Regulatory

Part:BBa_K4387000

Designed by: Jana Mehdy, Lea Bruellmann, Marine Mausy   Group: iGEM22_UZurich   (2022-09-28)
Revision as of 10:56, 8 October 2022 by Jmehdy (Talk | contribs)


Nitric Oxide Sensing Promoter pNorVβ

Usage and Biology

The inducible pNorVβ is an optimized nitric oxide sensitive promoter. It is an improved part of the 2016 ETH iGEM team pNorV promoter. In our constructs, this promoter is coupled to a superfolder GFP and the transcriptional regulator NorR. The corresponding integration host factor IHF2 binding site was removed from the promoter pNorV to exhibit good sensitivity and dosage response at a low range of inducer DETA/NO, the used nitric oxide source in our experiments, activating the downstream genes' transcription.

This promoter was tested in the bacterial strain E.coli Nissle 1917, and it was successfully coupled with other iGEM parts in the following constructs:

  • Nitric Oxide Sensing Genetic Circuit With One Ribosomal Binding Site (BBa_K4387005)
  • Nitric Oxide Sensing Genetic Circuit With Two Ribosomal Binding Site (BBa_K4387006)
  • Nitric Oxide Sensing Genetic Circuit With Three Ribosomal Binding Site (BBa_K4387007)
  • Nitric Oxide Sensing Genetic Circuit With Two Ribosomal Binding Site and Without BBa_K4387001 (BBa_K4387009)

Characterization

Nitric Oxide Sensing Genetic Circuit With One Ribosomal Binding Site

This composite part consists of the inducible pNorVβ promoter, superfolder GFP preceded by one strong ribosomal binding site, the NorR regulator, and a double forward terminator. We chose a high-copy backbone from Twist Bioscience for this assembly. 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.

Nitric Oxide Sensing Genetic Circuit With Two Ribosomal Binding Site

This composite part consists of the inducible pNorVβ promoter, superfolder GFP preceded by two strong ribosomal binding sites (BBa_B0029, BBa_B0034), the (NorR regulator), and a (double forward terminator). We chose a high-copy backbone from Twist Bioscience 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 had the highest GFP response.

Nitric Oxide Sensing Genetic Circuit With Three Ribosomal Binding Site

This composite part consists of the inducible pNorVβ promoter, superfolder GFP preceded by three strong ribosomal binding sites (BBa_K4387020, BBa_B0029, BBa_B0034), the (NorR regulator), and a (double forward terminator). 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 two more ribosomal binding sites to see if we could achieve a higher GFP response. According to the data below, we could prove that the construct with two ribosomal binding sites and the codon-optimized NorR BBa_K4387006 had the highest response.


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]


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


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