Part:BBa_K819007

Measurement device for Luminesensor

recA408 Promoter + B0030 + GFP + ssrA-tag

To measure the properties of our 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.

Cells exposed to different light intensity expressing Luminesensor showed manifest light-repressed reporter gene transcription. As shown in the Figure 1, all of the cells with dissimilar attenuators showed incredible repression efficiency (figure 1: luminance measurement of different attenuator).

Figure 1. luminance measurement of different attenuator

Cells exposed to different illumination time expressing Luminesensor indicated the repression efficiency. As shown in the Figure 2a, with the increase of illumination time (from group 1 to group 14), the expression of GFP increased. Figure 2b shows the quantitative data.

Figure 2a. Photo of GFP level to the illumination time

Figure 2b. luminance measurement of different attenuator

Peking iGEM 2012 has successfully demonstrated that the Luminesensor is able to sense the blue light produced by bacterial luciferase. This is the very first time that light-communication between cells has been achieved without direct physical contact. Quantitative data was obtained to evaluate the efficiency of light-communication.

GFP expression was repressed by Luminesensor expressed in light sensing cells under either bio-luminescence. To obtain more quantitative data, we measured the GFP expression level using a Tecan infinite 200 reader. As is shown in the graph below (Figure 3), the expression level of GFP in darkness (in our device with no glowing cells) is about 200-fold higher than that of the cells under bio-luminescence. The Figure 4 shows the quantitative data of GFP expression level to the dilution ratio of the light emitting cell.

Figure 3. Quantitative measurement of the repression effect of bio-luminescence

Figure 4. Measurement of relative GFP level to the dilution ratio of light emitting cell using a Tecan infinite 200 reader

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 850
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 850
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 850
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 850
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 745