![](https://parts.igem.org/images/partbypart/icon_regulatory.png)
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.
Applications of BBa_R0061
User Reviews
UNIQ0066beba218f85cc-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])
UNIQ0066beba218f85cc-partinfo-00000002-QINU
iGEM Chiba 2010
- To cheracterize R0061 biobrick part, we inserted a GFP reporter (E0240) under R0061 to make K396012.
- We tested whether the GFP expression is repressed by LuxR and 3OC6HSL.
Methods
- plamisd used
- K396012 on pSB2K3 (parts which GFP is inserted below R0061)
- K396011 on pSB1A3 (constitutive LuxR generator)
- J09250 on pSB2K3 (GFP under lac promoter) as a positive control
- R0061 on pSB2K3 as a negative control
- notes: pSB2K3 is known as a very-low-copy plasmid, only in a lacIq strain. In this experiment we used DH10B which does not have lacIq.
- 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 with a fluorescence microplate reader(excitation 485 nm,emission 527 nm).
Results & Discussion
![](/wiki/images/3/30/Chiba_R0061.png)
- 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, not log phase cells.
Reference
<biblio>
- Egland pmid=10633117
- Cox pmid=18004278
</biblio>