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.

    • Retrieved from "http://2012.igem.org/Team:Calgary/Notebook/Protocols/oleT_in_Validation_Assay"