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Desulfurization Assay Protocol

Media preparation

A modified M9 media is used for the desulfurization assay. The medium needs to be sulfur-free, as the presence of scarce amounts of sulfur represses the promoter controlling desulfurization genes expression. All the salts containing sulfate in the original recipe are replaced by equimolar amounts of chloride salts (e.g. MgCl 2 instead of MgSO 4). Prepare the media as follows:

  1. Dissolve the following salts in about 500mL of ddH2O:
    • 12.8 grams Na2HPO4
    • 3.0 grams KH2PO4
    • 0.5 grams NaCl
    • 1.0 gram NH4Cl
  2. Bring the media to approximately 950 ml with ddH2O and pH to 7.4 with NaOH.
  3. Autoclave for 20 minutes on slow exhaust. Store at 4°C when finished.
  4. The following salts were dissolved in 50mL of water and cold filtered with a sterilized 0.22 micron filter:
    • 10.8 grams glucose
    • 0.19g of MgCl2
    • 0.0152 grams CaCl2.2H2O
    • 0.0100 grams Thiamine
    • 0.007g of FeCl2.4H2O

To maintain the plasmid in the bacterial strain, 0.5g of kanamycin was added to the media. However, the sulfate from kanamycin sulfate (commercially available form) needs to be removed. Equimolar amounts of BaCl2. is added to a kanamycin sulfate solution (in distilled water) to precipitate and remove the sulfate in the form of BaSO4. The solution is then centrifuged to remove the precipitate from the solution. The solution is filtered through filter paper to remove all the precipitate. Finally, the solution was cold filtered into the stock media by a sterilized 0.22 micron filter.