Difference between revisions of "Part:BBa K2116015"
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__NOTOC__
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__NOTOC__
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<partinfo>BBa_K2116015 short</partinfo>
 
<partinfo>BBa_K2116015 short</partinfo>
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<p>
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We constructed a selection of AND gates responding to nitric oxide (NO) and 3OC6HSL (AHL). They were designed using the previously
 +
described NorV promoter [[Part:BBa_K1153000]]. This Promoter (from here on refered to as PnorV) is the native promoter controlling
 +
the nitric oxide reduction operon (norRVW) in <i>E. coli</i> [1]. Its transcriptional regulator, NorR, can bind to nitric
 +
oxide and activate gene expression. Using the distinct properties of esaboxes, PnorV was designed to also be responsive
 +
to AHL, giving it an AND gate behaviour. An esabox is an 18bp sequence to which the transcriptional regulator EsaR [[Part:BBa_K2116001]]
 +
can bind. Transcription can be initiated by the specific AHL EsaR responds to [N-(3-oxo-hexanoyl)-L-homoserine lactone].
 +
By placing one, two or three esaboxes at different positions in the vicinity of PnorV, different specificities for AHL and
 +
NO were reached. We created and characterized a collection of these kind of AND gates: <br>
  
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AND gate constructed by adding an esabox before the -24 on the sigma 54 binding site of PnorV. This esabox overlaps the sigma 54 binding site, creating competitive binding between the polymerase and EsaR.
 
  
 +
<ul>
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<li> [[Part:BBa_K2116004]]
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<li> [[Part:BBa_K2116005]]
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<li> [[Part:BBa_K2116006]]
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<li> [[Part:BBa_K2116012]]
 +
<li> [[Part:BBa_K2116013]]
 +
<li> [[Part:BBa_K2116014]]
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<li> [[Part:BBa_K2116007]]
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<li> [[Part:BBa_K2116008]]
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<li> [[Part:BBa_K2116068]]
 +
<li> [[Part:BBa_K2116015]]
 +
</ul>
 +
</p>
  
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<p>
 
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We constructed a selection of AND gates responding to nitric oxide (NO) and 3OC6HSL (AHL). They were designed using the previously described NorV promoter [[Part:BBa_K1153000]].
 
−
This Promoter (from here on refered to as PnorV) is the native promoter controlling the nitric oxide reduction operon (norRVW) in <i>E. coli</i> [1]. Its transcriptional regulator,
 
−
NorR, can bind to nitric oxide and activate gene expression.
 
−
Using the distinct properties of esaboxes, PnorV was designed to also be responsive to AHL, giving it an AND gate behaviour.
 
−
An esabox is an 18bp sequence to which the transcriptional regulator EsaR [[Part:BBa_K2116001]] can bind. Transcription can be initiated by the specific AHL EsaR responds
 
−
to [N-(3-oxo-hexanoyl)-L-homoserine lactone]. By placing one, two or three esaboxes at different positions in the vicinity of PnorV, different specificities for AHL and NO
 
−
were reached. We created and characterized a collection of these kind of AND gates: <br>
 
  
 +
<h1> Biology and Usage </h1>
  
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<ul>
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<p>Biological logic gates are useful for creating higher order genetic circuits. This AND gate has one esabox placed as a roadblock
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<li> [[Part:BBa_K2116004]]
+
after PnorV transcription start site. [[File:T--ETH Zurich--p23_2.png|right|100px;<b>Figure 1:</b> three esaboxes downstream of PnorV with 15bp spacer in between.]] It is regulated by a transcriptional
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<li> [[Part:BBa_K2116005]]
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activator, NorR, and a transcriptional repressor, EsaR. Transcription can be initiated by NO binding to NorR. EsaR sits
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<li> [[Part:BBa_K2116006]]
+
on the esabox and blocks RNA polymerase from advancing. As soon as 3OC6HSL binds EsaR it is released and transcription can
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<li> [[Part:BBa_K2116012]]
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continue. This design makes the AND gate modular. The esabox/EsaR system can be exchanged for another transcriptional repression
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<li> [[Part:BBa_K2116013]]
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system to create another AND gate.
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<li> [[Part:BBa_K2116014]]
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<li> [[Part:BBa_K2116007]]
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<li> [[Part:BBa_K2116008]]
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<li> [[Part:BBa_K2116068]]
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<li> [[Part:BBa_K2116015]]
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</ul>
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</p>
 
</p>
  
 +
<h1>Characterization</h1>
 +
 +
<p>
 +
 +
When characterizing our parts collection we initially confirmed functionality. Below you can see a graph depicting AND gate behaviour of this biobrick.
 +
    [[File:T--ETH_Zurich--THIS IS THE 8bp SPACER: p23_3.png|500px|thumb|center|<b>Figure 2:</b> AND gate behaviour. This part shows a desired behaviour. Optimization of the esabox positioning could improve the fold increase for full activation.]]
 +
   
 +
</p>
 +
 +
 +
<p>
 +
    The AND gate behaviour shown in Figure 2 can be explained bye the placing of three esaboxes after PnorV. This seems to be enough
 +
    to reach sufficient balance between repression and derepression. An improvement could be achieved by either increasing the number of esaboxes, their placement
 +
    or by decreasing the amount of EsaR production. We followed all these steps, and recommend you have a look at our favourite
 +
    AND gate [[Part:BBa_K2116011]], where an improvement compared to this part was shown.
 +
 +
 +
</p>
  
