Difference between revisions of "Part:BBa K4387009"
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__NOTOC__
 
 
<partinfo>BBa_K4387009 short</partinfo>
 
<partinfo>BBa_K4387009 short</partinfo>
  
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===Usage and Biology===
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==Usage and Biology==
  
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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.  
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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.  
  
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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.  
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Thus this 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 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 Bioscience for this part.
  
 
This construct was tested in the bacterial strain E.coli Nissle 1917.
 
This construct was tested in the bacterial strain E.coli Nissle 1917.
  
  
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===Sequence and Features===
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==Characterization==
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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:
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<ul>
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<li>Overnight growth and experiment were done in minimal M9 medium supplemented with Ampicillin at 37°C
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Start of experiment in 96 well plate at an OD600 of 0.05.</li>
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<li>Settings for GFP measurements: excitation at 485nm, emission at 520nm.</li>
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<li>Every condition was measured over three technical and three biological replicates.</li>
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<li>GFP emission was normalized to OD600.</li>
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</ul>
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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 GFP response.
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If high GFP expression is required, but some leakiness does not matter much, we recommend choosing <html><a href="https://parts.igem.org/Part:BBa_K4387006">BBa_K4387006</a></html>. If lower leakiness is essential, but GFP expression does not need to be very high, we recommend using parts <html><a href="https://parts.igem.org/Part:BBa_K4387005">BBa_K4387005</a></html> or <html><a href="https://parts.igem.org/Part:BBa_K4387007">BBa_K4387007</a></html> instead.
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===Measurements===
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==Sequence and Features==
 
<!-- -->
 
<!-- -->
 
<span class='h3bb'/span>
 
<span class='h3bb'/span>
 
<partinfo>BBa_K4387009 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4387009 SequenceAndFeatures</partinfo>
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==References==
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<ul>
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<li>[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 <html><a href="https://pubs.acs.org/doi/abs/10.1021/acssynbio.1c00223">DOI: 10.1021/acssynbio.1c00223</a></html></li>
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</ul>
  
  
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<partinfo>BBa_K4387009 parameters</partinfo>
 
<partinfo>BBa_K4387009 parameters</partinfo>
 
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===References===
 
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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
 

Revision as of 09:11, 9 October 2022

Nitric Oxide Sensing Genetic Circuit Without the NorR regulator BBa_K4387001

Usage and Biology

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.

Thus this part consists of the inducible pNorVβ promoter, a 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 Bioscience for this part.

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 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.

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 GFP response.

If high GFP expression is required, but some leakiness does not matter much, we recommend choosing BBa_K4387006. If lower leakiness is essential, but GFP expression does not need to be very high, we recommend using parts BBa_K4387005 or BBa_K4387007 instead.

Measurements

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]


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