Part:BBa_K381001
Nitrate reporter: PyeaR - GFP composite
Nitrate and Nitrite sensitive promoter PyeaR with a GFP coding device and strong RBS to create a nitrate-sensitive system which signals through expression of GFP.
PyeaR is normally repressed by NsrR, a protein native to most 'E. coli' cells. When Nitrate or Nitrite enter the cell it is converted to Nitric Oxide. This binds to NsrR, halting the repression and allowing the production of GFP.
Optimum use appears to be when sensing nitrate within a range of 0 - 20 mM (Calculated from Miller assays)
Activity between 0 - 10mM was investigated using an assay measuring change in GFP expression with increasing levels of Potassium Nitrate, the results of which can be seen below.
Additional characterization. Strain was NEB Turbo.
NCKU_Tainan 2017
BBa_K381001 was first designed by Katharine Coyte from team BCCS-Bristol in iGEM 2010. It is a nitrate reporter, PyeaR-GFP composite. The team BCCS-Bristol only test the BioBrick’s sensitivity at a higher concentration, but the nitrate level of aquatic water usually won’t reach up to 1 mM. Therefore, we carry out experiment by testing the fluorescence intensity with ppm scale of Potassium Nitrate, which is much more lower than all the teams before. The results are shown below.
It shows that the BioBrick BBa_K381001 have nice sensitivity not only in high nitrate concentration, but low concentration as well. Many teams had done improvements of K381001, but our team bring out the most beautiful data. And we also take X axis logarithm, which can make our data linear. Not only easier to read, but also more meaningful. The data is shown as below. ppm conc. 8 hours 12 hours 24 hours 0 454.623 546.841 620.402 2 689.961 754.085 821.113 4 766.835 866.846 922.813 6 816.472 916.119 972.653 8 831.843 931.43 985.14 10 900.114 998.935 1058.844 20 962.493 1093.076 1148.216 40 1090.926 1200.976 1252.106 60 1129.622 1253.052 1295.002 80 1240.331 1341.711 1373.601 100 1259.766 1385.946 1449.946
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 773
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