Difference between revisions of "Part:BBa K819007"

Revision as of 16:45, 26 September 2012

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 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 binds the SulA408 promoter to inhibit the transcription of the downstream GFP; if the environment is dark, the luminesensor will not dimerize and no supression of the promoter will occour, and GFP will be expressed.

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 1, 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 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 decrease 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.

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

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 Figure 4 shows the quantitative data of GFP expression level to the dilution ratio of the light emitting cell.

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

References

1. Shimizu-Sato, S., Huq, E., Tepperman, J.M., & Quail, P.H.(2002). A light-switchable gene promoter system. Nat. Biotechnol. 20: 1041: 1044
2. 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
3. Möglich, A., Ayers, R.A. & Moffat, K.(2009). Design and Signaling Mechanism of Light-Regulated Histidine Kinases. J. Mol. Biol., 385: 1433: 1444
4. Strickland, D., Moffat, K. & Sosnick, T.R.(2008). Light-activated DNA binding in a designed allosteric protein. Proc. Natl Acad. Sci.<i> USA, 105: 10709: 10714
5. Ohlendorf, R., Vidavski, R.R., Eldar, A., Moffat, K. & Möglich, A.(2012). From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. <i>J. Mol. Biol., 416: 534: 542
6. 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
7. Bacchus, W. & Fussenegger, M.(2011) The use of light for engineered control and reprogramming of cellular functions. Curr. Opin. Biotechnol., 23: 1: 8
8. Wang, X., Chen, X. & Yang, Y.(2012) spatiotemporal control of gene expression
by a light-switchable transgene system. Nat. Methods, 9: 266: 269
9. 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
10. Butalaa, M., Zgur-Bertokb, D., and Busby, S. J. W.(2009) The bacterial LexA transcriptional repressor. Cell. Mol. Life Sci., 66: 82: 93

Sequence and Features