Revision as of 16:41, 27 October 2017

PyeaR-NsrR binding site-B0030-GFP composite

A promoter PyeaR can be induced 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 can still be activated. In order to improve the biobrick K381001, we decided to add an additional nsrR 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 sequence to K381001 before RBS, we successfully decreased the fluorescence basal level. That is to say, under no nitrate induction, we can get lower fluorescence and maintain ability to do more accurate and easier nitrate detection without interference by other factors.

Figure above shows comparison between original PyeaR-GFP and our modified one. Our new biobricks can lower the fluorescence basal level because the fluorescence is lower under no nitrate induction after different culture time.

Our modified PyeaR-GFP composite were induced 12 hours in different nitrate concentration, and showed that they still sustain 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.

Sequence and Features