Difference between revisions of "Part:BBa K2587027"
 
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<partinfo>BBa_K2587027 short</partinfo>
 
<partinfo>BBa_K2587027 short</partinfo>
  
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This construct comprises parts from the Quorum sensing system from Vibrio fischeri. It contains the LuxI, the acyl homoserine lactone synthase, the LuxR, the regulator, which will bind to the promoter pLux, that will induce synthesis of GFP.  
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This construct comprises parts from the quorum sensing system of <i>Vibrio fischeri</i>. It contains LuxI, an acyl homoserine lactone synthase and LuxR, the corresponding regulator, which is able to bind to the promoter P<sub><I>lux</i></sub>, leading to induction of GFP synthesis.  
  
  
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<li> <strong> Acyl homoserine lactone synthase</strong>: LuxI</li>
 
<li> <strong> Acyl homoserine lactone synthase</strong>: LuxI</li>
 
<li> <strong>Regulator</strong>: LuxR</li>
 
<li> <strong>Regulator</strong>: LuxR</li>
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<li> <strong>Promoter</strong>: pLux, inducible by the quorum sensing molecule acyl homoserine lactone bound to LuxR</li>
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<li> <strong>Promoter</strong>: P<sub><i>lux</i></sub>, inducible by the quorum sensing molecule acyl homoserine lactone when bound to LuxR</li>
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<li> <strong>GFP</strong>fluorescent protein</li>
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<li> <strong>GFP</strong>: Green fluorescent protein</li>
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<li> GFP expression is induced after synthesis of the acyl homerine lactone by LuxI, which binds with LuxR to the pLux promoter </li>
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<li> <i>gfp</i> expression is induced after synthesis of the acyl homerine lactone by LuxI, which binds to the pLux promoter together with LuxR </li>
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<li> This construct shows functionality of promoter by increase of fluorescence upon time</li>
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<li> This construct shows functionality of promoter by increase of fluorescence over time</li>
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<li> This part contains Type II S restriction sites and can be further used for scarless assembly with other parts</li>
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<li> This part contains Type IIS restriction sites and can be further used for scarless assembly with other parts</li>
 
</ul>
 
</ul>
 
</p>
 
</p>
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<h4><strong>Characterization</strong></h4>
 
<h4><strong>Characterization</strong></h4>
 
<br>
 
<br>
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<strong>Characterisation of promoter by fluorescence measurement of GFP</strong>
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<strong>Characterization of promoter by fluorescence measurement of GFP</strong>
 
<br>
 
<br>
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To check whether the promoter, pLux, is really induced by the binding of the acyl homoserine lactone to the LuxR, a further construct (<strong>BBa_K2587027</strong>) is created which has the lysis gene E exchanged by a GFP gene. Here the relative fluorescence units are measured in comparison to the wild type E.coli cells.  
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To check whether the promoter P<sub><i>lux</i></sub> is really induced by the binding of the acyl homoserine lactone to the LuxR, this construct is created. Here the relative fluorescence units are measured in comparison to the wild type <i>E. coli</i> cells.  
 
<br>
 
<br>
  
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<strong>Results</strong> It can clearly be observed, that fluorescence increases upon growth of the cells. Wild type cells, as a negative control, show little or no fluorescence at all.  
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<strong>Results</strong> It can clearly be observed that fluorescence increases upon growth of the cells. Wild type cells (negative control) show little or no fluorescence at all.  
 
<br>
 
<br>
  
 
https://static.igem.org/mediawiki/parts/c/cd/T--Duesseldorf--promoter_functionality.PNG  
 
https://static.igem.org/mediawiki/parts/c/cd/T--Duesseldorf--promoter_functionality.PNG  
 
<br>
 
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<strong> Figure 1: Relative fluorescence measurement of lysis construct and <i>E.coli</i> wild type cells (negative control). </strong> Exctinction: 485nm; Emission: 520nm. Cells were measured only within a time span of 24h.  
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<strong> Figure 1: Relative fluorescence measurement of lysis construct and <i>E.coli</i> wild type cells (negative control). </strong> Exctinction: 485nm; Emission: 520nm. Cells were measured over the course of 24h.  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 18:13, 17 October 2018


luxI_luxR_Plux_gfp

This construct comprises parts from the quorum sensing system of Vibrio fischeri. It contains LuxI, an acyl homoserine lactone synthase and LuxR, the corresponding regulator, which is able to bind to the promoter Plux, leading to induction of GFP synthesis.


Usage and Biology


  • Acyl homoserine lactone synthase: LuxI
  • Regulator: LuxR
  • Promoter: Plux, inducible by the quorum sensing molecule acyl homoserine lactone when bound to LuxR
  • GFP: Green fluorescent protein
  • gfp expression is induced after synthesis of the acyl homerine lactone by LuxI, which binds to the pLux promoter together with LuxR
  • This construct shows functionality of promoter by increase of fluorescence over time
  • This part contains Type IIS restriction sites and can be further used for scarless assembly with other parts

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 829
    Illegal NheI site found at 852
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1082
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 892
    Illegal BsaI site found at 1681
    Illegal BsaI.rc site found at 1523
    Illegal BsaI.rc site found at 1790
    Illegal BsaI.rc site found at 2468


Characterization


Characterization of promoter by fluorescence measurement of GFP
To check whether the promoter Plux is really induced by the binding of the acyl homoserine lactone to the LuxR, this construct is created. Here the relative fluorescence units are measured in comparison to the wild type E. coli cells.

Results It can clearly be observed that fluorescence increases upon growth of the cells. Wild type cells (negative control) show little or no fluorescence at all.

T--Duesseldorf--promoter_functionality.PNG
Figure 1: Relative fluorescence measurement of lysis construct and E.coli wild type cells (negative control). Exctinction: 485nm; Emission: 520nm. Cells were measured over the course of 24h.