User:Eric M. Walters/Notebook/Spring 2012/2012/05/04

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Malonyl CoA reductase, why you gotta play me like that?

So it seems likely that the MCR is causing some sort of increased acrylate toxicity, likely due to the increase of CoA species in the cell. Before trying to transform again, I'm going to try to find the acrylate MIC in MCR-bearing 7002.

Today, I'm making plates of 5, 10, and 20 μM AA + Kan100 to grow them up on.

Gonna make 1 500 mL batch of kan+acrylate plates for growing up the transformants, once that happens

  • Each batch will be two separate bottles to autoclave
    • One 250 mL bottle of 2x media A (made new stock, recipe below)
    • One 250 mL bottle of 3% Bacto agar in milliQ H2O (7.5 g)
  • Once it cools, combine the bottles (gently!)
  • Add B12, 50μL per 1 L of plate liquid (so this time, 25 μL in each bottle)
  • Add acrylate, from the (0.05 M dilution of the .5 M stock on Matt's bench), to individual plates
    • 20 μM: 10 μL stock
    • 10 μM: 5 μL stock
    • 5 mM: 2.5 μL stock
  • Add kanamycin for kan100 (500 μL of the Kan50 stock per each 250 mL)
  • Label 'em and 3 blue stripes, then into fridge

Plating the culture

The wt MCR culture was .348 OD. Given what Erin told me about plating efficiency (1 ODmL is 1e7 colonies), this should be confluent (3.5e5) below MIC. Putting 100 μL of culture onto each of 6 plates, duplicates of the 5, 10, and 20 μM acrylate with kan100.

Media A recipe

For 1 L of 1x cyanobacteria growth media

  • 2.75 mL 3M Tris (pH 8.2)
  • 178 μL .4488 M EDTA
  • 18 g NaCl
  • 5 g MgSO4-7H2O
  • 0.6 g KCl
  • 0.37 g CaCl2-2H2O
  • 1 g NaNO3
  • 0.050 g KH2PO4
  • 10 mL trace element solution
  • Post-autoclave, add 22.2 μL B12 solution

FOR THE 2x CONCENTRATION FOR PLATES: Don't add B12 (will be added later), and all above values are doubled.