Part:BBa_K3733001

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.

Figure 1. The The nitrate response curve induced by different concentrations of potassium nitrate in E.coli BL21. The red vertical line shows the concentration of DSS induced in mouse, while the green vertical line shows the concentration of normal conditions. The DSS induced animal model often used as an inflammation model.

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

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]