Team:Peking/Modeling/Luminesensor/Orthogonality

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Our <i>Luminesensor</i> is expected to be orthogonal to endogenous SOS pathway. In order to remove this obstacle on the application prospects of our <i>Luminesensor</i>, we used LexA408 instead of wild-type LexA DNA Binding domain. LexA408 and LexA are bio-orthogonal with each other since the sequence of the binding sites have variations (See <a href="/Team:Peking/Project/Luminesensor/Characterization" title="">Characterization</a>).
Our <i>Luminesensor</i> is expected to be orthogonal to endogenous SOS pathway. In order to remove this obstacle on the application prospects of our <i>Luminesensor</i>, we used LexA408 instead of wild-type LexA DNA Binding domain. LexA408 and LexA are bio-orthogonal with each other since the sequence of the binding sites have variations (See <a href="/Team:Peking/Project/Luminesensor/Characterization" title="">Characterization</a>).
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  <img src="..." alt="Figure 10 <i>Luminesensor</i> LexA408-VVD" style="width:500px;"/>
 
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  <p class="description">Figure 10. Moecular Modeling for <i>Luminesensor</i> LexA408-VVD.</p>
 
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By adding several nodes into the network, we constructed modeling for orthogonality <i>in silica</i> simulation:
By adding several nodes into the network, we constructed modeling for orthogonality <i>in silica</i> simulation:
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   <img src="/wiki/images/2/2c/Peking2012_LuminesensorOrthogonal.png" alt="Figure 11" style="width:500px;"/>
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   <img src="/wiki/images/2/2c/Peking2012_LuminesensorOrthogonal.png" alt="Figure 10" style="width:500px;"/>
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   <p class="description">Figure 11. Kinetic Network for Orthogonal Analysis</p>
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   <p class="description">Figure 10. Kinetic Network for Orthogonal Analysis</p>
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   <img src="/wiki/images/7/7b/Peking2012_LuminesensorOrthogonalEffect.png" alt="Figure 12" />
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   <img src="/wiki/images/7/7b/Peking2012_LuminesensorOrthogonalEffect.png" alt="Figure 11" />
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   <p class="description">Figure 12. Orthogonality Simulation Result.
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   <p class="description">Figure 11. Orthogonality Simulation Result.
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Revision as of 15:44, 26 September 2012

Orthogonal Test in silico

Our Luminesensor is expected to be orthogonal to endogenous SOS pathway. In order to remove this obstacle on the application prospects of our Luminesensor, we used LexA408 instead of wild-type LexA DNA Binding domain. LexA408 and LexA are bio-orthogonal with each other since the sequence of the binding sites have variations (See Characterization).
By adding several nodes into the network, we constructed modeling for orthogonality in silica simulation:

Figure 10

Figure 10. Kinetic Network for Orthogonal Analysis

where

  • L denotes Luminesensor
  • I denotes the inner wild LexA
  • DL denotes the specific DNA binding site to Luminesensor
  • DI denotes the specific DNA binding site to wild LexA

The parameters are estimated as following:

ParameterValueUnitDescription
k61.x10-4s-1dimered LexA releasing rate constant from non-specific binding site
K61.x10-2(n mol/L)-1dimered non-specific binding equilibrium constant

Table 2. Reaction Parameters for Orthogonality Simulation

Figure 11

Figure 11. Orthogonality Simulation Result.

The result shows that the contrast is highly related to the orthogonality. As our Luminesensor is orthogonal to the endogerous LexA system, our system still works well in bacteria with endogenously expressed LexA (See Characterization).

Reference

  • 1. Zoltowski, B.D., Crane, B.R.(2008). Light Activation of the LOV Protein Vivid Generates a Rapidly Exchanging Dimer. Biochemistry, 47: 7012: 7019
  • 2. Mohana-Borges, R., Pacheco, A.B., Sousa, F.J., Foguel, D., Almeida, D.F., and Silva, J.L. (2000). LexA repressor forms stable dimers in solution. The role of specific DNA in tightening protein-protein interactions. J. Biol. Chem., 275: 4708: 4712
  • 3. Zoltowski, B.D., Vaccaro, B., and Crane, B.R. (2009). Mechanism-based tuning of a LOV domain photoreceptor. Nat. Chem. Biol. 5: 827: 834
  • 4. Dmitrova, M., Younes-Cauet, G., Oertel-Buchheit, P., Porte, D., Schnarr, M., Granger-Schnarr, M.(1998) A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli. Mol. Gen. Genet., 257: 205: 212
  • Totop Totop