.
That said, I think you're probably right, and the answer is not Zn.
Given that the standard mix already contains about 0.06mM histidine, I suspect that trace concentrations of Zn aren't that important (despite my previous statement about contaminants). Assuming the chelation is 1:1 (and I haven't looked this up), 0.1mM Zn woud have given 0.06mM chelate + 0.4mM free Zn.
ReplyDeleteI could get rid of the histidine, but it's present as part of supplementation with an amino acid mixture derived from a protein hydrolysate (casamino acids), and leaving this out will limit production of the DNA uptake machinery.
I didn't show the cells/ml data, but there was no evidence of toxicity below 3mM Zn.
I SHOULD probably try the same experiment with calcium and magnesium (vary the concentrations, chelate with EDTA). These cations are added to the competence mix, but I haven't found any data explicitly addressing their effects.