Part:BBa_K649001:Experience
Applications of BBa_K649001
Fluorescence intensity of BBa_K649001 was increased by 3OC12-HSL induction. GFP expression after induction of 3OC12-HSL is 170 times as high as before.
Generally, in the presence of 3OC12-HSL, LasR activates lasI promoter and the transcription level of downstream gene increases, but in the absence of 3OC12-HSL, LasR can't activate lasI promoter. To characterize BBa_K649000, we used Ptrc-rbs-lasR-TT as regulator part. Because LasR is constitutively expressed, the difference of fluorescence intensity by 3OC12-HSL induction indicates that BBa_K649000 is successfully regulated by 3OC12-HSL.
[Sample]
Ptrc-rbs-lasR-TT / PlasI(BBa_I649000)-rbs-gfp-TT
Ptrc-rbs-lasR-TT / Promoterless-rbs-gfp-TT (negative control)
[Method]
- Overnight cultures of sample strain grown at 37 °C in LB medium containing carbenicillin and kanamycin were diluted 1:100 in the medium, and overnight cultures of promoterless negative control strain grown at 37 °C in LB medium containing carbenicillin and kanamycin were diluted 1:200 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 3OC12-HSL (3OC12-HSL+) or 3 µL of DMSO (3OC12-HSL-) into the fresh cultures.
- After 3-hour incubation at 37 °C (OD reached approximately 1.80.), 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.
We improved previous las promoters.
To prove that the LasR regulator used in our lasI prmoter assay works, we did another assay. Details about this assay can be found [http://2011.igem.org/Team:Tokyo_Tech/Projects/RPS-game/assay#1. here].
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