Composite

Part:BBa_K381001

Designed by: Katharine Coyte   Group: iGEM10_BCCS-Bristol   (2010-10-05)

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.


GFPChar0to10.png


ParisBettencourt12NitrateReporter.png

Additional characterization. Strain was NEB Turbo.

HFLS_H2Z_hangzhou 2017

Improve the Characterization of BBa_K381001

K381001.png Team HFLS_H2Z_hanzhou's experience with this part. In our experience with this part, we found out that Pyear will yield strong transcription rate under the presence of high concentration of Nitrate. However, we found out, under high concentration of nitrite(40mM), the promoter will be repressed, unlike nitrite in low concentration. Furthermore, if nitrate(40mM) is present along with nitrite(40mM), the promoter will be inhibited.


NCKU_Tainan 2017

Improve the Characterization of BBa_K381001

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.

NCKU Tainan 2017Contribution fluorescence logarithm of nitrate concentration figure.png

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.

Sensing data of K381001.png

Improve the Function of BBa_K381001

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 and decrease the fluorescence basal level, 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.

To see more details about the construction and result, click the hyperlink below:

>> PyeaR-NsrR binding site-B0030-GFP composite : BBa_K2275011

>> PyeaR-B0030-NsrR binding site-GFP composite : BBa_K2275012

>> PyeaR-NsrR binding site-amplifier-rbs composite constructed by team HFLS_H2Z_hangzhou: [1]


Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 773


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