Difference between revisions of "Part:BBa K2116002"
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__NOTOC__
 
__NOTOC__
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<partinfo>BBa_K2116002 short</partinfo>
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<partinfo>BBa_K1153000 short</partinfo>
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 +
Our Biobrick has been designed to enable the detection of NO using the norV gene promoter, cloned from E.coli K12 TOP10 cells.
 +
In the presence of NOx it will promote the downstream genes in the plasmid, producing a protein in response to the environmental
 +
stimulus.
 +
 
 +
<h1>'''Contribution'''</h1>
 +
<ul>
 +
<li>'''Group:''' ETH Zurich 2016
 +
<li>'''Author:''' Asli Azizoglu
 +
<li>'''Summary:''' We cloned and characterised the norV promoter, and sent it to the registry as a biobrick. Our biobrick
 +
can be found here [[Part:BBa_K2116002]], and includes a spacer that is not found in this version.
 +
</ul>
 +
 
 +
<h1>Usage and Biology</h1>
 +
Promoter norV '''(PnorV)''' is the native promoter controlling the nitric oxide reduction operon (norRVW) in E. Coli. [1]
 +
It's transcriptional regulator, NorR, can bind to nitric oxide and activate gene expression.
 +
 
 +
 
 +
<h1>Characterisation of the Promoter</h1>
 +
<p>We cloned PnorV upstream of superfolder GFP for characterisation [[Part:BBa_K2116088]] This construct was expressed on a
 +
medium copy plasmid (ori:p15A). Since ''E.coli'' natively produces NorR, we relied on this to activate the promoter. Below
 +
is the dose response curve we obtained under the following experimental conditions, using a plate reader:</p>
 +
<ul>
 +
<li> Overnight growth and experiment in minimal M9 medium, with 25μg/μL chloramphenicol.
 +
<li> Plating at an OD<sub>600</sub> of 0.05, in M9, with 25μg/μL chloramphenicol.
 +
<li> Induction at OD<sub>600</sub>0.5.
 +
<li> Settings for GFP measurement: excitation-488nm, emission- 530nm.
 +
<li> Samples were always in three technical replicates, and the fluorescence measurements were normalized to OD<sub>600</sub>.
 +
</ul>
  
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Promoter norV '''(PnorV)''' is the native promoter controlling the nitric oxide reduction operon (norRVW) in E. Coli. [1] It's transcriptional regulator, NorR, can sense nitric oxide and activate gene expression.
+
[[File:PnorVdoseresponse.png|500px|thumb|center|PnorV dose response curve for a range of DETA/NO concentrations that corresponds
 +
to 7-70μM of NO. Above 15000uM of DETA/NO affects cell growth and is not included in the dose response. Measured 6 hours
 +
after induction. Error bars represent S.D. from three technical replicates.]]
  
−
We cloned  PnorV upstream of super folder GFP for characterisation. This construct was expressed on a medium copy plasmid (p15A). Since ''E.coli'' natively produce NorR, we relied on this to activate the promoter. Below is the dose response curve we obtained.  
+
<p>In order to show that the native NorR is essential for activating PnorV, we used a [http://cgsc.biology.yale.edu/KeioList.php
 +
Keio] norR knock out strain (&Delta;norR) and compared it to it's parent wild type strain (WT). It was shown that PnorV
 +
can be activated by DETA/NO in the parent strain, but not in the norR KO strain.</p>
  
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[[File:PnorVdoseresponse.png|500px|thumb|center|NorV promoter activated by nitric oxide, released from DETA/NO. Above 15000uM of DETA/NO affects cell growth and is not included in the dose response.]]
+
[[File:PnorV native norR functionality.png|500px|thumb|center|A norR KO strain was used as a negative control to demonstrate
 +
that the native norR can activate PnorV.Induced with 5000μM DETA/NO and measured 6 hours after induction. Error bars represent
 +
S.D. from three technical replicates.]]
  
−
In order to ascertain that we are using the native NorR and not another transcriptional regulator, we used a Keio norR knock-out strain (norR KO) [2] and compared it to it's parent wild type strain (WT). It was shown that PnorV can be activated by DETA/NO in the parent strain, but not in the norR KO strain.  
+
<h1> Materials </h1>
 +
We used DETA/NO to as a nitric oxide source. It has a half life of roughly 20h, and releases NO at a relatively constant
 +
rate [2].
  
