RRResearch: Phosphate levels and DNA controls for GFAJ-1

OK, it looks like my arsenic-bacteria experiments have advanced from "Why won't they grow" troubleshooting to real science.  My test of growth in medium with no added phosphorus (P- medium) succeeded nicely.  All of the cultures except the most dense grew to the same low density of about 4 x 10^6 cells/ml.  This tells me that their growth was indeed limited by the small amount of phosphorus contaminating the medium.  It also tells me that my P- medium contains less phorphate than the medium used by Wolfe-Simon et al., as the cells grew to about 2 x 10^7 in their P- medium.  This means that I should add a small amount of phosphate (~ 3 µM) to my P- medium to replicate the growth conditions they used.  I'll set up cultures with this medium tonight to check that growth is as expected.

I've also been thinking about controls for the DNA purification steps.  This weekend I'm going to make up my arsenic solution (100 ml of 1.0 M Na2HAsO4).  A colleague has given me her procedure for safely weighing out the powder in the fume hood (that's the only seriously risky step).

To check that my DNA-purification procedure does get rid of free arsenate from the medium, I'm going to do the following:  I'll start with about 1 mg of old H. influenzae chromosomal DNA that we no longer need for other experiments.  I'll mix it with some AML60 medium containing 40 mM arsenate, and then put it through the purification steps I plan to use on the GFAJ-1 DNA - two rounds of ethanol precipitation with the DNA collected by spooling, and a final spin-column purification.

I'll save a sample of the DNA after each step, and send all the steps to my collaborators for mass-spectrometry analysis of its arsenic content.  Ideally the DNA will not contain any detectable arsenic.  If it does, I can try more extensive purification, or just accept this level as the baseline, depending on how sensitive the mass-spec turns out to be.

Once I have the growth results from my medium with 3 µM phosphate, I'll also use my new sodium arsenate stock to make some 3 µM P medium that also has 40 mM arsenate, and then test whether the arsenate inhibits growth at all, or whether it enhances it as reported by Wolfe-Simon et al.  Then I can grow a big batch of cells in 3 µM P 40 mM As, purify DNA, and send the DNA to my collaborators for analysis.

And the Research Associate has already amplified the 16S-rRNA gene from the GFAJ-1 DNA I made and sent it off for sequencing (she's a whiz!).  So next week we'll be certain that I'm working with the right strain.  Or not.