Difference between revisions of "Part:BBa K819006"
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A fast degrading GFP ligated to the rear of a sulA promoter in 408 form (only recognizable by our LexA-VVD fusing protein, not by E.coli endogenous LexA). The sulA promoter promotes a gene which express SulA protein, a differentiation inhibitor, and was a member of the SOS regulon family. When co-transformed with our luminesensor plasmid into E.coli cell illuminated by blue light, the light will triger the dimerizaiton of the LexA408-VVD fusion protein and the dimerized LexA408 domain will bind the SOS box in 408 form  in the SulA408 promoter to inhibit the transcription of the downstream gene so that no GFP will be expressed; if the environment is dark, the luminesensor will not dimerize and no supression of the promoter will occour, and GFP will be expressed.<br/><br/>
 
A fast degrading GFP ligated to the rear of a sulA promoter in 408 form (only recognizable by our LexA-VVD fusing protein, not by E.coli endogenous LexA). The sulA promoter promotes a gene which express SulA protein, a differentiation inhibitor, and was a member of the SOS regulon family. When co-transformed with our luminesensor plasmid into E.coli cell illuminated by blue light, the light will triger the dimerizaiton of the LexA408-VVD fusion protein and the dimerized LexA408 domain will bind the SOS box in 408 form  in the SulA408 promoter to inhibit the transcription of the downstream gene so that no GFP will be expressed; if the environment is dark, the luminesensor will not dimerize and no supression of the promoter will occour, and GFP will be expressed.<br/><br/>
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In order to determine whether the LexA-VVD(M135I) Luminesensor has enhanced reversibility in comparison to the original LexA-VVD, the temporal change of GFP expression level of dilated overnight culture of the strains LV-WT and LV-135 under blue light were measured at 2 hour intervals for 26 hours. As shown in figure below, the GFP expression level of both of the two strains began to rise after incubating at dark for about 10 hour. We speculated that the GFP expression level of the two strains is mainly determined by the equilibrium between GFP production and degradation and the dimerized LexA-VVD(WT) and LexA-VVD(135) dissociate in a shorter time scale compared to the time needed to establish the equilibrium of the GFP production and degradation.
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In order to determine whether the LexA-VVD(M135I) Luminesensor has enhanced reversibility in comparison to the original LexA-VVD, the temporal change of GFP expression level of dilated overnight culture of the strains LV-WT and LV-135 under blue light were measured at 2 hour intervals for 26 hours. As shown in figure below, the GFP expression level of both of the two strains began to rise after incubating at dark for about 10 hour. We speculated that the GFP expression level of the two strains is mainly determined by the equilibrium between GFP production and degradation and the dimerized LexA-VVD(WT) and LexA-VVD(135) dissociate in a shorter time scale compared to the time needed to establish the equilibrium of the GFP production and degradation.<br />
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Cells exposed to different illumination time expressing Luminesensor indicated the repression efficiency. As shown in the Figure 1a, with the increase of illumination time (from group 1 to group 14), the expression of GFP increased. Figure 1b shows the quantitative data.
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<a href="https://static.igem.org/mediawiki/2012/1/1f/Peking2012_Timeline_sulA.jpg"target="blank"><img src="https://static.igem.org/mediawiki/2012/1/1f/Peking2012_Timeline_sulA.jpg" style="width:600px;margin-left:180px"  ></a>
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<p style="text-align:center">Figure.1a Photo of GFP level to the illumination time</p>
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<a href="https://static.igem.org/mediawiki/2012/b/ba/Peking2012_luminesensor_opt_time_rfp.jpg"target="blank"><img src="https://static.igem.org/mediawiki/2012/b/ba/Peking2012_luminesensor_opt_time_rfp.jpg" style="width:600px;margin-left:180px"  ></a>
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<a href="https://static.igem.org/mediawiki/2012/1/1f/Peking2012_Timeline_sulA.jpg"target="blank"><img src="https://static.igem.org/mediawiki/2012/1/1f/Peking2012_Timeline_sulA.jpg" style="width:600px;margin-left:180px"  ></a>
  
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<p style="text-align:center">Figure.1 The time course of RFP expression controlled by LV-WT and LV-135</p>
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<p style="text-align:center">Figure.1b The GFP expression intensity of different illumination time</p>
 
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Revision as of 14:17, 25 September 2012

Testing device for Luminesensor

A fast degrading GFP ligated to the rear of a sulA promoter in 408 form (only recognizable by our LexA-VVD fusing protein, not by E.coli endogenous LexA). The sulA promoter promotes a gene which express SulA protein, a differentiation inhibitor, and was a member of the SOS regulon family. When co-transformed with our luminesensor plasmid into E.coli cell illuminated by blue light, the light will triger the dimerizaiton of the LexA408-VVD fusion protein and the dimerized LexA408 domain will bind the SOS box in 408 form in the SulA408 promoter to inhibit the transcription of the downstream gene so that no GFP will be expressed; if the environment is dark, the luminesensor will not dimerize and no supression of the promoter will occour, and GFP will be expressed.

In order to determine whether the LexA-VVD(M135I) Luminesensor has enhanced reversibility in comparison to the original LexA-VVD, the temporal change of GFP expression level of dilated overnight culture of the strains LV-WT and LV-135 under blue light were measured at 2 hour intervals for 26 hours. As shown in figure below, the GFP expression level of both of the two strains began to rise after incubating at dark for about 10 hour. We speculated that the GFP expression level of the two strains is mainly determined by the equilibrium between GFP production and degradation and the dimerized LexA-VVD(WT) and LexA-VVD(135) dissociate in a shorter time scale compared to the time needed to establish the equilibrium of the GFP production and degradation.


Cells exposed to different illumination time expressing Luminesensor indicated the repression efficiency. As shown in the Figure 1a, with the increase of illumination time (from group 1 to group 14), the expression of GFP increased. Figure 1b shows the quantitative data.

Figure.1a Photo of GFP level to the illumination time



Figure.1b The GFP expression intensity of different illumination time



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 839
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 839
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 839
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 839
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 734