Team:NCKU Tainan/Improve
Improve
Concept
A promoter PyeaR can be activated when it senses the nitrate and nitrite (Lin, et al., 2007). The BBCS-Bristol 2010 iGEM team constructed the biobrick K381001, which is composed of a PyeaR promoter, a strong RBS B0030 and a reporter gene GFP. It can emit green fluorescence when detecting nitrate so that it can also be served as a nitrate sensor.
PyeaR is repressed by NsrR protein under no nitrate or nitric oxide condition, and is activated when nitrate or nitrite is existing. In theory, the biobrick K381001 can’t emit fluorescence under no nitrate or nitrite. However, the data showed that it could still be activated. In order to improve the biobrick K381001, we decided to add an additional nsrR binding sequence to it so as to reinforce repression and decrease interference. As a result, fluorescence basal level can be decreased. And detection will be enhanced.
Functional test
By adding an nsrR binding sequence to K381001, we successfully decreased the fluorescence basal level. That is to say, under no nitrate induction, we can get lower fluorescence and maintain detection ability to do more accurate nitrate detection without interference by other factors.
Figure 1. Comparing with original PyeaR-GFP, our new biobricks can lower the fluorescence basal level because the fluorescence is lower under no nitrate induction after different culture time. And the function of PyeaR-RBS-NsrR-GFP is better of the two new biobricks because it lowered much more fluorescence.
Figure 2. Our two new PyeaR-GFP were induced 12 hours in different nitrate concentration, and showed that they still sustained the ability to detect nitrate.
References
Lin, H. Y., Bledsoe, P. J., & Stewart, V. (2007). "Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen-responsive regulator Fnr in Escherichia coli K-12." Journal of bacteriology, 189(21), 7539-7548.
