Part:BBa_K4387007

Nitric Oxide Sensing Genetic Circuit With Three Ribosomal Binding Sites

Usage and Biology

This composite part consists of the inducible pNorVβ promoter (BBa_K4387000), superfolder GFP preceded by three strong ribosomal binding sites (BBa_K4387020, BBa_B0029, BBa_B0034, BBa_K2553008), the NorR regulator (BBa_K4387001), and a double forward (BBa_B0015). We chose a high-copy backbone from Twist for this part. Due to the competitive binding of the activated and inactivated NorR on the promoter, we decided on this construct with a positive feedback loop that adjusted the levels of NorR. The presence of nitric oxide would activate pNorVβ to induce GFP and NorR expression. Thereby, we ensure that high amounts of NorR will be produced in the presence of NO and in the presence of NO only.

In the frame of our project, we wanted to improve the construct BBa_K4387005 by adding one more ribosomal binding site to see if we could achieve a higher GFP response. According to the data below, we could prove that this construct with two ribosomal binding sites and the codon-optimized NorR was the best and had the highest response.

This construct was tested in the bacterial strain E.coli Nissle 1917.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 746
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Measurements

Figure 1: Induction response of the pNorVβ promoter to different DETA/NO concentrations with respect to time.

Figure 2: Dose response of the pNorVβ promoter at different DETA/NO concentrations.

References

Xiaoyu J. Chen, Baojun Wang, Ian P. Thompson, and Wei E. Huang et al. Rational Design and Characterization of Nitric Oxide Biosensors in E. coli Nissle 1917 and Mini SimCells ACS Synthetic Biology 2021 10 (10), 2566-2578 DOI: 10.1021/acssynbio.1c00223