Regulatory
luxR

Part:BBa_R0061:Experience

Designed by: Srini Devadas, David Gray, Ronny Krashinsky, Debra Lin, and Chris Zheng Liu   Group: Antiquity   (2004-01-28)
Revision as of 09:57, 6 November 2010 by Maiko (Talk | contribs) (→‎Results & Discussion)

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Applications of BBa_R0061

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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])


Tokyotech R0061 K395008 graph R0061.jpg

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iGEM Chiba 2010

  • To cheracterize R0061 biobrick part, we inserted a GFP 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 uM 3OC6HSL and antibiotics) at 37 ºC for 12 h.
    • OD600 was measured, and 200 µL of the cultures were used to measure GFP fluorescence (excitation 485 nm,emission 527 nm).

Results & Discussion

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.
  • Repression were not observed in the condition we tested (see right Figure).


  • Egland and Greenberg [1] (the original paper of this promoter) reported a strong repression of "Plux repression promoter" using log phase cells.
  • The results from iGEM Tokyo Tech 2010 (above in this page) shows about 1/3 repression also using log phase cells.
  • Cox et al. [2] reported that the "Plux repression promoter"' does not work. This paper measured the cells in a stationary phase (cultured at 25 ºC for 18 h) as our team did (at 37 ºC for 12 h).


  • From these information, we believe that the reason why we couldn't observe the repression is because we performed the experiment using stationary phase cells.



Reference

<biblio>

  1. Egland pmid=10633117
  2. Cox pmid=18004278

</biblio>