Difference between revisions of "Part:BBa J64010:Experience"
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Fluorescence intensity of BBa_K649001 was not increased by 3O-C12-HSL induction. | Fluorescence intensity of BBa_K649001 was not increased by 3O-C12-HSL induction. | ||
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| + | [[image:BBa_J64010_experience.png|thumb|center|400px|Effect of 3O-C12-HSL induction on fluorescence intensity ]] | ||
Revision as of 13:19, 1 October 2011
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Applications of BBa_J64010
Fluorescence intensity of BBa_K649001 was not increased by 3O-C12-HSL induction.
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.
[Sample]
pSB3K3-PlasI(BBa_J64010)-rbs-gfp-TT / pBR-Ptrc-rbs-lasR-TT
[Method]
①Overnight culture of Sample 1 grown at 37 in LB medium containing carbenicillin and kanamycin were diluted 1:1000 in the medium, and overnight cultures of Sample 2 grown at 37 °C in LB medium containing carbenicillin and chloramphenicol were diluted 1:100 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.
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