Part:BBa_K3733027

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

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.

Figure 1. The sequence of EBS- P_yeaR.

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.

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]