Team:British Columbia/DSZNotebook

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To obtain a control for our project which distributes the DszABC operon amongst three different auxotrophs , we needed to show the rate of desulfurization (removal of dibenzothiophene) when the entire DszABC pathway is transformed into a single host. To perform this experiment, we prepared a culture of E.coli containing an optimized plasmid for the DszABC pathway cloned from Rhodococcus erythropolis IGTS8. This construct was obtained from Dr.Keasling and was the source for our DszC biobrick.

An overnight culture for this E.coli cloned with the DszABC operon was grown overnight in 5mL culture of LB. We innoculated the overnight culture in 200mL culture of LB in a 500mL flask so that we have a starting O.D600 is 0.05

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.

Identify retention time and DBT.