Team:Tokyo Tech/Projects/RPS-game/assay
From 2011.igem.org
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<ul> | <ul> | ||
<li> | <li> | ||
- | <a href="# | + | <a href="#1.">1. Previous lasI promoter activity</a> |
<ul> | <ul> | ||
- | <li><a href="# | + | <li><a href="#1.1.">1.1. Sample</a></li> |
- | <li><a href="# | + | <li><a href="#1.2.">1.2. Method</a></li> |
+ | <li><a href="#1.3.">1.3. Result</a></li> | ||
+ | </ul> | ||
+ | <a href="#2.">2. LasR repression</a> | ||
+ | <ul> | ||
+ | <li><a href="#2.1.">2.1. Sample</a></li> | ||
+ | <li><a href="#2.2.">2.2. Method</a></li> | ||
+ | <li><a href="#2.3.">2.3. Result</a></li> | ||
</ul> | </ul> | ||
</li> | </li> | ||
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<h1> RPS detailed method and result </h1> | <h1> RPS detailed method and result </h1> | ||
- | <h2 id=" | + | <h2 id="1.">1. Previous lasI promoter activity</h2> |
- | <h3 id=" | + | <h3 id="1.1.">1.1. Sample</h3> |
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<ul> | <ul> | ||
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</ul> | </ul> | ||
</p> | </p> | ||
- | <h3 id=" | + | <h3 id="1.2.">1.2. Method</h3> |
<p> | <p> | ||
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</ol> | </ol> | ||
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- | <h3 id=" | + | <h3 id="1.3.">1.3. Result</h3> |
<img src="https://static.igem.org/mediawiki/2011/8/80/BBa_J64010_graph3.png" alt="Previous lasI promoter activity" width="500px" align="center" /> | <img src="https://static.igem.org/mediawiki/2011/8/80/BBa_J64010_graph3.png" alt="Previous lasI promoter activity" width="500px" align="center" /> | ||
+ | |||
+ | <h2 id="2.">2. LasR repression</h2> | ||
+ | |||
+ | <h3 id="2.1.">2.1. Sample</h3> | ||
+ | <p> | ||
+ | <ul> | ||
+ | <li>Ptrc-rbs-lasR-TT-PlasI-rbs-cI on pBR / Pλ-rbs-gfp on pAC</li> | ||
+ | <li>Ptrc-rbs-lasR-TT on pBR / promoterless-rbs-gfp-TT on pSB3K3 (negative control)</li> | ||
+ | </ul> | ||
+ | </p> | ||
+ | <h3 id="2.2.">2.2. Method</h3> | ||
+ | <p> | ||
+ | <ol> | ||
+ | <li>Overnight culture of sample strain grown at 37 °C in LB medium containing carbenicillin and chloramphenicol were diluted 1:20 in the medium, and overnight culture of promoterless negatgive 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.</li> | ||
+ | <li>After 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.</li> | ||
+ | <li>After 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).</li> | ||
+ | <li>We 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.</li> | ||
+ | </ol> | ||
+ | </p> | ||
+ | <h3 id="2.3.">2.3. Result</h3> | ||
+ | <img src="https://static.igem.org/mediawiki/2011/6/60/LasR_repression.png" alt="LasR repression" width="500px" align="center" /> | ||
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Revision as of 10:51, 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
- Overnight 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.
- After 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.
- After 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).
- We 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
2. LasR repression
2.1. Sample
- Ptrc-rbs-lasR-TT-PlasI-rbs-cI on pBR / Pλ-rbs-gfp on pAC
- Ptrc-rbs-lasR-TT on pBR / promoterless-rbs-gfp-TT on pSB3K3 (negative control)
2.2. Method
- Overnight culture of sample strain grown at 37 °C in LB medium containing carbenicillin and chloramphenicol were diluted 1:20 in the medium, and overnight culture of promoterless negatgive 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.
- After 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.
- After 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).
- We 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.