Difference between revisions of "Part:BBa K819006"
 
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<partinfo>BBa_K819006 short</partinfo>
 
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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.<br/><br/>
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===Sequence and Features===
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<partinfo>BBa_K819006 SequenceAndFeatures</partinfo><br />
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===Characterization===
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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/>
  
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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. 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.<br />
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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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<a href="https://static.igem.org/mediawiki/parts/a/a7/Peking2012_wild%2Boptimized1.png"target="blank"><img src="https://static.igem.org/mediawiki/parts/a/a7/Peking2012_wild%2Boptimized1.png" style="width:600px;margin-left:180px"  ></a>
 
<a href="https://static.igem.org/mediawiki/parts/a/a7/Peking2012_wild%2Boptimized1.png"target="blank"><img src="https://static.igem.org/mediawiki/parts/a/a7/Peking2012_wild%2Boptimized1.png" style="width:600px;margin-left:180px"  ></a>
  
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<p style="text-align:center">Figure.1 Time course of GFP expression level of the two strains</p>
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<p style="text-align:center">Figure.1 The time course of GFP expression level controlled by <i>Luminesensor</i> before and after optimization</p>
 
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Cells with luminesenser under different illumination shows different repression level. As shown in the Figure 2, with the decrease of illumination time (from group 1 to group 14), the expression of GFP increased. <br />
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<i>Luminesensor</i> treated with decreasing illumination time shows decreasing repression, and thereby, increasing GFP expression. <br />
  
 
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<a href="https://static.igem.org/mediawiki/2012/1/1f/Peking2012_Timeline_sulA.jpg"target="blank"><img src="https://static.igem.org/mediawiki/2012/1/1f/Peking2012_Timeline_sulA.jpg" style="width:600px;margin-left:180px"  ></a>
 
<a href="https://static.igem.org/mediawiki/2012/1/1f/Peking2012_Timeline_sulA.jpg"target="blank"><img src="https://static.igem.org/mediawiki/2012/1/1f/Peking2012_Timeline_sulA.jpg" style="width:600px;margin-left:180px"  ></a>
  
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<p style="text-align:center">Figure.2 Photo of GFP level to the illumination time</p>
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<p style="text-align:center">Figure.2 <i>Luminesensor</i> treated with decreasing illumination time shows increasing GFP expression.</p>
 
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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/>
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We tested the sensitivity of <i>Luminesensor</i> 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.<br/>
  
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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.<br/>
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Cells expressing <i>Luminesensor</i> 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.<br/>
  
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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/>
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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. <br/>
  
 
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<a href="https://static.igem.org/mediawiki/2012/2/22/Peking2012_Luminesensor_sensitivity_2.jpg"target="blank"><img src="https://static.igem.org/mediawiki/2012/2/22/Peking2012_Luminesensor_sensitivity_2.jpg" style="width:600px;margin-left:180px"  ></a>
 
<a href="https://static.igem.org/mediawiki/2012/2/22/Peking2012_Luminesensor_sensitivity_2.jpg"target="blank"><img src="https://static.igem.org/mediawiki/2012/2/22/Peking2012_Luminesensor_sensitivity_2.jpg" style="width:600px;margin-left:180px"  ></a>
  
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<p style="text-align:center">Figure.3 Luminance measurement of different attenuator.</p>
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<p style="text-align:center">Figure.3 luminance attenuation using different attenuators could also be sensed by <i>Luminesensor</i>, which is much dimmer than natural light.</p>
 
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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 />
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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 />
  
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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.<br />
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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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The photo below shows the GFP level to the dilution ratio of light emitting cell.<br />
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<a href="https://static.igem.org/mediawiki/parts/d/dc/Peking2012_light_communication_dilution51.png"target="blank"><img src="https://static.igem.org/mediawiki/parts/d/dc/Peking2012_light_communication_dilution51.png" style="width:600px;margin-left:180px"  ></a>
 
<a href="https://static.igem.org/mediawiki/parts/d/dc/Peking2012_light_communication_dilution51.png"target="blank"><img src="https://static.igem.org/mediawiki/parts/d/dc/Peking2012_light_communication_dilution51.png" style="width:600px;margin-left:180px"  ></a>
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<p style="text-align:center">Figure 4. Photo of GFP level to the dilution ratio of light emitting cell.</p>
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<p style="text-align:center">Figure 4. . Measurement of relative GFP level to the dilution ratio of light emitting cell using a Tecan infinite 200 reader.</p>
 
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===References===
 
===References===
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1. Cole, S.T.(1983) Charaeterisation of the Promoter
for the LexA Regulated sulA Gene of <i>Escherichia coli.</i> <i>Mol. Gen. Genet.</i>, 189: 400: 404 <br />
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1. Cole, S.T.(1983) Characterization of the Promoter
for the LexA Regulated sulA Gene of <i>Escherichia coli.</i> <i>Mol. Gen. Genet.</i>, 189: 400: 404 <br />
−
2. Wang, X., Chen, X. & Yang, Y.(2012) spatiotemporal control of gene expression
by a light-switchable transgene system. <i>Nat. Methods</i>, 9: 266: 269<br />
+
2. Wang, X., Chen, X. & Yang, Y.(2012) Spatiotemporal control of gene expression
by a light-switchable transgene system. <i>Nat. Methods</i>, 9: 266: 269<br />
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3. Zhang, A.P.P., Pigli, Y.Z & Rice, P.A.(2010) Structure of the LexA–DNA complex and implications for SOS box measurement.<i>Nature</i>, 466: 883: 886 <br />
+
3. Zhang, A.P.P., Pigli, Y.Z & Rice, P.A.(2010) Structure of the LexA–DNA complex and implications for SOS box measurement. <i>Nature</i>, 466: 883: 886 <br />
 
4. Butalaa, M., Zgur-Bertokb, D., and Busby, S. J. W.(2009) The bacterial LexA transcriptional repressor. <i>Cell. Mol. Life Sci.</i>, 66: 82: 93<br />
 
4. Butalaa, M., Zgur-Bertokb, D., and Busby, S. J. W.(2009) The bacterial LexA transcriptional repressor. <i>Cell. Mol. Life Sci.</i>, 66: 82: 93<br />
 
5. Shimizu-Sato, S., Huq, E., Tepperman, J.M., & Quail, P.H.(2002). A light-switchable gene promoter system. <i>Nat. Biotechnol.</i> 20: 1041: 1044<br />
 
5. Shimizu-Sato, S., Huq, E., Tepperman, J.M., & Quail, P.H.(2002). A light-switchable gene promoter system. <i>Nat. Biotechnol.</i> 20: 1041: 1044<br />
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===Usage and Biology===
 
===Usage and Biology===
  
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K819006 SequenceAndFeatures</partinfo>
 
  
  

Latest revision as of 07:56, 18 October 2012

Testing device for Luminesensor

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 839
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 839
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 839
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 839
  • 1000
    INCOMPATIBLE 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