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Team:Calgary/Notebook/Protocols/AmidaseAssay
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| - | In order to upgrade the hydrocarbons produced by OSCAR, we validated the ability of the <i>amdA</i> gene from <i>Rhodococcus</i> to be able to degrade nitrogen containing compounds. This was validated via gas chromatography/mass spectrometry (GC/MS). | + | <p>In order to upgrade the hydrocarbons produced by OSCAR, we validated the ability of the <i>amdA</i> gene from <i>Rhodococcus</i> to be able to degrade nitrogen containing compounds. This was validated via gas chromatography/mass spectrometry (GC/MS). |
| - | < | + | <ol> |
| - | <li>Cells containing the <i>amdA</i> gene or a vector control (pSB1C3) were grown overnight at 37<sup>o</sup>C with shaking. | + | <li>Cells containing the <i>amdA</i> gene or a vector control (pSB1C3) were grown overnight at 37<sup>o</sup>C with shaking. </li> |
| - | <li>Cells were washed in M9 minimal media without ammonia chloride two times and spun at 4,000 RPM 4<sup>o</sup>C, for 10 min. | + | <li>Cells were washed in <a href="https://2012.igem.org/Team:Calgary/Notebook/Protocols/m9media">M9 minimal media</a> without ammonia chloride two times and spun at 4,000 RPM 4<sup>o</sup>C, for 10 min. |
<li>Cells were incubated with benzamide at a concentration of 1 mM in 5mL of M9 minimal media without ammonia chloride (no nitrogen source). Cells were incubated in this media at an OD<sub>600</sub> of approximately 0.100 and incubated at 37<sup>o</sup>C for 48 hours with shaking. | <li>Cells were incubated with benzamide at a concentration of 1 mM in 5mL of M9 minimal media without ammonia chloride (no nitrogen source). Cells were incubated in this media at an OD<sub>600</sub> of approximately 0.100 and incubated at 37<sup>o</sup>C for 48 hours with shaking. | ||
<li> Cultures were then sonicated with a Misonix Cell Disruptor at Dial 11 (output of 18 watts) for 1 min. | <li> Cultures were then sonicated with a Misonix Cell Disruptor at Dial 11 (output of 18 watts) for 1 min. | ||
<li> 3 mL of ethyl acetate was added to each tube shaken vigorously and spun down at 4000 RPM for 5 min. at 4<sup>o</sup>C | <li> 3 mL of ethyl acetate was added to each tube shaken vigorously and spun down at 4000 RPM for 5 min. at 4<sup>o</sup>C | ||
<li> 1 mL of the ethyl acetate layer was concentrated to 250 uL and ran onto the GC-MS. | <li> 1 mL of the ethyl acetate layer was concentrated to 250 uL and ran onto the GC-MS. | ||
| - | + | </p> | |
| - | + | </ol> | |
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</html> | </html> | ||
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Latest revision as of 03:41, 27 October 2012
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Amidase Degradation Assay
In order to upgrade the hydrocarbons produced by OSCAR, we validated the ability of the amdA gene from Rhodococcus to be able to degrade nitrogen containing compounds. This was validated via gas chromatography/mass spectrometry (GC/MS).
- Cells containing the amdA gene or a vector control (pSB1C3) were grown overnight at 37oC with shaking.
- Cells were washed in M9 minimal media without ammonia chloride two times and spun at 4,000 RPM 4oC, for 10 min.
- Cells were incubated with benzamide at a concentration of 1 mM in 5mL of M9 minimal media without ammonia chloride (no nitrogen source). Cells were incubated in this media at an OD600 of approximately 0.100 and incubated at 37oC for 48 hours with shaking.
- Cultures were then sonicated with a Misonix Cell Disruptor at Dial 11 (output of 18 watts) for 1 min.
- 3 mL of ethyl acetate was added to each tube shaken vigorously and spun down at 4000 RPM for 5 min. at 4oC
- 1 mL of the ethyl acetate layer was concentrated to 250 uL and ran onto the GC-MS.
