Difference between revisions of "Part:BBa J64010:Experience"

Tokyo-Tech iGEM 2011

Fluorescence intensity of PlasI(BBa_J64010)-gfp did not change before and after 3O-C12-HSL induction.

Effect of 3O-C12-HSL induction on fluorescence intensity
This work is done by Takuya Tsubaki.

Generally, in the presence of 3O-C12-HSL, lasR activates lasI promoter and the transcription level of downstream gene increases, but in the absence of 3O-C12-HSL, lasR can't activate lasI promoter. To characterize BBa_J64010, we used PlasI(BBa_J64010)-rbs-gfp-TT on pSB3K3 as reporer part and Ptrc-rbs-lasR-TT on pBR 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 3O-C12-HSL indicates that BBa_K649000 is not regulated by 3O-C12-HSL.

If you want to know why the lasR is a working part, click [here] and visit our team's wiki.

We improved this part. LasI promoter(BBa_K649000) which we constructed was successfully regulated by 3O-C12-HSL.

[Sample]

pSB3K3-PlasI(BBa_J64010)-rbs-gfp-TT / pBR-Ptrc-rbs-lasR-TT

[Method]

①Overnight culture of grown at 37 in LB medium containing carbenicillin and kanamycin were diluted 1:1000 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 (3O-C12-HSL+) or 3µL of DMSO (3O-C12-HSL-) into the fresh cultures.

③After 3-hour incubation at 37 °C, 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.