Difference between revisions of "Part:BBa K819007"

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recA408 Promoter + B0030 + GFP + ssrA-tag<br /><br />
 
recA408 Promoter + B0030 + GFP + ssrA-tag<br /><br />
  
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.<br /><br />
+
To measure the properties of our Luminesensor we placed a fast degrading GFP under control of a sulA promoter in 408 form (only recognizable by our Luminesensor, not by E.coli endogenous LexA). The sulA promoter belongs to the SOS regulon family and promotes a gene which expresses SulA protein, a cell division inhibitor. After co-transforming our luminesensor with this measurement device into E.coli cell and illuminating the cell with blue light, the light will induce luminesensor to dimerize and bind to the SulA408 promoter to inhibit the transcription of the downstream GFP; if left in dark, the luminesensor will not dimerize and no repression of the promoter will occour, and GFP will be expressed.<br /><br />
  
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.<br />
+
We tested the sensitivity of Luminesensor by examining the light-dependent transcription activity of a GFP-ssrA reporter. ssrA is a protein tag that induces fast degradation of protein, which in our case facilitates the observation of transcription activity.<br />
  
Cells with luminesenser under different illumination shows different repression level. As shown in the Figure 1, all of the cells with dissimilar attenuators showed incredible repression efficiency. <br />
+
Cells with luminesensor cultivated under different light intensity showed different GFP expression level. As shown in the Figure 1, all of the cells with different attenuators showed incredible repression efficiency. <br />
  
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.<br />
+
It proves that once the cells are exposed to natural light, the transcription of reporter gene will be strongly repressed, although still possessing a dose-response curve. Besides, in the negative control group, which was cultivated in total darkness, 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.<br />
  
 
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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.<br />
+
Cells exposed to light for different amount of time showed different levels of GFP expression. As shown in Figure 2a, as the light-exposure time decreased (from group 1 to group 14), the expression of GFP increased. Figure 2b shows the quantitative data.<br />
  
 
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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.<br />
+
GFP expression was repressed under either bio-luminescence or dim LED. To obtain more quantitative data, we measured the GFP expression level using a Tecan infinite 200 reader. As 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 exposed to bio-luminescence.<br />
  
 
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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.<br />
+
The response threshold of our luminesensor, BBa_K819005, can't be found by simply using blue LED and attenuating filters, because the luminesensor is so sensitive that it can even respond to light that cannot be detected by our photometer. And it’s difficult to control light with extremely low intensity simply by using attenuating filters. So, we managed to find the threshold response using bio-luminescence as light source based on our light-communication system.<br />
  
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.<br />
+
Light emitting cell broth was diluted to create different light intensity. We used the dilution ratio as an indicator of light intensity. (e.g. 0.001 means the cell broth was diluted 1000 times, and indicates the weakest light intensity) Using this method, we managed to approach the linear area of our sensor’s dose-response curve. And we succeeded in regulating the gene expression level by changing the light intensity. Figure 4 shows the quantitative data demonstating the relationship between GFP expression level and the dilution ratio of the light emitting cell.<br />
  
 
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Revision as of 18:20, 26 September 2012

Measurement device for Luminesensor

recA408 Promoter + B0030 + GFP + ssrA-tag

To measure the properties of our Luminesensor we placed a fast degrading GFP under control of a sulA promoter in 408 form (only recognizable by our Luminesensor, not by E.coli endogenous LexA). The sulA promoter belongs to the SOS regulon family and promotes a gene which expresses SulA protein, a cell division inhibitor. After co-transforming our luminesensor with this measurement device into E.coli cell and illuminating the cell with blue light, the light will induce luminesensor to dimerize and bind to the SulA408 promoter to inhibit the transcription of the downstream GFP; if left in dark, the luminesensor will not dimerize and no repression of the promoter will occour, and GFP will be expressed.

We tested the sensitivity of Luminesensor by examining the light-dependent transcription activity of a GFP-ssrA reporter. ssrA is a protein tag that induces fast degradation of protein, which in our case facilitates the observation of transcription activity.

Cells with luminesensor cultivated under different light intensity showed different GFP expression level. As shown in the Figure 1, all of the cells with different 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 possessing a dose-response curve. Besides, in the negative control group, which was cultivated in total darkness, 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 light for different amount of time showed different levels of GFP expression. As shown in Figure 2a, as the light-exposure time decreased (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




GFP expression was repressed under either bio-luminescence or dim LED. To obtain more quantitative data, we measured the GFP expression level using a Tecan infinite 200 reader. As 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 exposed to bio-luminescence.

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



The response threshold of our luminesensor, BBa_K819005, can't be found by simply using blue LED and attenuating filters, because the luminesensor is so sensitive that it can even respond to light that cannot be detected by our photometer. And it’s difficult to control light with extremely low intensity simply by using attenuating filters. So, we managed to find the threshold response using bio-luminescence as light source based on our light-communication system.

Light emitting cell broth was diluted to create different light intensity. We used the dilution ratio as an indicator of light intensity. (e.g. 0.001 means the cell broth was diluted 1000 times, and indicates the weakest light intensity) Using this method, we managed to approach the linear area of our sensor’s dose-response curve. And we succeeded in regulating the gene expression level by changing the light intensity. Figure 4 shows the quantitative data demonstating the relationship between GFP expression level and 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


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