Zinc? I just realized that we've been neglecting the possible role in DNA uptake of divalent cations, especially zinc.
There are lots of reasons why we shouldn't have. A paper discussing DNA's ability to deform under stress showed that addition of zinc allowed kinks to form. There's a whole family of DNA-binding proteins called zinc finger proteins, because zinc mediates an interaction between histidines in the protein and DNA. Some of the ancient (45 years old) classic papers about competence point out that divalent cations influence DNA uptake. And every beginning biochemist learns that divalent cations help proteins interact with DNA.
The standard medium for inducing competence (which we also use for assaying DNA uptake and transformation), contains Mg++ and Ca++. The ingredients list doesn't include zinc, but it may be present as a contaminant, and variation in the level of contamination might even explain the long-term variations we see in transformation frequency. And it should be easy to test - just supplement the usual DNA-uptake solution with different amounts of a zinc salt or the zinc-chelating amino acid histidine. We have a couple of grams of histidine on the shelf, and for some reason a whole kg of ZnSO4. And it's high time I got back into the lab and did an experiment.
Experiment plan for tomorrow:
I think I have lots of competent cells in the freezer. Oops, no I don't, so I will need to grow some wildtype cells and make them competent. We have lots of DNA with a gene giving resistance to the antibiotic novobiocin, to use for the transformations. I'll need to make some agar plates, both plain sBHI (the usual nutrients) and with novobiocin added to select for my transformants.
I'll need to make up solutions of ZnSO4 (50 ml of 100mM) and histidine (small volume of 100mM?) and filter-sterilize them. The zinc-kink paper used 1mM Zn, but said 0.1mM worked too, so I'll try both. Maybe I should try a wide range of values, in case too much zinc is as bad as too little. The standard competence solution contains a little bit of histidine (0.013mg/ml - at 210 grams/mole that's 0.062mM). I'll also try adding histidine at 0.1mM and 1mM.
If zinc does help cells take up DNA, I should see higher transformation frequencies when the uptake solution is supplemented with zinc, and/or lower transformation frequencies when it is supplemented with histidine. The 'and/or' is because I don't know whether the uptake solution already contains significant amounts of zinc.
This will be fine old-fashioned microbiology experiment, and I'm pretty sure I can do it in one day. It's the weekend so there shouldn't be a lot of interuptions.
What mutation rate do I want for my experiment?
18 hours ago in RRResearch