Growth of GFAJ-1 under phosphate limitation

Before we can test whether the bacterium GFAJ-1 can really incorporate arsenic into its DNA when phosphorus is scarce but arsenic is abundant, I need to find out how it behaves when phosphorus is scarce and no arsenic is available.  Here are the results.

The first graph shows how dense cultures got when no phosphate was added to the basic growth medium.  I described this experiment in two posts a few days ago (planning, results), but I didn't show the data.  I started the cultures with cells that were either phosphate-replete or phosphate-depleted, and at densities ranging from 10^4 to 10^7 cells per ml.  All of the cultures except the most dense phosphate-replete one ended up at densities of 1-3 x 10^6 cells/ml.  This tells me that, even though I didn't add any phosphate to the basic medium, each ml contained enough phosphate to produce about 2 x 10^6 cells.  This is about ten-fold lower than the growth observed by Wolfe-Simon et al, and so my experiments should use medium with enough phosphate added to produce about 2 x 10^7 cells/ml.

The second graph shows how small amounts of added phosphate increase the cell density.  These cultures all had the same initial cell density (10^4 cells/ml), but they had different amounts of added phosphate.  As before, medium with no added phosphate yielded about 2 x 10^6 cells/ml, and medium with 1500 µM phosphate yielded about 3 x 10^9 cells/ml.  Media with 3 µM phosphate (the concentration of contaminating phosphorus in most of the media used by Wolfe-Simon) gave a cell density just below 2 x 10^7, and 6 µM phosphate gave about 4 x 10^7.  So I think I'll use 4 µM phosphate as my final phosphate-limiting medium.

Yesterday I also (very carefully) made up my 1.0 M stock of sodium arsenate.  I put on all the safety gear I usually eschew (lab coat, gloves, dust mask, full face shield) and moved our top-loading balance into the fume hood so any arsenic dust would be sucked away rather than entering the lab air supply.  (I even put the balance into a plastic bag so no arsenic dust could get into its innards.)  The sodium arsenate turned out to be in the form of granular crystals (like coarse salt or sugar), not fine powder, so dust wasn't a problem.  But when I carefully unscrewed the cap a lot of grains fell out of it (maybe 0.5 g!), so I was really glad to be working in the hood where cleanup was easy.

Now the arsenate solution is sitting on my lab bench, conspicuously labeled POISON.  Beside it sits a big bottle to discard the waste culture medium into  - this will eventually be sent to our chemical waste facility for disposal.  I'll set up another bottle for waste tips and other solids contaminated with significant levels of arsenic.