Part:BBa_K819007

Measurement device for Luminesensor

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
  

Characterization

To measure the properties of our Luminesensor we placed a fast degrading GFP under control of a sulA promoter in 408 form (408 form means the promoter can only be recognized by our Luminesensor but not by E.coli endogenous LexA, making our reporter system orthogonal to the host genetic context). 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 expression of the GFP-ssrA reporter. ssrA is a protein tag that induces fast degradation of protein, which in our case facilitates the observation of expression level.

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 attenuation using different attenuators could also be sensed by the Luminesensor, which is much dimmer than natural light.

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. GFP expression level decreases with increasing illumination time

Figure 2b. The time course of GFP expression controlled by Luminesensor

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 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 dose response of GFP expression 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. 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. 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