Difference between revisions of "Part:BBa R0061:Experience"
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We confirm LuxR repression. | We confirm LuxR repression. | ||
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Revision as of 20:59, 5 November 2010
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iGEM Tokyo_Tech 2010
iGEM Tokyo_Tech 2010
In order to characterize R0061, Plux repression promoter, we constructed K395101 combining R0061 and K121013, which is a promoter-less gfp reporter (rbs-gfp-ter-ter) on pSB6A1 and used a fusion of PlacIq (I14032) to gfp (K121013) as a positive control and used promoterless gfp (K121013) as a negative control.
Overnight cultures of reporter strains grown at 37 °C containing appropriated antibiotics were diluted at least 1:100 and incubated at 37 °C as fresh cultures. After their OD590 reached 0.6, added 3OC6HSL. After 3 hours of induction, fluorescence intensity was measured with flow cytometry.
After 3 hours of induction by 3OC6HSL, the expression of GFP with 3OC6HSL dropped to 1/3 comparing with the expression without 3OC6HSL.
We confirmed that this promoter works correctly.
(→[http://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep more information])
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iGEM Chiba 2010
We cheracterized about R0061.We constructed Plux inv-GFP combining R0061 (Plux inv) and E0240 (RBS and GFP).
Plux inv-GFP and Plac-LuxR was cotransformed by strain of DH10B.The strain was incubeted in LB liquid medium (AHL 0 nM and AHL1000 nM) for 12 h at 37゜C.
After incubation,the sample was spin-dawned and we observed the pellet.
We confirm LuxR repression.
