"
Team:Tokyo Tech/Construction
From 2008.igem.org
| Home | Construction | Acrylic container | Development of promoter | Genetic toggle switch | Parts Submitted to the Registry | Result | The Team | Acknowledgements |
|---|
Confirmatory experiment
Construction
For confirming pressure-response ability of pressure-inducible promoter, we experimented under 0.1MPa and 30MPa pressure. We chose TetR promoter(PtetR) as pressure-inducible promoter and we constructed two plasmids - one is PtetR-GFP on pSB6(BBa_K121010), the other is promoter less-GFP on pSB6(BBa_K121013) as a negative control.
Experiment under high pressure
Dilute this culture medium by 1% by adding fresh medium and suitable antibiotic (ampicilin; 50㎍/ml). Next, seal this culture medium with oxygen-saturated fluorinert (25% volume of medium) in polypropylene tubes with parafilm (figure3). Put this tube into pressure vessel filled with water (figure4). Next cup pressure vessel. Finally, pressurize pressure vessel by pressure device(figure5). and then incubation was started at 37℃ immediately at each pressure vessel(original designed) for 16h at 37℃.After cultivation(figure6), the cells were examined by fluorescence microscope.
 figure3 Polypropylene tubes with parafilm |
 figure4 Pressure vessel |
 |figure5 Pressure device |
 figure6 Constant-temperature bath |
Result
The result was that PtetR activity under 30MPa pressure is about 2.5 times stronger than PtetR activity under 0.1MPa pressure. Therefore, we confirmed that PtetR was induced under 30MPa pressure.
