Part:BBa_K4387004

Nitric Oxide Sensing Genetic Circuit with Promoter BBa_K2116002

Usage and Biology

This composite 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.

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

Characterization

We measured the GFP expression upon NO induction with a plate reader over 16 hours. Below is the dose-response curve of pNorV, 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.

Measurements

Figure 1: Induction response of the 2016 ETH Team NorV promoter to different DETA/NO concentrations with respect to time.

Figure 2: Dose response of the 2016 ETH Team NorV promoter at different DETA/NO concentrations.

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 742
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]