−
[[File:PnorV native norR functionality.png|500px|thumb|center|A norR KO strain was used as a negative control to demonstrate that the native norR of ''E.coli'' can activate PnorV in the AND gate.]]
+
<h1>References:</h1>
 +
<ul>
 +
<li> [1] Gardner, A. M. "Regulation Of The Nitric Oxide Reduction Operon (Norrvw) In Escherichia Coli. ROLE OF Norr AND Sigma
 +
54 IN THE NITRIC OXIDE STRESS RESPONSE". Journal of Biological Chemistry 278.12 (2003): 10081-10086.
 +
<li>[2] Roselle, Dominick C and Daniel J Smith. "Characterization And Nitric Oxide Release Studies Of Lipophilic 1-Substituted
 +
Diazen-1-Ium-1,2-Diolates". Journal of Controlled Release 51.2-3 (1998): 131-142. Web.
  
−
References:
 
−
[1] Gardner, A. M. "Regulation Of The Nitric Oxide Reduction Operon (Norrvw) In Escherichia Coli. ROLE OF Norr AND Sigma 54 IN THE NITRIC OXIDE STRESS RESPONSE". Journal of Biological Chemistry 278.12 (2003): 10081-10086.
 
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[2] Baba, Tomoya, et al. "Construction of Escherichia coli K‐12 in‐frame, single‐gene knockout mutants: the Keio collection." Molecular systems biology 2.1 (2006).
 
  
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<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
  
 
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2116002 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K1153000 SequenceAndFeatures</partinfo>
  
  
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K2116002 parameters</partinfo>
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<partinfo>BBa_K1153000 parameters</partinfo>
 
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Revision as of 15:01, 22 October 2016

NorV promoter. NOx detector.

Our Biobrick has been designed to enable the detection of NO using the norV gene promoter, cloned from E.coli K12 TOP10 cells. In the presence of NOx it will promote the downstream genes in the plasmid, producing a protein in response to the environmental stimulus.

Contribution

  • Group: ETH Zurich 2016
  • Author: Asli Azizoglu
  • Summary: We cloned and characterised the norV promoter, and sent it to the registry as a biobrick. Our biobrick can be found here Part:BBa_K2116002, and includes a spacer that is not found in this version.

Usage and Biology

Promoter norV (PnorV) is the native promoter controlling the nitric oxide reduction operon (norRVW) in E. Coli. [1] It's transcriptional regulator, NorR, can bind to nitric oxide and activate gene expression.


Characterisation of the Promoter

We cloned PnorV upstream of superfolder GFP for characterisation Part:BBa_K2116088 This construct was expressed on a medium copy plasmid (ori:p15A). Since E.coli natively produces NorR, we relied on this to activate the promoter. Below is the dose response curve we obtained under the following experimental conditions, using a plate reader:

  • Overnight growth and experiment in minimal M9 medium, with 25μg/μL chloramphenicol.
  • Plating at an OD600 of 0.05, in M9, with 25μg/μL chloramphenicol.
  • Induction at OD6000.5.
  • Settings for GFP measurement: excitation-488nm, emission- 530nm.
  • Samples were always in three technical replicates, and the fluorescence measurements were normalized to OD600.
PnorV dose response curve for a range of DETA/NO concentrations that corresponds to 7-70μM of NO. Above 15000uM of DETA/NO affects cell growth and is not included in the dose response. Measured 6 hours after induction. Error bars represent S.D. from three technical replicates.

In order to show that the native NorR is essential for activating PnorV, we used a [http://cgsc.biology.yale.edu/KeioList.php Keio] norR knock out strain (ΔnorR) and compared it to it's parent wild type strain (WT). It was shown that PnorV can be activated by DETA/NO in the parent strain, but not in the norR KO strain.

A norR KO strain was used as a negative control to demonstrate that the native norR can activate PnorV.Induced with 5000μM DETA/NO and measured 6 hours after induction. Error bars represent S.D. from three technical replicates.

Materials

We used DETA/NO to as a nitric oxide source. It has a half life of roughly 20h, and releases NO at a relatively constant rate [2].

References:

  • [1] Gardner, A. M. "Regulation Of The Nitric Oxide Reduction Operon (Norrvw) In Escherichia Coli. ROLE OF Norr AND Sigma 54 IN THE NITRIC OXIDE STRESS RESPONSE". Journal of Biological Chemistry 278.12 (2003): 10081-10086.
  • [2] Roselle, Dominick C and Daniel J Smith. "Characterization And Nitric Oxide Release Studies Of Lipophilic 1-Substituted Diazen-1-Ium-1,2-Diolates". Journal of Controlled Release 51.2-3 (1998): 131-142. Web. 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]