Difference between revisions of "Part:BBa K819006"
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===Characterization=== | ===Characterization=== | ||
| − | A fast degrading GFP placed under the control of sulA promoter with a specific mutation (only recognizable by our <i>Luminesensor</i> | + | A fast degrading GFP placed under the control of sulA promoter with a specific mutation (only recognizable by our <i>Luminesensor</i>). After it is co-transformed with <i>Luminesensor</i> plasmid into <i>E.coli</i> cells, illuminating the cells by blue light, the light will triger the dimerizaiton of <i>Luminesensor</i>, making dimers bind to this promoter, thus to inhibit the transcription of downstream GFP; if the environment is dark, the <i>Luminesensor</i> will not dimerize and no repression of the promoter will occour.<br/><br/> |
The time course of GFP expression level controlled by <i>Luminesensor</i> before and after optimization at 2 hour intervals for 26 hours. As shown in figure below, the GFP expression began to rise after incubating at dark for about 10 hours. <br /> | The time course of GFP expression level controlled by <i>Luminesensor</i> before and after optimization at 2 hour intervals for 26 hours. As shown in figure below, the GFP expression began to rise after incubating at dark for about 10 hours. <br /> | ||
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| − | The response threshold value of our <i>Luminesensor</i> | + | The response threshold value of our <i>Luminesensor</i>, was not found simply using blue LED and attenuating filters, because the <i>Luminesensor</i> is so sensitive to very dim light which cannot be detected by the photometer we used. And it’s difficult to control the intensity of very dim light simply using attenuating filters. As a matter of fact, when we serially diluted light-emitting cells which expresses bacterial luciferase, the receiver cells expressing <i>Luminesensor</i> presents significant dose response. Taking everything into account, our <i>Luminesensor</i> does possess high sensitivity across several orders of magnitude.<br /> |
Light-emitting cell broth was diluted to create a light intensity gradient, 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 dose response curve. <br /> | Light-emitting cell broth was diluted to create a light intensity gradient, 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 dose response curve. <br /> | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
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Latest revision as of 07:56, 18 October 2012
Testing device for Luminesensor
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
Characterization
A fast degrading GFP placed under the control of sulA promoter with a specific mutation (only recognizable by our Luminesensor). After it is co-transformed with Luminesensor plasmid into E.coli cells, illuminating the cells by blue light, the light will triger the dimerizaiton of Luminesensor, making dimers bind to this promoter, thus to inhibit the transcription of downstream GFP; if the environment is dark, the Luminesensor will not dimerize and no repression of the promoter will occour.
The time course of GFP expression level controlled by Luminesensor before and after optimization at 2 hour intervals for 26 hours. As shown in figure below, the GFP expression began to rise after incubating at dark for about 10 hours.
Figure.1 The time course of GFP expression level controlled by Luminesensor before and after optimization
Luminesensor treated with decreasing illumination time shows decreasing repression, and thereby, increasing GFP expression.
Figure.2 Luminesensor treated with decreasing illumination time shows increasing GFP expression.
We tested the sensitivity of Luminesensor by examining 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 expressing Luminesensor exposed to different light intensity 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.
Figure.3 luminance attenuation using different attenuators could also be sensed by Luminesensor, which is much dimmer than natural light.
The response threshold value of our Luminesensor, 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. And it’s difficult to control the intensity of very dim light simply using attenuating filters. As a matter of fact, when we serially diluted light-emitting cells which expresses bacterial luciferase, the receiver cells expressing Luminesensor presents significant dose response. Taking everything into account, our Luminesensor does possess high sensitivity across several orders of magnitude.
Light-emitting cell broth was diluted to create a light intensity gradient, 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 dose response curve.
Figure 4. . Measurement of relative GFP level to the dilution ratio of light emitting cell using a Tecan infinite 200 reader.
References
1. Cole, S.T.(1983) Characterization 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