Difference between revisions of "Part:BBa K2116013"
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<partinfo>BBa_K2116013 short</partinfo>
 
<partinfo>BBa_K2116013 short</partinfo>
  
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AND gate constructed by adding an esabox that starts after the -12 binding site for sigma 54, and replaces some of the sigma 54 binding site.  
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<b>Summary:</b> AND gate constructed by placing 2 esaboxes after the transcription start site of PnorV, native spacer has been removed.  
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<p>
 
<p>
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</p>
 
</p>
  
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<h1> Biology and Usage </h1>
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    [[File:T--ETH Zurich--p22.png|thumb|500px|<b>Figure 1:</b> Design of AND gate. The spacing between the esaboxes is 8bp long. NRB;NorR binding site, Sigma54B; RNA polymerase binding site. The native spacer has been removed.]]
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<p>
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    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.
 +
   
 +
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    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.
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</p>
  
 
<h2> Characterization of this part </h2>
 
<h2> Characterization of this part </h2>
  
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Data will be here before the final presentation at the jamboree. Apologies for the delay.  
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<p> When characterizing our parts collection we initially confirmed functionality. Below you can see a graph depicting AND gate behaviour of this biobrick.
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    [[File:T--ETH_Zurich--XX.png|500px|thumb|center|<b>Figure 2:</b> AND gate behaviour. This part does not resemble an optimal AND gate behaviour.]]
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</p>
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<p>
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    The AND gate behaviour shown in Figure 2 can be explained due to the placing of only two esabox after PnorV. This seems not to be enough
 +
    to reach optimal 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>
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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>
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[[File:T--ETH_Zurich--XX.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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<p></p>
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<h2>References:</h2>
 
<h2>References:</h2>

Revision as of 02:32, 28 October 2016


AND gate p25

Summary: AND gate constructed by placing 2 esaboxes after the transcription start site of PnorV, native spacer has been removed.


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

Figure 1: Design of AND gate. The spacing between the esaboxes is 8bp long. NRB;NorR binding site, Sigma54B; RNA polymerase binding site. The native spacer 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 of this part

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--XX.png
Figure 2: AND gate behaviour. This part does not resemble an optimal AND gate behaviour.

The AND gate behaviour shown in Figure 2 can be explained due to the placing of only two esabox after PnorV. This seems not to be enough to reach optimal 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.

File:T--ETH Zurich--XX.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.</i>
  • [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]