Regular readers will know that my attempts to grow GFAJ-1 in medium with 40 mM arsenate have given very inconsistent results (Expts. 1-3, Expts. 1-4, Expt. 5, Expt. 6).
The Wolfe-Simon et al paper reported that these cells were resistant to 40 mM arsenate, but in my experiments so far the only time the cells really appeared to be resistant to 40 mM arsenate was Expt. 5, when I grew them in screw-capped glass tubes.
Even if I set aside all the other experiments (in foil-capped flasks or in screw-capped plastic tubes), this experiment was compromised by tube-to-tube inconsistencies in final cell density, probably due to the presence of some limiting nutrient (perhaps not phospate) contaminating some of the tubes. So I've now acid-washed all the glass tubes and caps, rinsed them lavishly in distilled water, and re-autoclaved them, so I can see if this experiment's result is reproducible. These conditions are closest to those used by Wolfe-Simon et al., so if I can consistently get growth in 40 mM arsenate I can prepare DNA from arsenate-grown and control cultures for mass-spectrometry analysis.
I'm going to streamline the number of conditions (just ± 40 mM arsenate, combined with no added phosphate, 3 µM phosphate or 1500 µM phosphate) and do two replicates in glass tubes and one in plastic tubes. I'll make up 50 ml of no-phosphate medium, add cells (from my frozen stock of phosphate-depleted GFAJ-1) to about 10^4 cfu/ml, and split this in two. I'll add arsenate to one half (and water to the other). Then I'll put 5 ml into each no-phosphate tube, add phosphate to 3 µM to the rest (both parts), and put 5 ml into each 3 µM PO4 tube (using 3 replicate glass tubes). Then I'll add more phosphate to the rest, to 1500 µM, and put 5 ml into each 1500 µM tube. Then I'll put all the cells gently rocking in the 28 °C incubator.
Why I'm Marching for Science
18 hours ago in Angry by Choice