Part:BBa_R0061:Experience

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

User Reviews

UNIQ3067a2cd7f7fe2f0-partinfo-00000000-QINU

No review score entered.

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])

UNIQ3067a2cd7f7fe2f0-partinfo-00000002-QINU

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