Part:BBa_K395008:Experience

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No review score entered. iGEM Tokyo_Tech 2010

We designed a new promoter which is repressed by AHL and LuxR complex by changing one base of the existing BioBrick parts (BBa_R0061).

In order to characterize K395008, Plux repression promoter, we constructed K395105 combining K395008 and K121013, which is a promoter-less gfp reporter (rbs-gfp-ter-ter) and this backbone is pSB6A1. We 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.

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