Part:BBa_R0061:Experience

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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

under construction!

• To cheracterize R0061 biobrick part, we inserted an RFP reporter (E0240 below R0061 to make K396012. We tested whether the RFP expression is repressed by LuxR and 3OC6HSL.

Methods

• used parts
  • K396012 on pSB2K3 (parts which RFP is inserted below R0061)
  • K396011 on pSB1A3 (constitutive LuxR generator)
  • GFP regulated by lac promoter on pSB2K3 as a positive control
• 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,We measured GFP fluorescence(excitation 485 nm,emission 527 nm) and OD600.
  

Results=

Fig. 1