Difference between revisions of "Part:BBa K2116007"
 
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__NOTOC__
 
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<partinfo>BBa_K2116007 short</partinfo>
 
<partinfo>BBa_K2116007 short</partinfo>
  
 
AND gate constructed by placing 2 esaboxes with 15bp spacing in between, after the transcription start site of PnorV.  
 
AND gate constructed by placing 2 esaboxes with 15bp spacing in between, after the transcription start site of PnorV.  
−
[[File:T--ETH Zurich--p22.png|thumb|500px|Design of AND gate. The spacing between the esaboxes is 15bp long. NRB;NorR binding site, Sigma54B; RNA polymerase binding site]]
+
 
 +
 
 +
 
 +
<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>
 +
 
 +
 
 +
<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>
 +
 
 +
 
 +
<h1> Biology and Usage </h1>
 +
 
 +
<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. [[File:T--ETH Zurich--p22.png|thumb|500px|Design of AND gate. The spacing between the esaboxes is 15bp long. NRB;NorR binding site, Sigma54B; RNA polymerase binding site]]
 +
</p>
 +
 +
<p>
 +
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>When characterizing our parts collection we initially confirmed functionality. Below you can see a graph depicting AND gate behaviour of this biobrick.
 +
    [[File:ANDgate_BBa_K2116007.png|500px|thumb|center|<b>Figure 1:</b> AND gate behaviour. This part does not resemble an optimal AND gate behaviour.]]
 +
   
 +
</p>
 +
 
 +
 
 +
<p>
 +
    The AND gate behaviour shown in Figure 1 can be explained bye the placing of only two esaboxes after PnorV. This seems not 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]].
 +
 
 +
</p>
 +
 
 +
<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--BBa_K2116007.png|500px|thumb|center|<b>Figure 2:</b> AND gate behaviour compared to cells containing an EsaR expressing plasmid vs non expressing.
 +
Concentrations of DETA/NO: 0,0,0,5,5,5 mM and the concentrations of AHL: 0,0.1,10, 0,0.1,10 µM. The difference in repression suggests that EsaR is the major protein acting, producing the desired AND gate function. ]]
 +
 
 +
 
 +
<p></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
 
<!-- Add more about the biology of this part here

Latest revision as of 17:57, 28 October 2016

AND gate regulated by norR and esaR (two esaboxes)

AND gate constructed by placing 2 esaboxes with 15bp spacing in between, after the transcription start site of PnorV.


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.

Design of AND gate. The spacing between the esaboxes is 15bp long. NRB;NorR binding site, Sigma54B; RNA polymerase binding 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

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

Figure 1: AND gate behaviour. This part does not resemble an optimal AND gate behaviour.


The AND gate behaviour shown in Figure 1 can be explained bye the placing of only two esaboxes after PnorV. This seems not 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.

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.

Figure 2: AND gate behaviour compared to cells containing an EsaR expressing plasmid vs non expressing. Concentrations of DETA/NO: 0,0,0,5,5,5 mM and the concentrations of AHL: 0,0.1,10, 0,0.1,10 µM. The 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]