Part:BBa_K4387000

Nitric Oxide Sensing Promoter pNorVβ

Usage and Biology

The inducible pNorVβ is an optimized nitric oxide sensitive promoter controlling the NorRVW operon in E. coli. 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 [1]. In our constructs below, this promoter was coupled to a superfolder GFP and the transcriptional regulator NorR which creates a feedback loop to finetune the amount of NorR to the number of free NO molecules. It is also an improved part of the 2016 ETH iGEM team pNorV promoter.

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

Characterization and Improvement

We measured the GFP expression upon NO induction with a plate reader over 16 hours. Below is the dose-response curve of pNorVβ, measured in a plate reader. For all measurements, we used the following conditions:

• Overnight growth and experiment were done in minimal M9 medium supplemented with Ampicillin at 37°C Start of experiment in 96 well plate at an OD600 of 0.05.
• Settings for GFP measurements: excitation at 485nm, emission at 520nm.
• Every condition was measured over three technical and three biological replicates.
• GFP emission was normalized to OD600.

Nitric Oxide Sensing Genetic Circuit With the ETH promoter pNorV

This part consists of the ETH promoter pNorV, a superfolder GFP preceded by one strong ribosomal binding site (BBa_B0034), the NorR regulator, and a double forward terminator. We chose a high-copy backbone from Twist Bioscience for this part. We wanted to compare this ETH NorV promoter to the pNorVβ promoter and see which one was better suited for sensing nitric oxide at lower concentration ranges. According to figure__, when tested at different concentration levels, the pNorVβ had higher responses to DETA/NO induction than the NorV promoter of the ETH 2016 team.

Nitric Oxide Sensing Genetic Circuit With One Ribosomal Binding Site

This part consists of the inducible pNorVβ promoter, a superfolder GFP preceded by one strong ribosomal binding site (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 based on the amount of nitric oxide present [1]. 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 only when NO is present.

Nitric Oxide Sensing Genetic Circuit With Two Ribosomal Binding Sites

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.

Thus this 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 based on the amount of nitric oxide present [1]. 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 only when NO is present.

According to figure__, we could prove that this construct with two ribosomal binding sites and the codon-optimized NorR had the highest GFP response.

While this part has higher overall GFP expression values, it is also leakier than the constructs with one or three ribosomal binding sites. If high GFP expression is required, but some leakiness does not matter much, we recommend choosing BBa_K4387006. If lower leakiness is essential, but GFP expression does not need to be very high, we recommend using parts BBa_K4387005 or BBa_K4387007 instead.

Nitric Oxide Sensing Genetic Circuit With Three Ribosomal Binding Sites

In the frame of our project, we wanted to further improve the construct BBa_K4387005 by adding two more ribosomal binding sites to see if we could achieve a higher GFP response.

Thus this part consists of the inducible pNorVβ promoter, superfolder GFP preceded by three strong ribosomal binding sites (BBa_K4387020 [2], 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 based on the amount of nitric oxide present [1]. 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 only when NO is present.

According to figure__, we could prove that the construct with two ribosomal binding sites and the codon-optimized NorR BBa_K4387006 had the highest GFP response.

Nitric Oxide Sensing Genetic Circuit Without the NorR regulator

In the frame of our project, we wanted to improve the sensitivity of our construct BBa_K4387005.

For this purpose, we removed the codon-optimized NorR, creating a circuit that would rely on endogenous NorR. This part consists of the inducible pNorVβ promoter, superfolder GFP preceded by two strong ribosomal binding sites (BBa_B0029, BBa_B0034), and a double forward terminator. We chose a high-copy backbone from Twist Bioscience for this part.

According to figure__, we could prove that the construct with two ribosomal binding sites and the presence of the codon-optimized NorR BBa_K4387006 had the highest response.

Exchanging the sfGFP with a Single Domain Antibody

TBA

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

• [1] 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
• [2] Ayelet Levin-Karp, Uri Barenholz, Tasneem Bareia, Michal Dayagi, Lior Zelcbuch, Niv Antonovsky, Elad Noor, and Ron Milo et al. Quantifying Translational Coupling in E.coli Synthetic Operons Using RBS Modulation and Fluorescent Reporters ACS Synthetic Biology 2013 2 (6), 327-336 DOI: 10.1021/sb400002n