Field of Science

Starting to work with GFAJ-1!

The GFAJ-1 bacteria I requested should arrive today, so I'd better get some medium made.  My plan is to initially grow them in medium supplemented with different levels of phosphate but no arsenate.  This should let me see the extent to which phosphate availability limits growth in the absence of arsenate. Once I've carefully characterized their growth I'll test the effect of adding arsenate to medium that's phosphate-limited, and purify DNA from various cultures.  The levels of P and As in the DNA and the various culture media will be determined by mass spectrometry, by Leonid Kruglyak and Josh Rabinowitz.

The medium-salts base contains 1 M NaCl,  with ammonium sulfate, magnesium sulfate and sodium carbonate and bicarbonate; it's pH 9.8.  I'm going to make this up as a 5X stock, then dilute it into water and add the glucose, phosphate (at 0 µM, 3 µM, 30 µM, 300 µM and 1.5 mM) and some but not all of the vitamins in the mix that Wolfe-Simon et al. used.  (The paper never tested whether any of the vitamins were needed, and I don't have any thiotic acid (= thioctic acid = alpha lipoic acid).  I'll test diluting the medium salts into both distilled water and into tap water, because I don't think I need to fuss with the trace element mix the authors added to their medium (they didn't use analytical-purity reagents and neither will I).

I think the cells will arrive streaked on a phosphate-,medium agar plate.  I'll scrape up some cells, dilute them in a bit of the no-phoshate medium, and check the cell density under the microscope.  Then I'll dilute them into the various media, aiming for a cell concentration that's detectable but not high.  I'll incubate them in our glass culture tubes (using new tubes) in a 28 °C incubator, and check the cell counts every day.

Time to go turn down the 33 °C incubator, and find the chemicals and the new tubes.

3 comments:

  1. NotAnAstrobiologistJune 21, 2011 at 4:08 PM

    Looking forward to your results!

    ReplyDelete
  2. AnAstrobiologistJune 21, 2011 at 6:27 PM

    Me too, good luck!

    ReplyDelete
  3. Looking forward, if you get the expected results (no As in DNA - maybe even not reproducing FWS' growth curves), it's worth anticipating the author's response. How might they argue why you didn't see what they saw? Thinking about this response may help you to design some additional controls.

    ReplyDelete

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