Difference between revisions of "Part:BBa K2587024"
Line 3: Line 3:
 
<partinfo>BBa_K2587024 short</partinfo>
 
<partinfo>BBa_K2587024 short</partinfo>
  
−
This part is an assembly of different constructs, which then yield a final plasmid, able to self lyse <i>Escherichia coli</i> cells. It is a construct based on the Quorum sensing system, which induces cell lysis upon synthesis of the quorum sensing molecule acyl homoserine lactone. Upon synthesis of this by the <i>LuxI</i> gene, it will bind to the regulator LuxR and then to the promoter pLux, which will activate synthesis of the lysis protein E from the bacteriophage phiX174E.  
+
This part is an assembly of different constructs, which then yield a final plasmid, able to self lyse <i>Escherichia coli</i> cells. It is a construct based on the Quorum sensing system of <i>Vibrio fischeri</i>. With the addition of a lysis gene it will induce cell lysis upon synthesis of the quorum sensing molecule acyl homoserine lactone. Upon synthesis of this by the LuxI gene, it will bind to the regulator LuxR and then to the promoter pLux, which will activate synthesis of the lysis protein E from the bacteriophage phiX174E.
 
   
 
   
  
  
−
Usage and Biology
+
<strong><h4>Usage and Biology</h4></strong>
−
This construct can be used to regulate cell density. Compared to the wildtype cells, cells harbouring this plasmid grow slower. Observation of their growth shows a remissive exponential phase, reaching the stationary phase with a lower cell density compared to wild type celly.  
+
<br>
 +
<p><ul>
 +
<li> <strong> Acyl homoserine lactone synthase</strong> : LuxI</li>
 +
<li> <strong>Regulator</strong>: LuxR</li>
 +
<li> <strong>Promoter</strong>: pLux, inducible by the quorum sensing molecule acyl homoserine lactone bound to LuxR</li>
 +
<li> <strong>Lysis gene</strong>: Lysis gene E from bacteriophage phiX174E</li>
 +
<li> Lysis protein induces host cell lysis by inhibiting host translocase MraY activity, which is necessary for catalysis of lipid I , a factor for cell wall synthesis (<strong>Cytolysis</strong>) (1) </li>
 +
<li> This construct is able to modify <i>E.coli´s</i> growth, by slowing it down</li>
 +
<li> This part contains Type II S restriction sites and can be further used for scarless assembly with other parts</li>
 +
</ul>
 +
</p>
  
 
<!-- -->
 
<!-- -->
Line 14: Line 24:
 
<partinfo>BBa_K2587024 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2587024 SequenceAndFeatures</partinfo>
  
 +
<br>
 +
<h4><strong>Characterization</strong></h4>
 +
<br>
 +
<strong>Characterisation of promoter by fluorescence measurement of GFP</strong>
 +
<br>
 +
To check whether the promoter, pLux, is really induced by the binding of the acyl homoserine lactone to the LuxR, a further construct (BBa_K2587027) 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.
 +
<br>
 +
 +
<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.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 08:17, 1 October 2018


luxI_luxR_Plux_E

This part is an assembly of different constructs, which then yield a final plasmid, able to self lyse Escherichia coli cells. It is a construct based on the Quorum sensing system of Vibrio fischeri. With the addition of a lysis gene it will induce cell lysis upon synthesis of the quorum sensing molecule acyl homoserine lactone. Upon synthesis of this by the LuxI gene, it will bind to the regulator LuxR and then to the promoter pLux, which will activate synthesis of the lysis protein E from the bacteriophage phiX174E.


Usage and Biology


  • Acyl homoserine lactone synthase : LuxI
  • Regulator: LuxR
  • Promoter: pLux, inducible by the quorum sensing molecule acyl homoserine lactone bound to LuxR
  • Lysis gene: Lysis gene E from bacteriophage phiX174E
  • Lysis protein induces host cell lysis by inhibiting host translocase MraY activity, which is necessary for catalysis of lipid I , a factor for cell wall synthesis (Cytolysis) (1)
  • This construct is able to modify E.coli´s growth, by slowing it down
  • This part contains Type II S 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 site found at 1832
    Illegal BsaI.rc site found at 1523
    Illegal BsaI.rc site found at 1790


Characterization


Characterisation 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, a further construct (BBa_K2587027) 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.

Results 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.