Difference between revisions of "Part:BBa K4387009"

Revision as of 13:25, 4 October 2022

Nitric Oxide Sensing Genetic Circuit Without the NorR regulator BBa_K4387001

Usage and Biology

This composite part consists of the inducible pNorVβ promoter (BBa_K4387000), superfolder GFP preceded by two strong ribosomal binding sites (BBa_B0029, BBa_B0034, BBa_K2553008), and a double forward terminator (BBa_B0015). 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 a 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 sensitivity of our construct BBa_K4387005. For this purpose, we removed the codon-optimized NorR, creating a circuit that would rely on endogenous NorR. According to the data below, we could prove that the construct with two ribosomal binding sites and the presence of the codon-optimized NorR BBa_K4387006 was the best and had the highest response.

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 726
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 ===Usage and Biology===
-This composite part consists of the inducible pNorVβ promoter (<html><a href="https://parts.igem.org/Part:BBa_K4387000">BBa_K4387000</a></html>), superfolder GFP preceded by two strong ribosomal binding sites (<html><a href="https://parts.igem.org/Part:BBa_B0029">BBa_B0029</a></html>, <html><a href="https://parts.igem.org/Part:BBa_B0034">BBa_B0034</a></html>, <html><a href="https://parts.igem.org/Part:BBa_K2553008">BBa_K2553008</a></html>), and a double forward terminator (<html><a href="https://parts.igem.org/Part:BBa_B0015">BBa_B0015</a></html>). 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 sensitivity of our construct BBa_K4387005. For this purpose, we removed the codon-optimized NorR, creating a circuit that would rely on endogenous NorR. We then tested this system in E.coli Nissle 1917. According to the data below, we could prove that the construct with two ribosomal binding sites and the presence of the codon-optimized NorR was the best and had the highest response.
+This composite part consists of the inducible pNorVβ promoter (<html><a href="https://parts.igem.org/Part:BBa_K4387000">BBa_K4387000</a></html>), superfolder GFP preceded by two strong ribosomal binding sites (<html><a href="https://parts.igem.org/Part:BBa_B0029">BBa_B0029</a></html>, <html><a href="https://parts.igem.org/Part:BBa_B0034">BBa_B0034</a></html>, <html><a href="https://parts.igem.org/Part:BBa_K2553008">BBa_K2553008</a></html>), and a double forward terminator (<html><a href="https://parts.igem.org/Part:BBa_B0015">BBa_B0015</a></html>). 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 a 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 sensitivity of our construct <html><a href="https://parts.igem.org/Part:BBa_K4387005">BBa_K4387005</a></html>. For this purpose, we removed the codon-optimized NorR, creating a circuit that would rely on endogenous NorR. According to the data below, we could prove that the construct with two ribosomal binding sites and the presence of the codon-optimized NorR <html><a href="https://parts.igem.org/Part:BBa_K4387006">BBa_K4387006</a></html> was the best and had the highest response.