Difference between revisions of "Part:BBa K649001:Experience"

Revision as of 05:34, 3 October 2011

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Applications of BBa_K649001

Fluorescence intensity of BBa_K649001 was increased by 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_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]

①Overnight 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.

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 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).

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.

We improved previous lasI promoter(BBa_J64010).our assay of BBa_J64010

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