Improvement and Characterize
Group: Tokyo Tech 2016
Author: Yoshio Takata
Summary of Improvement and Characterization:
I. Improved Prhl by iGEM 2014 Tokyo_Tech and characterize RhlR assay
II. Improvement of the wild type Prhl
III. Comparison of the improved Prhl by iGEM 2014 Tokyo_Tech to our original improved Prhl.
The sequences of these two chosen mutants are shown in below.
Native Prhl (BBa_R0071)
If you want more information, you see our work in Tokyo_Tech 2016 wiki!
iGEM 2014 Tokyo_Tech
We, Tokyo Tech 2014, improved Prhl Promoter (BBa_R0071) by changing LuxR binding site of Plux Promoter to RhlR binding site.
We dsigned new Prhl promoter We measured the GFP expression with the four different promoters (Prhl : BBa_R0071, Prhl(RR) : BBa_K1529320, Prhl(LR) : BBa_K1529310 and Prhl(RL) : BBa_K1529300) by flow cytometer. Each promoter was tested in the presence and also in the absence of C4HSL.
Fig. 1 shows the fluorescence intensity detected by flow cytometer.
From the result, when induced by C4HSL, Prhl(RR) promoter showed higher maximum expression level and higher leak than the original Prhl promoter (BBa_R0071).
Although Prhl(RL) promoter had lower maximum expression level compared to Prhl(RR) promoter, it had the highest induced/not-induced ratio.
This means Prhl(RL) promoter has little leak.
Therefore, we can say that Prhl(RL):BBa_K1529300 promoter is the best improved Prhl promoter due to the advantages of less leak and higher expression level.
For more information, see our work in Tokyo_Tech 2014 wiki.
Applications of BBa_R0071
No review score entered. Northwestern 2011
The 2011 Northwestern iGEM team used this promoter as a unit within our Pseudomonas Aeruginosa biosensor. When this RhlR/C4-HSL regulated promoter is induced at varying concentrations of C4-HSL, we observed GFP fluorescence in accordance to the graph below.