Now that I have good growth conditions for GFAJ-1, I need to plan the rest of the work in more detail.
Frozen cell stocks: I'll want to start my phosphate-limited cultures with cells that have been pre-grown under phosphate-limited conditions, so that their subsequent growth will be limited by the phosphate in the medium, rather than by the phosphorus they have accumulated in RNA and other molecules. I have about 50 ml of dense culture in +P medium, so I'll wash these cells, resuspend them in a large volume of -P medium, and incubate them just until growth stalls**. To make monitoring easy I'll start the cultre at the final OD reached by my original -P culture; at this density I expect the cells to run out of phosphate within a few doublings.
(** I need to be careful that apparent cell density isn't being influenced by accumulation of poly-hydroxybutyrate, as it was in the Wolfe-Simon paper. So I'll also plate the cells to check that numbers are no longer increasing.)
Once I have a phosphate-limited culture I'll collect the cells (by filtration or centrifugation), resuspend them at high density, and freeze 1 ml aliquots of them in 15% glycerol. I'll check the cell density by plating before and after freezing, to make sure that freezing doesn't kill the cells.
DNA prep: I should have enough cells to also make my first DNA prep - if not I'll inoculate one of my freezer-stock tubes into a new overnight culture. I'll put this DNA through the same multiple purification steps I plan to use for DNA from arsenic-grown cells, checking the DNA concentration at each step, and running a gel to confirm the quality of the DNA and the absence of contaminating RNA. This will let me estimate the loss in each purification step, so I'll know how many cells I'll need for the arsenic-grown DNA prep.
Elemental analysis: I'll send aliquots of the media and some of the DNA to my collaborators, so they can check levels of phosphorus and arsenic. We can consider this to be our control DNA - if the DNA from arsenic-grown cells has no more arsenic than this DNA, we can conclude that arsenic-grown cells do not put arsenic into their DNA. It will be good to test this DNA now, as it sets the limit of detection we need for the arsenic-grown DNA.
However, when I purify the DNA from the arsenic-grown cells I'll also do a second control, by briefly incubating phosphate-grown cells in the arsenic medium before DNA purification. This will control for any carryover of arsenic in the DNA purification. If my purification is adequate I expect this DNA to have no more arsenic than the first control.
Strain identification: I'll give some of the DNA to the RA. She has the primers for the 16S rDNA amplification and will get it sequenced to confirm that these bacteria are GFAJ-1.
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