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Team:British Columbia/DSZNotebook
From 2012.igem.org
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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 grew a culture of E.coli containing the 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. | 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 grew a culture of E.coli containing the 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 inoculated the overnight culture in 200mL culture of LB in a 500mL flask so that we have a starting O.D600 is 0.05. Prior to inoculation, dibenzothiophene was dissolved in DMSO at a concentration 100ppm. This was added to the 200mL culture such that it contains 2% DMSO. For a control, we inoculated an E.coli K12 strain without the plasmid, which should not contain the necessary enzyme of biodesulfurization. | + | An overnight culture for this E.coli cloned with the DszABC operon was grown overnight in 5mL culture of LB. We inoculated the overnight culture in 200mL culture of LB in a 500mL flask so that we have a starting O.D600 is 0.05. Prior to inoculation, dibenzothiophene (DBT) was dissolved in DMSO at a concentration 100ppm. This was added to the 200mL culture such that it contains 2% DMSO. For a control, we inoculated an E.coli K12 strain without the plasmid, which should not contain the necessary enzyme of biodesulfurization. |
| - | Next, we incubated the | + | Next, we incubated both cultures at 37°C shaking at 200 rpm. To obtain a representation of DBT removal over time, we extracted 25mL of each culture at O.D 600 of 0.3, 0.7, and 1.0 intervals. Once obtained, we prepared our sample for HPLC analysis. |
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| + | Supernatant was extracted by spinning down cells at 1600g for 10min. The supernatant was then acidified to pH of 2.0 with 6N HCl for efficient extraction of solutes. We then extracted the supernatant with equal volume of ethyl acetate. The extract was then dried with nitrogen gas and re-suspended in mobile phase with 80% acetonitrile. The resuspended extract was lastly filtered using a 0.45 μm PTFE or nylon filter. These samples were then run on an HPLC using a C18 column (150 x 3 mm) at a flow rate of 0.8 ml/min. The samples were monitored at 280 nm with identification of DBT and its retention time. | ||
Latest revision as of 03:48, 4 October 2012
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 grew a culture of E.coli containing the 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 inoculated the overnight culture in 200mL culture of LB in a 500mL flask so that we have a starting O.D600 is 0.05. Prior to inoculation, dibenzothiophene (DBT) was dissolved in DMSO at a concentration 100ppm. This was added to the 200mL culture such that it contains 2% DMSO. For a control, we inoculated an E.coli K12 strain without the plasmid, which should not contain the necessary enzyme of biodesulfurization.
Next, we incubated both cultures at 37°C shaking at 200 rpm. To obtain a representation of DBT removal over time, we extracted 25mL of each culture at O.D 600 of 0.3, 0.7, and 1.0 intervals. Once obtained, we prepared our sample for HPLC analysis.
Supernatant was extracted by spinning down cells at 1600g for 10min. The supernatant was then acidified to pH of 2.0 with 6N HCl for efficient extraction of solutes. We then extracted the supernatant with equal volume of ethyl acetate. The extract was then dried with nitrogen gas and re-suspended in mobile phase with 80% acetonitrile. The resuspended extract was lastly filtered using a 0.45 μm PTFE or nylon filter. These samples were then run on an HPLC using a C18 column (150 x 3 mm) at a flow rate of 0.8 ml/min. The samples were monitored at 280 nm with identification of DBT and its retention time.
