Difference between revisions of "Part:BBa K2116014"
 
(4 intermediate revisions by the same user not shown)
Line 1: Line 1:
 +
__NOTOC__
 +
<partinfo>BBa_K2116014 short</partinfo>
 +
<p>
 +
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>
  
−
__NOTOC__
 
−
<partinfo>BBa_K2116067 short</partinfo>
 
  
−
AND gate designed by adding an esabox before the first norR binding site, to create competitive binding between EsaR and NorR.
+
<ul>
 +
<li> [[Part:BBa_K2116004]]
 +
<li> [[Part:BBa_K2116005]]
 +
<li> [[Part:BBa_K2116006]]
 +
<li> [[Part:BBa_K2116012]]
 +
<li> [[Part:BBa_K2116013]]
 +
<li> [[Part:BBa_K2116014]]
 +
<li> [[Part:BBa_K2116007]]
 +
<li> [[Part:BBa_K2116008]]
 +
<li> [[Part:BBa_K2116068]]
 +
<li> [[Part:BBa_K2116015]]
 +
</ul>
 +
</p>
  
−
<!-- Add more about the biology of this part here
+
 
 +
<h1> Biology and Usage </h1>
 +
[[File:T--ETH Zurich--p23_2.png|right|100px;<b>Figure 1:</b> the native 8bp spacer downstream of PnorV has been removed.]]
 +
<p>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. 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.
 +
</p>
 +
 
 +
<h1>Characterization</h1>
 +
<p> This part has not been properly characterized yet. Due to the similarity with [[Part:BBa_K2116006]] a similar behaviour is expected with a slight increase in efficiency of EsaR repression due to the removal of a native spacer between PnorV and esabox. This results in competitive binding between EsaR and polymerase. This part shows a desired AND gate behaviour. Optimization of the esabox positioning could improve the fold increase for full activation.
 +
 
 +
 
 +
</p>
 +
<h2>References:</h2>
 +
 
 +
 
 +
<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>
 +
<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>
 +
 
 +
 
 +
 
 +
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
  
 
<!-- -->
 
<!-- -->
−
<span class='h3bb'>Sequence and Features</span>
+
<span class='h3bb'>Sequence and Features</span>
−
<partinfo>BBa_K2116014 SequenceAndFeatures</partinfo>
+
<partinfo>BBa_K2116014 SequenceAndFeatures</partinfo>
  
  
−
<!-- Uncomment this to enable Functional Parameter display  
+
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2116014 parameters</partinfo>
 
<partinfo>BBa_K2116014 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Latest revision as of 14:56, 30 October 2016

PnorV_esabox_(8bp)spacer_esabox_(8bp)spacer_esabox_spacer(p26)

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

100px;Figure 1: the native 8bp spacer downstream of PnorV has been removed.

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. 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

This part has not been properly characterized yet. Due to the similarity with Part:BBa_K2116006 a similar behaviour is expected with a slight increase in efficiency of EsaR repression due to the removal of a native spacer between PnorV and esabox. This results in competitive binding between EsaR and polymerase. This part shows a desired AND gate behaviour. Optimization of the esabox positioning could improve the fold increase for full activation.

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]