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Team:Tokyo Tech/Projects/RPS-game/assay
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<ul> | <ul> | ||
| - | <li><a href="#Overall1">1.Previous lasI promoter activity</a></li> | + | <li><a href="#Overall1">1. Previous lasI promoter activity</a></li> |
| - | + | ||
</ul> | </ul> | ||
| + | <u2> | ||
| + | <li><a href="#Overall2">1.1. Sample</a></li> | ||
| + | <li><a href="#Overall3">1.2. Method</a></li> | ||
| + | </u2> | ||
</div> | </div> | ||
</div> | </div> | ||
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<h1> RPS detailed method and result </h1> | <h1> RPS detailed method and result </h1> | ||
| - | <h2 id=" | + | <h2 id="Overall1">1. Previous lasI promoter activity</h2> |
| - | + | ||
| + | <h3 id="Overall2">1.1. Sample</h3> | ||
<p> | <p> | ||
| - | + | PlasI(BBa_J64010)-rbs-gfp-TT on pSB3K3 / Ptrc-rbs-lasR-TT on pBR<br> | |
| + | promoterless-gfp on pSB3K3 / Ptrc-rbs-lasR-TT-PlasI-rbs-cI-TT on pBR (negative control) | ||
</p> | </p> | ||
| + | <h3 id="Overall3">1.2. Method</h3> | ||
| + | <p> | ||
| + | ①Overnight culture of sample strain grown at 37 °C in LB medium containing carbenicillin and kanamycin, and overnight culture of promoterless negative control grown at 37 °C in LB medium containing carbenicillin and kanamycin were diluted 1:300 in the medium, and then they were incubated at 37 °C as fresh cultures.<br> | ||
| + | ②After their OD600 reached 0.2, we added 3 µL of 500 µM 3O-C12-HSL (3OC12-HSL+) or 3µL of DMSO (3OC12-HSL-) into the fresh cultures.<br> | ||
| + | ③After 3-hour incubation at 37 °C (OD approximately reached 1.50.), 0.25 mL of each culture was harvested by centrifugalization and suspended by adding 1 mL of PBS (phosphate-buffered saline). <br> | ||
| + | ④We dispensed 500 µL of each suspension into a disposable tube through a cell strainer, and its fluorescence intensity was measured with a flow cytometer of Becton, Dickinson and Company.<br> | ||
| + | </p> | ||
| + | <h3 id="Overall4">1.3. Result</h3> | ||
| + | <img src="https://static.igem.org/mediawiki/2011/8/80/BBa_J64010_graph3.png" alt="Previous lasI promoter activity" width=600px /> | ||
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Revision as of 10:17, 4 October 2011
RPS detailed method and result
1. Previous lasI promoter activity
1.1. Sample
PlasI(BBa_J64010)-rbs-gfp-TT on pSB3K3 / Ptrc-rbs-lasR-TT on pBR
promoterless-gfp on pSB3K3 / Ptrc-rbs-lasR-TT-PlasI-rbs-cI-TT on pBR (negative control)
1.2. Method
①Overnight culture of sample strain grown at 37 °C in LB medium containing carbenicillin and kanamycin, and overnight culture of promoterless negative control grown at 37 °C in LB medium containing carbenicillin and kanamycin were diluted 1:300 in the medium, and then they were incubated at 37 °C as fresh cultures.
②After their OD600 reached 0.2, we added 3 µL of 500 µM 3O-C12-HSL (3OC12-HSL+) or 3µL of DMSO (3OC12-HSL-) into the fresh cultures.
③After 3-hour incubation at 37 °C (OD approximately reached 1.50.), 0.25 mL of each culture was harvested by centrifugalization and suspended by adding 1 mL of PBS (phosphate-buffered saline).
④We dispensed 500 µL of each suspension into a disposable tube through a cell strainer, and its fluorescence intensity was measured with a flow cytometer of Becton, Dickinson and Company.
1.3. Result
