Difference between revisions of "Part:BBa K4387000"

Revision as of 11:09, 8 October 2022

Nitric Oxide Sensing Promoter pNorVβ

Usage and Biology

The inducible pNorVβ is an optimized nitric oxide sensitive promoter. It is an improved part of the 2016 ETH iGEM team pNorV promoter. In our constructs, this promoter is coupled to a superfolder GFP and the transcriptional regulator NorR. 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.

This promoter was tested in the bacterial strain E.coli Nissle 1917, and it was successfully coupled with other iGEM parts in the following constructs:

Nitric Oxide Sensing Genetic Circuit With One Ribosomal Binding Site (BBa_K4387005)
Nitric Oxide Sensing Genetic Circuit With Two Ribosomal Binding Site (BBa_K4387006)
Nitric Oxide Sensing Genetic Circuit With Three Ribosomal Binding Site (BBa_K4387007)
Nitric Oxide Sensing Genetic Circuit With Two Ribosomal Binding Site and Without BBa_K4387001 (BBa_K4387009)

Characterization

Nitric Oxide Sensing Genetic Circuit With One Ribosomal Binding Site

This composite part consists of the inducible pNorVβ promoter, superfolder GFP preceded by one strong ribosomal binding site, 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. 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.

Nitric Oxide Sensing Genetic Circuit With Two Ribosomal Binding Site

This composite 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. 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 construct BBa_K4387005 by adding one more ribosomal binding site to see if we could achieve a higher GFP response. According to figure__, we could prove that this construct with two ribosomal binding sites and the codon-optimized NorR had the highest GFP response.

Nitric Oxide Sensing Genetic Circuit With Three Ribosomal Binding Site

This composite part consists of the inducible pNorVβ promoter, superfolder GFP preceded by three strong ribosomal binding sites (BBa_K4387020, BBa_B0029, BBa_B0034), the NorR regulator, and a double forward terminator. 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 construct BBa_K4387005 by adding two more ribosomal binding sites to see if we could achieve a higher GFP response. According to figure__, we could prove that the construct with two ribosomal binding sites and the codon-optimized NorR BBa_K4387006 had the highest 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 composite 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 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.

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

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

@@ Line 14: / Line 14: @@
 	<li>Nitric Oxide Sensing Genetic Circuit With Two Ribosomal Binding Site and Without <html><a href="https://parts.igem.org/Part:BBa_K4387001">BBa_K4387001</a></html> [https://parts.igem.org/Part:BBa_K4387009 (BBa_K4387009)]</li>
 </ul>
 ===Characterization===
@@ Line 25: / Line 26: @@
 This composite part consists of the inducible pNorVβ promoter, 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>), 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 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 construct <html><a href="https://parts.igem.org/Part:BBa_K4387005">BBa_K4387005</a></html> by adding one more ribosomal binding site to see if we could achieve a higher GFP response. According to the data below, we could prove that this construct with two ribosomal binding sites and the codon-optimized NorR had the highest GFP response.
+In the frame of our project, we wanted to improve the construct <html><a href="https://parts.igem.org/Part:BBa_K4387005">BBa_K4387005</a></html> by adding one more ribosomal binding site to see if we could achieve a higher GFP response. According to figure__, we could prove that this construct with two ribosomal binding sites and the codon-optimized NorR had the highest GFP response.
 ==Nitric Oxide Sensing Genetic Circuit With Three Ribosomal Binding Site==
-This composite part consists of the inducible pNorVβ promoter, superfolder GFP preceded by three strong ribosomal binding sites (<html><a href="https://parts.igem.org/Part:BBa_K4387020">BBa_K4387020</a></html>, <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>), 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 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.
+This composite part consists of the inducible pNorVβ promoter, superfolder GFP preceded by three strong ribosomal binding sites (<html><a href="https://parts.igem.org/Part:BBa_K4387020">BBa_K4387020</a></html>, <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>), 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 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 construct <html><a href="https://parts.igem.org/Part:BBa_K4387005">BBa_K4387005</a></html> by adding two more ribosomal binding sites to see if we could achieve a higher GFP response. According to figure__, we could prove that the construct with two ribosomal binding sites and the codon-optimized NorR <html><a href="https://parts.igem.org/Part:BBa_K4387006">BBa_K4387006</a></html> had the highest response.
+==Nitric Oxide Sensing Genetic Circuit Without the NorR regulator==
-In the frame of our project, we wanted to improve the construct <html><a href="https://parts.igem.org/Part:BBa_K4387005">BBa_K4387005</a></html> by adding two more ribosomal binding sites to see if we could achieve a higher GFP response. According to the data below, we could prove that the construct with two ribosomal binding sites and the codon-optimized NorR <html><a href="https://parts.igem.org/Part:BBa_K4387006">BBa_K4387006</a></html> had the highest response.
+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. This composite part consists of the inducible pNorVβ promoter, 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>), 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 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 <html><a href="https://parts.igem.org/Part:BBa_K4387006">BBa_K4387006</a></html> had the highest response.