Team:Tokyo Tech/Construction

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!align="center"|[[Team:Tokyo_Tech/Result|Result]]
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!align="center"|[[Team:Tokyo_Tech/Acknowledgements|Acknowledgements]]
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== '''Construction''' ==
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== '''Confirmatory experiment''' ==
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[[Image:Construction.png|260px|thumb|left|figure1 We constructed A.PtetR-GFP, B.promoter less-GFP for confirmatory experiment]]
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'''Construction'''
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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.
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<p class=MsoNormal>For checking pressure-response ability of Plac, we constructed three plasmids.Those are showed at figure 1.Plac stands for a promoter repressed by lacI protein. Ptet stands for promoter repressed by tetR protein.∆p stands for promoter less.JM109 is constitutive expression lacI protein so that Plac is always repressed by lacI protein.And JM109 isn’t expression tetR. Therefore, we used Ptet as a positive control.  </font></p>
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<p class=MsoNormal>Fig1</p></td>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<IMG src="https://static.igem.org/mediawiki/2008/c/c8/Tech_const_fig1.jpg
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<p class=MsoNormal>Each parts is constructed on low-copy plasmid <a herf=http://partsregistry.org/Part:BBa_I739201>“pSB6”</a> ,and inserted into E.coli strain JM109.</p></td>
 
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For confirming pressure-response ability of pressure-inducible promoter, we
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experimented under 30MPa pressure. We chose TetR promoter(Ptet) as a pressure-inducible promoter. we constructed two plasmids - one is Ptet-GFP on pSB6([http://partsregistry.org/Part:BBa_I739201 BBa_I739201]), the other is promoter less-GFP on pSB6.
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{{clear}}
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'''Experiment under high pressure'''
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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.  
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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.
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<gallery widths = "150px" heights="150px" perrow="4" align="center" >
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Image:Tech_tube_2.JPG|figure3 Polypropylene tubes with parafilm
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Image:Tech_equip1.jpg|figure4 Pressure vessel
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Image:Tech_equip2.jpg||figure5 Pressure device
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Image:Tech_epuip3.jpg|figure6 Constant-temperature bath 
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[[Image:Pressure-response_ability_of_PtetR.png|350px|thumb|right|figure2 Result of experiment]]
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'''Result'''
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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.

Latest revision as of 13:04, 26 October 2008

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.






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

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.

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