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

UNIQ60e70ae77741c73a-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])

UNIQ60e70ae77741c73a-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

• plamisd used
  1. K396012 on pSB2K3 (parts which RFP is inserted below R0061)
  2. K396011 on pSB1A3 (constitutive LuxR generator)
  3. J09250 on pSB2K3 (GFP under lac promoter) as a positive control
  4. R0061 on pSB2K3 as a negative control
• strain
  • DH10B

• procedure
  • plasmid 1 and 2 was cotransformed into DH10B. (plasmid 2 and 3 was used as a positive control, 3 and 4 as a negative control)
  • Colonies were cultured overnight in LB media with appropriate antibiotics.
  • Overnight cultures were diluted to 1:100 and incubated in a fresh LB medium (containing 0 or 1 nM 3OC6HSL and antibiotics) at 37 ºC for 12 h.
  • OD₆₀₀was measured, and 200 µL of the cultures were used to measure GFP fluorescence (excitation 485 nm,emission 527 nm).

Results

Figure. Fluorescence of lux repressor - GFP with/without AHL. p.c. stands for positive control, n.c. stands for negative control. Bar heights are normalized to OD₆₀₀ and represent the averages of three replicates; error bars, standard deviations.