Difference between revisions of "Part:BBa K4387003"

Revision as of 09:20, 9 October 2022

Negative Control of Nitric Oxide Sensing Genetic Circuit

Usage and Biology

This composite part consists of a superfolder GFP, the transcriptional regulator NorR, and a double forward terminator. We chose a high-copy backbone from Twist Bioscience for this part. To assess the proper induction of our construct by nitric oxide, we removed the pNorV promoter to create a negative control. With the removal of the promoter, the presence of nitric oxide should not affect the expression of GFP. Therefore, this part was used for the autofluorescence of the EcN cultures in the following constructs:

• Nitric Oxide Sensing Genetic Circuit With the ETH promoter pNorV
• Nitric Oxide Sensing Genetic Circuit With One Ribosome Binding Site
• Nitric Oxide Sensing Genetic Circuit With Two Ribosome Binding Sites
• Nitric Oxide Sensing Genetic Circuit With Three Ribosome Binding Sites
• Nitric Oxide Sensing Genetic Circuit Without the NorR regulator BBa_K4387001

This construct 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.

Sequence and Features