Team:Calgary/Notebook/Protocols/PetroBrick Validation Assay


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PetroBrick Validation Assay

In order to identify if there were any hydrocarbons that could be produced with the PetroBrick. A validation assay was performed based on the 2011 Washington Team Wiki. Their protocol was modified slightly as we had difficulty replicating their results with the conditions they described.

  1. Prepare LB broth and Washington Production Media.
  2. Grow up PetroBrick construct overnight at 37oC shaking in LB broth.
  3. Make a 20 mL 50:50 LB:Washington Production Media solution in a 125 mL Erlenmeyer flask. Add 5 mL of the grown PetroBrick into the culture and incubate for 72 hours. (in subsequent experiments other medias were used containing naphthenic acids or cyclohexane pentanoic acid at a concentration of 50 mg/L).
  4. Sonicate the culture using a Misonix Cell Disrupter at dial 11 for 2 min.
  5. Extract the solution with 5 mL of ethyl acetate, shaking vigorously, and spinning down the solution at 4000 RPM for 5 min.
  6. Take off 1.5 mL of solution.

    Following organic extraction of the samples and controls for Petrobrick experiments, samples were concentrated to 0.3 mL and placed into autosampler vials. The samples are introduced via autoinjection on an Agilent GC-MS system (model 7890A GC, model 5975C inert XL MSD) equipped with an HP-1 capillary column (50 m x 0.32 mm i.d. x 0.2 μm film, Agilent Technologies, Inc.). The samples were analyzed in splitless mode, with the inlet held at 270°C. The oven was initially held at 100°C for 5 min, then increased at a rate of 8°C/min to a final temperature of 300°C that was held for 5 min.