Team:Tokyo Tech/Projects/RPS-game/assay

From 2011.igem.org

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<a href="#Overall1">1. Previous lasI promoter activity</a>
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<a href="#1.">1. Previous lasI promoter activity</a>
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<li><a href="#Overall2">1.1. Sample</a></li>
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<li><a href="#1.1.">1.1. Sample</a></li>
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<li><a href="#Overall3">1.2. Method</a></li>
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<li><a href="#1.2.">1.2. Method</a></li>
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                                <li><a href="#1.3.">1.3. Result</a></li>
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                        <a href="#2.">2. LasR repression</a>
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                        <ul>
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<li><a href="#2.1.">2.1. Sample</a></li>
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<li><a href="#2.2.">2.2. Method</a></li>
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                                <li><a href="#2.3.">2.3. Result</a></li>
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<h1> RPS detailed method and result </h1>
<h1> RPS detailed method and result </h1>
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<h2 id="Overall1">1. Previous lasI promoter activity</h2>
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<h2 id="1.">1. Previous lasI promoter activity</h2>
          
          
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         <h3 id="Overall2">1.1. Sample</h3>
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         <h3 id="1.1.">1.1. Sample</h3>
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         <h3 id="Overall3">1.2. Method</h3>
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         <h3 id="1.2.">1.2. Method</h3>
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         <h3 id="Overall4">1.3. Result</h3>
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         <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" />
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        <h2 id="2.">2. LasR repression</h2>
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        <h3 id="2.1.">2.1. Sample</h3>
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        <p>
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        <ul>
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<li>Ptrc-rbs-lasR-TT-PlasI-rbs-cI on pBR / Pλ-rbs-gfp on pAC</li>
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<li>Ptrc-rbs-lasR-TT on pBR / promoterless-rbs-gfp-TT on pSB3K3 (negative control)</li>
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</ul>
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</p>
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        <h3 id="2.2.">2.2. Method</h3>
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        <p>
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        <ol>
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        <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>
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        <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>
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        <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>
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        <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>
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        </ol>
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        </p>
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        <h3 id="2.3.">2.3. Result</h3>
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        <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

Tokyo Tech 2011

Tokyo Tech 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

  1. 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.
  2. 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.
  3. 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).
  4. 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

Previous lasI promoter activity

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

  1. 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.
  2. 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.
  3. 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).
  4. 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.

2.3. Result

LasR repression

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