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Team:Tokyo Tech
From 2008.igem.org
| Parts Submitted to the Registry | Our Team | Acknowledgements |
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Contents |
Our project
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We want to make a Coli Touch!!
To make a Coli Touch, we use input style of pressure. |
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Why pressure?
First, there were various ways as input.
For example, small molecule, heat, and light were used until iGEM 2007.
But no one used pressure as input.
Moreover, past way(small molecule, heat and light) are difficult to induct uniformly.
Pressurize can induct unifomly.It's prospect of technological application in confirmatory experiment. |
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We use pressure sensitive parts.
One paper says "One promoter is sensitive to pressure.". So we focused on it. |
Pressure induction
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.
Touch display
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We create devices for confirming pressure response of lac promoter. This is the first step of creating touch display. This device made of Acrylic glasses and has two holes (show figure). Each hole contains tubes and water. Inside tubes E. coli is cultivated. Pressure can travel to inside tubes. One hole (A) is covered with a plastic tape (show figure). Therefore the hole is pressurized.The other (B) is covered with a block made of an acrylic glass. (show figure) Therefore the hole is not pressurized by water. |
E.coli type in tubes  |
* “Ptet” on pSB6 plasmid (E.coli strain; JM109) |
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After pressurized the container, we observed the E.coli by a fluorescence microscope. The result shows below. |
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IPTG assay
Objective
By testing how LacI represses the lac promoter, Hill coefficient of lac promoter should be decided. In order to adjust effective concentration of LacI, IPTG was added.
Result & Conclusion
GFP fluorescence intensity was enhanced in an IPTG-dose dependent manner. It indicates that the LacI repression was getting weak by adding IPTG. Taken together with the graph shown in figure 1 and the formulation of Hill function fitting described above, the characteristics of the lac promoter expressed in Hill function was determined. Finally, we obtained nPlac = 2.15.
Calculate the domain of appropriate parameters
It is known that Plac activity under 30MPa pressure is 94.0 times stronger than Plac activity under atmospheric pressure, so, β30Plac = 94.0. If 30MPa additional activity rate of PL (β30PL) or the ratio of Plac - RBS strength (αPlac) and PL-RBS strength(αPL) are not appropriate value, we can't construct PIGTS. So we calculated the domain of appropriate β30PL and the ratio of αPlac and αPL.
We identified β30PL = 1.42 by our experiment under 30MPa pressure(figure 5-6). Therefore, we can implement rewritable function if we choose PL - RBS strength range from 2.4 to 7.4.
