Difference between revisions of "Part:BBa K649001"
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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. | ||
Revision as of 17:22, 1 October 2011
GFP regulated by 3OC12-HSL and LasR
GFP reporter regulated by 3OC12-HSL and LasR.
Fluorescence intensity of BBa_K649001 was 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_K649000, we used Ptrc-rbs-lasR-TT on pBR as regulator part. Because lasR is constitutively expressed, the difference of fluorescence intensity by 3O-C12-HSL induction indicates that BBa_K649000 is successfully regulated by 3O-C12-HSL.
[Sample]
Ptrc-rbs-lasR-TT on pBR / PlasI(BBa_I649000)-rbs-gfp-TT on pSB3K3
[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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 118
Illegal BamHI site found at 106 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 805