Team:Tokyo Tech/Projects/RPS-game/assay
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<h3 id="Overall2">1.1. Sample</h3> | <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> | <h3 id="Overall3">1.2. Method</h3> | ||
<p> | <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> | 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> | 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> |
Revision as of 10:23, 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.