Team:Calgary/Notebook/Protocols/oleT in Validation Assay


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OleT Validation Assay

oleT is a gene that has been demonstrated to be able to convert fatty acids into terminal olefins (alkene based compounds) through reduction of the carboxylic acid component. This assay was performed with palmitic acid (a C16 fatty acid) as it has been previously demontrated this can be converted to i-C20 olefins.

  1. Micrococcus candicans was grown at 26oC in tryptic soy broth supplemented with 0.5% yeast extract, overnight with shaking.
  2. Incubate a culture of 20 mL with the same media composition with 1 mL of the grown micrococcus and 1mM palmitic acid.
  3. Note to get the palmitic acid to dissolve it needed to be heated in filter sterilized water to about 45oC.
  4. After 72 hours cultures were sonicated with a Misonix Cell Disruptor for 1 min at Dial 11 and extracted with 5 mL of ethyl acetate
  5. Cultures were shaken well, and spun down at 4000 RPM for 5 min at 4oC. 1.5 mL of the ethyl acetate layer was removed and processed further.

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.