Difference between revisions of "Part:BBa K2116014"
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<h1>Characterization</h1>
 
<h1>Characterization</h1>
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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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<p> This part has not been properly characterized yet. Due to the similarity with [[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.
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    [[File:T--ETH_Zurich--XX.png|500px|thumb|center|<b>Figure 1:</b> AND gate behaviour. This part shows a desired behaviour. Optimization of the esabox positioning could improve the fold increase for full activation.]]
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    The AND gate behaviour shown in Figure 1 can be explained bye the placing of three esaboxes after PnorV. This seems to be enough
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    to reach sufficient balance between repression and derepression. An improvement could be achieved by either increasing the number of esaboxes, their placement
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    or by decreasing the amount of EsaR production. We followed all these steps, and recommend you have a look at our favourite
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    AND gate [[Part:BBa_K2116011]], where an improvement compared to this part was shown.  
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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 2:</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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<h2>References:</h2>
 
<h2>References:</h2>
  

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 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]