Difference between revisions of "Part:BBa K3733020"
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The NarXL is a the two-component signal system in E.coli, 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. And then the phosphorylated NarL will binding the site and activate the transcription of PyeaR promoter. So in this nitrate sensor system, we use J23110 (medium promoter) and B0033 (weak RBS) to produce NarX and NarL. When we add different concentrations of nitrate, and measurement the fluorescence intensity to reflect the expression level of PyeaR promoter.
 
The NarXL is a the two-component signal system in E.coli, 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. And then the phosphorylated NarL will binding the site and activate the transcription of PyeaR promoter. So in this nitrate sensor system, we use J23110 (medium promoter) and B0033 (weak RBS) to produce NarX and NarL. When we add different concentrations of nitrate, and measurement the fluorescence intensity to reflect the expression level of PyeaR promoter.
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===Usage and Biology===
 
===Usage and Biology===
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<p>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. </p>
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<p>In this part, we use the combination is J23110 -B0033. The promoter J23110 is the medium promoter, but B0033 is a weak RBS. </p>
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<p>To learn more about nitrate sensor system, a collection of this kind of nitrate sensor system were constructed. </p>
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<p>BBa_K3733015: https://parts.igem.org/Part:BBa_K3733015 </p>
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<p>BBa_K3733016: https://parts.igem.org/Part:BBa_K3733016 </p>
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<p>BBa_K3733017: https://parts.igem.org/Part:BBa_K3733017 </p>
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<p>BBa_K3733018: https://parts.igem.org/Part:BBa_K3733018 </p>
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<p>BBa_K3733019: https://parts.igem.org/Part:BBa_K3733019 </p>
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<p>BBa_K3733020: https://parts.igem.org/Part:BBa_K3733020 </p>
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<p>BBa_K3733021: https://parts.igem.org/Part:BBa_K3733021 </p>
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<p>BBa_K3733022: https://parts.igem.org/Part:BBa_K3733022 </p>
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<p>BBa_K3733023: https://parts.igem.org/Part:BBa_K3733023 </p>
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<p>BBa_K3733024: https://parts.igem.org/Part:BBa_K3733024 </p>
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<p>BBa_K3733028: https://parts.igem.org/Part:BBa_K3733028 </p>
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<p>BBa_K3733029: https://parts.igem.org/Part:BBa_K3733029 </p>
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<p>BBa_K3733030: https://parts.igem.org/Part:BBa_K3733030 </p>
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<p>BBa_K3733031: https://parts.igem.org/Part:BBa_K3733031 </p>
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===Functional Parameters===
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<p>For the nitrate sensor sensitivity testing, we transformed the constructed plasmid with nitrate sensor into <i>E.coli</i> DH5α and 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><img src=https://static.igem.org/mediawiki/parts/8/8a/T--HZAU-China--BBa_K3733020_img1.png style="width:60% "></center>
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<center><b>Figure 1. </b>The The nitrate response curve with J23110- B0033 in <i>E.coli</i> DH5α and 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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<p> In this combination, nitrate sensor system does not seem to have biological function in <i>E.coli </i> BL21, but in <i>E.coli</i> DH5α, the performance of the nitrate sensor system is still effective. Based on the results of <i>E.coli </i>BL21, we infer that the adjustable range of nitrate sensor system is very small and the activation threshold is very low. Then the lowest induction concentration has saturated it. </p>
  
 
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Revision as of 03:05, 20 October 2021


The nitrate sensor(J23110-B0033) system with neGFP

The NarXL is a the two-component signal system in E.coli, 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. And then the phosphorylated NarL will binding the site and activate the transcription of PyeaR promoter. So in this nitrate sensor system, we use J23110 (medium promoter) and B0033 (weak RBS) to produce NarX and NarL. When we add different concentrations of nitrate, and measurement the fluorescence intensity to reflect the expression level of PyeaR promoter.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2017
    Illegal BglII site found at 2185
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2769
    Illegal AgeI site found at 2892
  • 1000
    COMPATIBLE WITH RFC[1000]