Difference between revisions of "Part:BBa K3733001"
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<p>For the nitrate sensor sensitivity testing, we transformed the constructed plasmid with nitrate sensor into <i>E.coli</i> BL21. The <i>E.coli</i> strain was cultured at 37°C overnight, and then diluted to OD<sub>600</sub> = 0.4. And then, culture bacteria at 37°C for 2 hours, the potassium nitrate solution that has been serially diluted were add. After 8 hours inducing, we detected the expression of the neGFP by microplate reader. </p> | <p>For the nitrate sensor sensitivity testing, we transformed the constructed plasmid with nitrate sensor into <i>E.coli</i> BL21. The <i>E.coli</i> strain was cultured at 37°C overnight, and then diluted to OD<sub>600</sub> = 0.4. And then, culture bacteria at 37°C for 2 hours, the potassium nitrate solution that has been serially diluted were add. After 8 hours inducing, we detected the expression of the neGFP by microplate reader. </p> | ||
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<center><b>Figure 1. </b>The The nitrate response curve induced by different concentrations of potassium nitrate in <i>E.coli</i> BL21. The red vertical line shows the concentration of <b>DSS induced in mouse</b>, while the green vertical line shows the concentration of <b>normal conditions</b>. The DSS induced animal model often used as an inflammation model. </center> | <center><b>Figure 1. </b>The The nitrate response curve induced by different concentrations of potassium nitrate in <i>E.coli</i> BL21. The red vertical line shows the concentration of <b>DSS induced in mouse</b>, while the green vertical line shows the concentration of <b>normal conditions</b>. The DSS induced animal model often used as an inflammation model. </center> | ||
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Revision as of 18:01, 19 October 2021
NarX(C415R): A point mutation of NarX
The histidine kinase is a part of two-component signal system, which each constitute a sensor histidine kinase and a response regulator. The NarX is sensor histidine kinase, which can phosphorylate NarL in response to nitrate and nitrite.
Usage and Biology
The NarX/NarL two component system(TCS) can be activated if the concentration of nitrate is enough. So this NarX/NarL TCS can be designed the nitrate sensor with the PyeaR promoter. When there is enough nitrate, the NarX will change configuration and then phosphorylate. After that, the phosphorylated NarX will transport the phosphate group to NarL. Last, the phosphorylated NarL will bind to the certain sequence of PyeaR, and it acts as a transcription factor, which can active the downstream of PyeaR to transcription.
The NarX (C415R) is a mutant of NarX (BBa_K3733000), which amino acid at position of 415 changed from cysteine to arginine. For this change, we can make the nitrate sensing system more sensitive.
Functional Parameters
For the nitrate sensor sensitivity testing, we transformed the constructed plasmid with nitrate sensor into E.coli BL21. The E.coli strain was cultured at 37°C overnight, and then diluted to OD600 = 0.4. And then, culture bacteria at 37°C for 2 hours, the potassium nitrate solution that has been serially diluted were add. After 8 hours inducing, we detected the expression of the neGFP by microplate reader.
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Although the mutation makes the nitrate sensor system more sensitive, it is still activated under normal circumstances that we do not want to see.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]