Team:British Columbia/Protocols/Biodesulfurization

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<h1>Detection of Biodesulfurization Intermediates by Reversed Phase HPLC</h1>
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#Grow overnight cultures of cells containing ''dsz'' operon in 5mL culture of LB.  
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<h1>Biodesulfurization with HPLC</h1>
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#Measure O.D 600 with a spectrometer. Inoculate in 200 mL culture of LB in a 500 mL flask such that the starting O.D 600 is 0.05
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#Dibenzothiphene at 100ppm dissolved in 2% DMSO is added to the culture
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1. Grow overnight cultures of cells containing dsz operon in 5mL culture of LB.  
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#Incubate at 37°C shaking at 200 rpm.
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#Extract 25mL at O.D 600 intervals of 0.3, 0.7, and 1.0.
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2. Measure O.D 600 with a spectrometer. Inoculate in 200mL culture of LB in a 500mL flask such that the starting O.D 600 is 0.05.
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#Spin down cells using a centrifuge (1600 g, 10 min) and extract 25mL of supernatant.
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#Acidify supernatant to pH of 2.0 with 6N HCl for efficient extraction of solutes.
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3. Incubate at 37°C shaking at 200rpm.
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#Extract with equal volume of ethyl acetate.
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#Dry extract with nitrogen gas and re-suspend in mobile phase (80% acetonitrile).  
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4. Extract 25mL at O.D 600 intervals of 0.3, 0.7, and 1.0.
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#Filter the resuspended extract using a 0.45 μm PTFE or nylon filter.  
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#Run the sample on an HPLC using a C18 column (150 x 3 mm) and a flow rate of 0.8 ml/min. Monitor the sample at 280 nm.
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5. Spin down cells using a centrifuge (1600g, 10min) and extract 25mL of supernatant.
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6. Acidify supernatant to pH of 2.0 with 6N HCl to inactivate enzymes.
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7. Extract with equal volume of ethyl acetate.
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8. Dry extract with nitrogen gas and re-suspend in mobile phase with 80% acetonitrile.  
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9. Filter extract resuspended in acetonitrile using reverse phase chromatography and C-18 column.  
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10. Elute mobile phase at a flow rate of 0.8 ml/min and peaks of DBT were detected at 280nm by HPLC.
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Latest revision as of 04:03, 4 October 2012

British Columbia - 2012.igem.org
UBC iGEM 2012 protocols

Detection of Biodesulfurization Intermediates by Reversed Phase HPLC

  1. Grow overnight cultures of cells containing dsz operon in 5mL culture of LB.
  2. Measure O.D 600 with a spectrometer. Inoculate in 200 mL culture of LB in a 500 mL flask such that the starting O.D 600 is 0.05
  3. Dibenzothiphene at 100ppm dissolved in 2% DMSO is added to the culture
  4. Incubate at 37°C shaking at 200 rpm.
  5. Extract 25mL at O.D 600 intervals of 0.3, 0.7, and 1.0.
  6. Spin down cells using a centrifuge (1600 g, 10 min) and extract 25mL of supernatant.
  7. Acidify supernatant to pH of 2.0 with 6N HCl for efficient extraction of solutes.
  8. Extract with equal volume of ethyl acetate.
  9. Dry extract with nitrogen gas and re-suspend in mobile phase (80% acetonitrile).
  10. Filter the resuspended extract using a 0.45 μm PTFE or nylon filter.
  11. Run the sample on an HPLC using a C18 column (150 x 3 mm) and a flow rate of 0.8 ml/min. Monitor the sample at 280 nm.
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