Part:BBa_K3733002

EBS-PyeaR: The promoter responsive to nitrate with extra NarL binding site

The NarL protein can activate transcription at many promoters include PyeaR in response to nitrate and nitrite. When the NarX protein phosphorylates by nitrate, the phosphate groups can be transferred to NarL. The phosphorylated NarL will binding the site and activate the transcription 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
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]