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- Get the GFAJ-1 cells growing in liquid ML60 medium (with vitamins and trace elements) at least as well as they grew for Wolfe-Simon et al. That means at least two doublings a day.
- Do preliminary growth curves to find out what phosphate concentrations limit growth.
- Grow a big batch of cells under phosphate-limiting conditions. Collect them and freeze them when they are still growing exponentially (so no complications due to accumulation of poly-hydroxybutyrate granules).
- Do meticulous growth curves with different concentrations of added phosphate with and without 40 mM arsenate. Also try media with no added phosphate, in case my phosphate-contamination levels are like those in the paper.
- Grow enough cells at the lowest phosphate level, with and without arsenate, that I can purify enough DNA for analysis.
- Send the DNA and samples of the various media to Leonid Krugliak and Josh Rabinowitz, who will use mass spectrometry to measure phosphorus and arsenic levels. I'm waiting for them to tell me the sample sizes they'll need.
I'll also ask the RA to order the universal 16S rDNA primers so we can use PCR and DNA sequencing to confirm that the cells I'm growing are indeed GFAJ-1.
It's a shame Felicia never grew a phosphate-limited chemostat really - seems like such an obvious way to study phosphate limitation, afterall, and is known to limit DNA synthesis - to the point that plasmids are lost, so if the bug really does take arsenate into the DNA, it would do so very well under such conditions.
ReplyDeleteOh well, one of the many things on the "Why didn't they do..." list!
rich
PS: Your planned expts look perfectly sensible to me - big clincher being "get the bloody thing to grow". Once you've got the medium 100% the same, it'll probably help a lot. If you can't get it to grow even in that and it is the right bug, will you try and optimise growth or stop there?
@Rich: Given how nicely the GFAJ-1 cells grow on plates, I'm pretty confident I can get them growing well in liquid medium. But if I can't I'll stop, because the scientific value of this project is much lower than the other work I could be doing.
ReplyDeleteDo the cells grow in stationary liquid medium or as a biofilm?
ReplyDeleteAny change you can run LC-MS/MS analysis of the DNA?
www.pnas.org/cgi/doi/10.1073/pnas.1017261108 PNAS | February 15, 2011 | vol. 108 | no. 7 | 2963–2968