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

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We improved previous lasI promoter(BBa_J64010).[https://parts.igem.org/Part:BBa_J64010:Experience our assay of BBa_J64010]
 
We improved previous lasI promoter(BBa_J64010).[https://parts.igem.org/Part:BBa_J64010:Experience our assay of BBa_J64010]
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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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===User Reviews===
 
===User Reviews===

Revision as of 01:04, 5 October 2011

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

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


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].



User Reviews

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