pLux OmpR hybrid promoter
In order to optimize the function of the lux promoter, We creat a hybrid promoter which combine plux with OmpR, and in this case, plux can be lead to expression by AHL and obtain by phosphorylation of OmpR protein. According to the research of it before, we camp up with a program . Keep the sequencing of Lux Box, -35box and -10box. Then put the sequencing of C3 point of OmpR promoter into -35box and -10box.
Figure structural representation of Plux promoter
Figure : the analysis of conserved sequence of pLux by Weblogo
Plux promoter is organized from the promoter the shining gene of V. fischeri(R0062). Through analyzing common hybrid promoter of Lux by Weblogo , we found that the sequence of Lux Box is conserved. In benefit of LuxR Binding Site, -35box and -10box of the promoter the shining gene of V. fischeri(R0062).
Figure : Our design of pLux-OmpR hybrid promoter
We link the hybrid promoter to the test circuit. For control group experiment, we put strain A to LB medium adding 20% Sucrose solution and not adding any sucrose solution to cultivate in 37% shake flask , and detect the change of OD value and fluorescence value between 12h.
Though high concentration sucrose solution restrains the grain of E.coli, it doesn’t have effect on the work of circuit. In conclusion, we find that high osmotic pressure has no effect on quorum sensing circuit and the activated of plux promoter. Strain A could serve as control group of pLux-OmpR hybrid promoter.
Measurement of pLux/OmpR hybrid promoter's expression property
Place strain B in 100 mL LB medium with gradient osmotic pressure stress and culture in shake-flask in 37°C.Calculate RFU of each time point ,and we get a surprising result!
Experimental Group Relative Fluorescence Unit--Time Scatter graph
From the figure we can see that when stressed by 7% sucrose, GFP's expression is stronger than those cultured in normal condition. However,when stressed by 14% sucrose, GFP's RFU is 3 times as high as normal culture condition.This indicates that sucrose stress promotes GFP's expression via gene circuit.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC