Difference between revisions of "Part:BBa K819006"

Revision as of 14:44, 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. 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.Figure 2 shows the GFP level to the dilution ratio of light emitting cell.

Figure.1 Photo of GFP level to the illumination time

Figure.2 Luminance measurement of different attenuator.

Light emitting cell broth was diluted to create different light intensity, represented by the dilution ratio. (e.g. 0.001 indicates the weakest light intensity) And with this method we managed to get closer to the linear area of our sensor’s response. And we succeeded in regulating the gene expression level by changing the light intensity.

The photo below shows the GFP level to the dilution ratio of light emitting cell.

Figure 3. Photo of GFP level to the dilution ratio of light emitting cell.

Sequence and Features