Part:BBa_J64010:Experience

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UNIQcfc35eda42a0ac51-partinfo-00000000-QINU UNIQcfc35eda42a0ac51-partinfo-00000001-QINU

• Tokyo-Tech iGEM 2011

Fluorescence intensity of PlasI(BBa_J64010)-gfp did not change before and after 3OC12-HSL induction. Effect of 3OC12-HSL induction on fluorescence intensity This work is done by Takuya Tsubaki. 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_J64010, we used PlasI(BBa_J64010)-rbs-gfp-TT as reporer part and Ptrc-rbs-lasR-TT as regulator part. Since Ptrc is a constitutive promoter, lasR is constitutively expressed and, since it is know that this lasR is a working part, the fact that the fluorescence intensity levels do not change before and after induction of BBa_K649000 by 3OC12-HSL indicates that BBa_K649000 is not regulated by 3OC12-HSL. We improved this part. LasI promoter(BBa_K649000) which we constructed was successfully regulated by 3OC12-HSL. 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]. [Sample] PlasI(BBa_J64010)-rbs-gfp-TT / Ptrc-rbs-lasR-TT promoterless-gfp / Ptrc-rbs-lasR-TT-PlasI-rbs-cI-TT (negative control) [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 3OC12-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.

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