I've now tested whether the growth of GFAJ-1 is indeed stimulated by arsenate, as was suggested by the yields of my DNA-prep cultures. This time I was very careful to keep the ionic strength constant, giving each culture tube the same volume of varying mixtures of 1 M NaAsO4 and 1 M NaCl. The arsenic concentrations ranged from 5 µM to 60 mM in 2-fold or 2.5-fold steps (also 0 mM).
To make sure that all culture tubes started with the same medium and the same density of cells, I mixed up a big batch of no-phosphate medium, added cells (2 x 10^5 cfu/ml), and divided the culture into thirds. I then added NaPO4 to two of these parts, to give 3 µM and 1500 µM (in addition to whatever phosphate might be contaminating the medium), and added 5 ml of each part to each of 15 screw-cap galss tubes to which I had already added the appropriate NaAsO4/NaCl mixture. So I had 45 tubes in all, 15 with no added phosphate, 15 with 3 µM, and 15 with 1500 µM.
I incubated the tubes at 28 °C with gentle rocking, and checked the optical density after 24 and 48 hr.
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To make sure that all culture tubes started with the same medium and the same density of cells, I mixed up a big batch of no-phosphate medium, added cells (2 x 10^5 cfu/ml), and divided the culture into thirds. I then added NaPO4 to two of these parts, to give 3 µM and 1500 µM (in addition to whatever phosphate might be contaminating the medium), and added 5 ml of each part to each of 15 screw-cap galss tubes to which I had already added the appropriate NaAsO4/NaCl mixture. So I had 45 tubes in all, 15 with no added phosphate, 15 with 3 µM, and 15 with 1500 µM.
I incubated the tubes at 28 °C with gentle rocking, and checked the optical density after 24 and 48 hr.
Conclusion: Arsenate stimulated growth, but didn't affect the final densities of the cultures. The stimulation is not because the arsenate is contaminated with phosphate, because the effect was strong only in the cultures with 1500 µM added phosphate, and because it didn't affect final density in the phosphate-limited cultures.
There's still much more variation in final culture density than I'd like to see. This might be due to minor differences in trace contaminants in the tubes, although they were all last used for similar cultures and all thoroughly washed the same way. One solution would be to use only new tubes, but these tubes are not cheap and I don't want to take money from our transformation work.
I'm not going to do any more work on this - not going to do experiments to find out why arsenate stimulates growth, unless the mass spec shows that there really is arsenic in the DNA of arsenate-grown cells. The growth stimulation I'm seeing isn't a replication of Wolfe-Simon et al's report that their cultures grew with arsenate but not without it, but it might reflect the same biological process.
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