Difference between revisions of "Part:BBa K3733027"

 
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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 J23115 (weak promoter) and J61100 (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 J23115 (weak promoter) and J61100 (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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===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(BBa_K216005). 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>The EBS-PyeaR is the PyeaR promoter with an extra NarL binding region (Figure 1). The extra NarL binding region is inserted to the upstream of the original region, which has the 20bp spacer. For this attempt is done, the biological function has no significantly effect. Moreover, the expression has a slight improvement. </p>
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<center><img src=https://static.igem.org/mediawiki/parts/4/4c/T--HZAU-China--EBS_img1.png style="width:80%"></center>
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<center><b>Figure 1.</b> The sequence of EBS- P<sub>yeaR</sub>.</center>
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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. 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://parts.igem.org/wiki/images/a/a9/T--HZAU-China--EBS_img2.png style="width:60%"></center>
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<center><b>Figure 2.</b> The nitrate response curve with J23115-J61100 in <i>E.coli</i> DH5α. 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>.</center>
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<p>For this result (Figure 2), we can find that when the concentration is higher than 0.1mM, the nitrate sensor will be activated. Meanwhile, for our knowledge, the concentration of nitrate is approximately to 620μM with DSS induced in mouse. And the DSS induced animal model often used as an inflammation model, for this we can infer that our system can achieve its function in this condition.</p>
  
 
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Revision as of 07:03, 20 October 2021


The nitrate sensor(J23115-J61100) system with EBS-PyeaR and 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 J23115 (weak promoter) and J61100 (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.

Usage and Biology

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(BBa_K216005). 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 EBS-PyeaR is the PyeaR promoter with an extra NarL binding region (Figure 1). The extra NarL binding region is inserted to the upstream of the original region, which has the 20bp spacer. For this attempt is done, the biological function has no significantly effect. Moreover, the expression has a slight improvement.

无标题文档

Figure 1. The sequence of EBS- PyeaR.

Functional Parameters

For the nitrate sensor sensitivity testing, we transformed the constructed plasmid with nitrate sensor into E.coli DH5α. 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.

无标题文档

Figure 2. The nitrate response curve with J23115-J61100 in E.coli DH5α. The red vertical line shows the concentration of DSS induced in mouse, while the green vertical line shows the concentration of normal conditions.


For this result (Figure 2), we can find that when the concentration is higher than 0.1mM, the nitrate sensor will be activated. Meanwhile, for our knowledge, the concentration of nitrate is approximately to 620μM with DSS induced in mouse. And the DSS induced animal model often used as an inflammation model, for this we can infer that our system can achieve its function in this condition.

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 337
    Illegal BglII site found at 505
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1089
    Illegal AgeI site found at 1212
  • 1000
    COMPATIBLE WITH RFC[1000]