Difference between revisions of "Part:BBa K4387005"

Revision as of 17:57, 8 October 2022

Nitric Oxide Sensing Genetic Circuit With One Ribosomal Binding Site

Usage and Biology

This composite 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 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 [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 in the presence of NO and in the presence of NO only.

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

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.

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 703
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

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 ==Usage and Biology==
-This composite part consists of the inducible <html><a href="https://parts.igem.org/Part:BBa_K4387000">pNorVβ promoter</a></html>, <html><a href="https://parts.igem.org/Part:BBa_K2553008">superfolder GFP</a></html> preceded by one strong ribosomal binding site (<html><a href="https://parts.igem.org/Part:BBa_B0034">BBa_B0034</a></html>), the <html><a href="https://parts.igem.org/Part:BBa_K4387001">NorR regulator</a></html>, and a <html><a href="https://parts.igem.org/Part:BBa_B0015">double forward terminator</a></html>. 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 [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 in the presence of NO and in the presence of NO only.
+This composite part consists of the inducible <html><a href="https://parts.igem.org/Part:BBa_K4387000">pNorVβ promoter</a></html>, a <html><a href="https://parts.igem.org/Part:BBa_K2553008">superfolder GFP</a></html> preceded by one strong ribosomal binding site (<html><a href="https://parts.igem.org/Part:BBa_B0034">BBa_B0034</a></html>), the <html><a href="https://parts.igem.org/Part:BBa_K4387001">NorR regulator</a></html>, and a <html><a href="https://parts.igem.org/Part:BBa_B0015">double forward terminator</a></html>. 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 [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 in the presence of NO and in the presence of NO only.
 This construct was tested in the bacterial strain E.coli Nissle 1917.