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<h2> Characterization of this part </h2>
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<p>Secondly we tested the system with and without EsaR present, in order to show that the AND gate behaviour is due to repression by EsaR.  </p>
 +
[[File:T--ETH_Zurich--THIS IS THE 8BP SPACER: p23_1.png|500px|thumb|center|<b>Figure 3:</b> AND gate behaviour compared to cells containing an EsaR expressing plasmid vs non expressing.
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Lighter colors depict cells lacking EsaR. This difference in repression suggests that EsaR is the major protein acting, producing the desired AND gate function]]
  
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Data will be here before the final presentation at the jamboree. Apologies for the delay.
 
  
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<p></p>
 
<h2>References:</h2>
 
<h2>References:</h2>
  
  
 
<ul>
 
<ul>
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<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.</i>
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<li> [1] Gardner, A. M. "Regulation Of The Nitric Oxide Reduction Operon (Norrvw) In Escherichia Coli. ROLE OF Norr AND Sigma
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<li> [2] Shong, Jasmine and Cynthia H. Collins. "Engineering The Esar Promoter For Tunable Quorum Sensing-Dependent Gene Expression". ACS Synth. Biol. 2.10 (2013): 568-575. </li>
+
54 IN THE NITRIC OXIDE STRESS RESPONSE". Journal of Biological Chemistry 278.12 (2003): 10081-10086.</li>
 +
<li> [2] Shong, Jasmine and Cynthia H. Collins. "Engineering The Esar Promoter For Tunable Quorum Sensing-Dependent Gene Expression".
 +
ACS Synth. Biol. 2.10 (2013): 568-575. </li>
  
  
  
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<!-- Add more about the biology of this part here
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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_K2116015 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K2116015 SequenceAndFeatures</partinfo>
  
  
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<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2116015 parameters</partinfo>
 
<partinfo>BBa_K2116015 parameters</partinfo>
 
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Revision as of 03:12, 28 October 2016

PnorV_esabox_(15bp)spacer_esabox_spacer_esabox (p30)

We constructed a selection of AND gates responding to nitric oxide (NO) and 3OC6HSL (AHL). They were designed using the previously described NorV promoter Part:BBa_K1153000. This Promoter (from here on refered to as PnorV) is the native promoter controlling the nitric oxide reduction operon (norRVW) in E. coli [1]. Its transcriptional regulator, NorR, can bind to nitric oxide and activate gene expression. Using the distinct properties of esaboxes, PnorV was designed to also be responsive to AHL, giving it an AND gate behaviour. An esabox is an 18bp sequence to which the transcriptional regulator EsaR Part:BBa_K2116001 can bind. Transcription can be initiated by the specific AHL EsaR responds to [N-(3-oxo-hexanoyl)-L-homoserine lactone]. By placing one, two or three esaboxes at different positions in the vicinity of PnorV, different specificities for AHL and NO were reached. We created and characterized a collection of these kind of AND gates:


Biology and Usage

Biological logic gates are useful for creating higher order genetic circuits. This AND gate has one esabox placed as a roadblock after PnorV transcription start site.

100px;Figure 1: three esaboxes downstream of PnorV with 15bp spacer in between.
It is regulated by a transcriptional

activator, NorR, and a transcriptional repressor, EsaR. Transcription can be initiated by NO binding to NorR. EsaR sits on the esabox and blocks RNA polymerase from advancing. As soon as 3OC6HSL binds EsaR it is released and transcription can continue. This design makes the AND gate modular. The esabox/EsaR system can be exchanged for another transcriptional repression system to create another AND gate.

Characterization

When characterizing our parts collection we initially confirmed functionality. Below you can see a graph depicting AND gate behaviour of this biobrick.

File:T--ETH Zurich--THIS IS THE 8bp SPACER: p23 3.png
Figure 2: AND gate behaviour. This part shows a desired behaviour. Optimization of the esabox positioning could improve the fold increase for full activation.


The AND gate behaviour shown in Figure 2 can be explained bye the placing of three esaboxes after PnorV. This seems to be enough to reach sufficient balance between repression and derepression. An improvement could be achieved by either increasing the number of esaboxes, their placement or by decreasing the amount of EsaR production. We followed all these steps, and recommend you have a look at our favourite AND gate Part:BBa_K2116011, where an improvement compared to this part was shown.

Secondly we tested the system with and without EsaR present, in order to show that the AND gate behaviour is due to repression by EsaR.

File:T--ETH Zurich--THIS IS THE 8BP SPACER: p23 1.png
Figure 3: AND gate behaviour compared to cells containing an EsaR expressing plasmid vs non expressing. Lighter colors depict cells lacking EsaR. This difference in repression suggests that EsaR is the major protein acting, producing the desired AND gate function


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] Shong, Jasmine and Cynthia H. Collins. "Engineering The Esar Promoter For Tunable Quorum Sensing-Dependent Gene Expression". ACS Synth. Biol. 2.10 (2013): 568-575.


    • 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]