Difference between revisions of "Part:BBa K649001:Experience"
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[[Image:K649001_experience.png|thumb|center|400px|Effect of 3O-C12-HSL induction on fluorescence intensity ]] | [[Image:K649001_experience.png|thumb|center|400px|Effect of 3O-C12-HSL induction on fluorescence intensity ]] | ||
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Fluorescence intensity of BBa_K649001 was increased by 3O-C12-HSL induction. | Fluorescence intensity of BBa_K649001 was increased by 3O-C12-HSL induction. | ||
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Generally, in the presence of 3O-C12-HSL lasI promoter is activated and the transcription level of downstream gene increases. To characterize this part, we used Ptrc-lasR on pBR as regulator part. | Generally, in the presence of 3O-C12-HSL lasI promoter is activated and the transcription level of downstream gene increases. To characterize this part, we used Ptrc-lasR on pBR as regulator part. | ||
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'''Method''' | '''Method''' | ||
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①Overnight cultures of sample strain grown at 37 °C in LB medium containing carbenicillin and kanamycin were diluted 1:100 in the medium, and then they were incubated at 37 °C as fresh cultures. | ①Overnight cultures of sample strain grown at 37 °C in LB medium containing carbenicillin and kanamycin were diluted 1:100 in the medium, and then they were incubated at 37 °C as fresh cultures. | ||
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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. | 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. | ||
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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). | 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). | ||
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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. | 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. | ||
Revision as of 10:33, 1 October 2011
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Applications of BBa_K649001
Fluorescence intensity of BBa_K649001 was increased by 3O-C12-HSL induction.
Generally, in the presence of 3O-C12-HSL lasI promoter is activated and the transcription level of downstream gene increases. To characterize this part, we used Ptrc-lasR on pBR as regulator part.
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 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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