Part:BBa_K819006
Testing device for Luminesensor
A fast degrading GFP ligated to the rear of a sulA promoter in 408 form (only recognizable by our luminesensor, 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. After co-transformed with our luminesensor plasmid into E.coli cell，illuminating the cell by blue light,the light will triger the dimerizaiton of luminesensor, making dimer bind the SulA408 promoter to inhibit the transcription of the downstream GFP; if the environment is dark, the luminesensor will not dimerize and no suppression 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.
Figure.1 the GFP expression level of the two strains
Cells exposed to different illumination time expressing Luminesensor indicated the repression efficiency. As shown in the Figure 2, with the increase of illumination time (from group 1 to group 14), the expression of GFP increased.
Figure.2 Photo of GFP level to the illumination time
We tested the sensitivity of Luminesensor by examine the light-dependent transcriptional activity of a GFP-ssrA reporter. ssrA is a protein tag that induces fast degradation of protein, which in our case facilitated the observation of transcriptional activity.
Cells exposed to different light intensity expressing Luminesensor showed manifest light-repressed reporter gene transcription. As shown in the Figure 3, all of the cells with dissimilar attenuators showed incredible repression efficiency.
It proves that once the cells are exposed to natural light, the transcription of reporter gene will be strongly repressed, although still presents as a dose response. Besides, in the negative control group, which was entirely in the dark state, the expression of GFP ran up to a high degree of 50,000. As a matter of fact, as you can see later in "Results of light communication between cells", when we serially diluted light-emitting cells which expresses bacterial luciferase, the cells expressing Luminesensor presents significant dose response. Taking everything into account, our luminesensor does possess high sensitivity across several orders of magnitude.
Figure.3 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. Figure 3 shows the GFP level to the dilution ratio of light emitting cell.
The response threshold value of our luminesensor, BBa_K819005, was not found simply using blue LED and attenuating filters, because the luminesensor is so sensitive to very dim light which cannot be detected by the photometer we used.(see characterization:sensitivity) And it’s difficult to control the intensity of very dim light simply using attenuating filters. So, we managed to find the threshold and therefore regulate the response intensity using bio-luminescence as light source based on our light-communication system.
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 4. Photo of GFP level to the dilution ratio of light emitting cell.
References
1. Cole, S.T.(1983) Charaeterisation of the Promoter
for the LexA Regulated sulA Gene of Escherichia coli. Mol. Gen. Genet., 189: 400: 404
2. Wang, X., Chen, X. & Yang, Y.(2012) spatiotemporal control of gene expression
by a light-switchable transgene system. Nat. Methods, 9: 266: 269
3. Zhang, A.P.P., Pigli, Y.Z & Rice, P.A.(2010) Structure of the LexA–DNA complex and implications for SOS box measurement.Nature, 466: 883: 886
4. Butalaa, M., Zgur-Bertokb, D., and Busby, S. J. W.(2009) The bacterial LexA transcriptional repressor. Cell. Mol. Life Sci., 66: 82: 93
5. Shimizu-Sato, S., Huq, E., Tepperman, J.M., & Quail, P.H.(2002). A light-switchable gene promoter system. Nat. Biotechnol. 20: 1041: 1044
6. Levskaya, A., Weiner, O.D., Lim, W.A. & Voigt, C.A.(2009). Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature, 461: 997: 1001
7. Möglich, A., Ayers, R.A. & Moffat, K.(2009). Design and Signaling Mechanism of Light-Regulated Histidine Kinases. J. Mol. Biol., 385: 1433: 1444
8. Strickland, D., Moffat, K. & Sosnick, T.R.(2008). Light-activated DNA binding in a designed allosteric protein. Proc. Natl Acad. Sci. USA, 105: 10709: 10714
9. Ohlendorf, R., Vidavski, R.R., Eldar, A., Moffat, K. & Möglich, A.(2012). From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. J. Mol. Biol., 416: 534: 542
10. Toettcher, J.E., Voigt, C.A., Weiner, O.D. & Lim, W.A.(2010). The promise of optogenetics in cell biology: interrogating molecular circuits in space and time. Nat. Methods, 8: 35: 38
11. Bacchus, W. & Fussenegger, M.(2011) The use of light for engineered control and reprogramming of cellular functions. Curr. Opin. Biotechnol., 23: 1: 8
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 839
- 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 839
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 839
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 839
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 734
//function/reporter/fluorescence
| emission | 507nm |
| excitation | 470nm |