Team:Tokyo Tech/Construction

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Home Construction Acrylic container Development of promoter Genetic toggle switch Parts Submitted to the Registry Result The Team Acknowledgements

Confirmatory experiment

figure1 We constructed A.PtetR-GFP, B.promoter less-GFP for 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([http://partsregistry.org/Part:BBa_K121010 BBa_K121010]), the other is promoter less-GFP on pSB6([http://partsregistry.org/Part:BBa_K121013 BBa_K121013]) as a negative control.








figure3 Polypropylene tubes with parafilm

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.



figure2 Result of experiment


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.