Part:BBa_K3733029

The nitrate sensor(J23100-RiboJ-B0033) system with RiboJ 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 J23100 (strong promoter) and B0033 (weak RBS) to produce NarX and NarL. And RiboJ is an insulator between promoter and RBS. 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. 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.

In this part, we use the combination is J23100 -B0033. The promoter J23100 is the strong promoter, and B0033 is a weak RBS. RiboJ is an insulator between promoter and RBS, which can affect translation of the transcript is via the hairpin at the end of the RiboJ sequence, downstream of the ribozyme. It has been suggested that this appended hairpin aids in exposing the RBS.

To learn more about nitrate sensor system, a collection of this kind of nitrate sensor system were constructed.

BBa_K3733015: https://parts.igem.org/Part:BBa_K3733015

BBa_K3733016: https://parts.igem.org/Part:BBa_K3733016

BBa_K3733017: https://parts.igem.org/Part:BBa_K3733017

BBa_K3733018: https://parts.igem.org/Part:BBa_K3733018

BBa_K3733019: https://parts.igem.org/Part:BBa_K3733019

BBa_K3733020: https://parts.igem.org/Part:BBa_K3733020

BBa_K3733021: https://parts.igem.org/Part:BBa_K3733021

BBa_K3733022: https://parts.igem.org/Part:BBa_K3733022

BBa_K3733023: https://parts.igem.org/Part:BBa_K3733023

BBa_K3733028: https://parts.igem.org/Part:BBa_K3733028

BBa_K3733029: https://parts.igem.org/Part:BBa_K3733029

BBa_K3733030: https://parts.igem.org/Part:BBa_K3733030

BBa_K3733031: https://parts.igem.org/Part:BBa_K3733031

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 1. The The nitrate response curve with J23100- B0033 with RiboJ 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. The DSS induced animal model often used as an inflammation model.

In this combination, RiboJ did not have a particularly significant effect on the nitrate sensing system.

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 2092
  Illegal BglII site found at 2260
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2844
  Illegal AgeI site found at 2967
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

Clifton K P, Jones E M, Paudel S, et al. The genetic insulator RiboJ increases expression of insulated genes[J]. Journal of biological engineering, 2018, 12(1): 1-6.