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Team:Peking/Modeling/Luminesensor/Orthogonality
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<h3 id="title1">Orthogonal Test <i>in silico</i></h3> | <h3 id="title1">Orthogonal Test <i>in silico</i></h3> | ||
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| - | + | Our <i>Luminesensor</i> is expected to be orthogonal to endogenous SOS pathway. LexA, a natural element from the lactin-SOS system in bacteria may cause unexpected crosstalk. In order to remove this obstacle on the application prospects of our <i>Luminesensor</i>, we use LexA408 instead of the wild-type LexA. 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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</p> | </p> | ||
Revision as of 15:38, 26 September 2012
Orthogonal Test in silico
Our Luminesensor is expected to be orthogonal to endogenous SOS pathway. LexA, a natural element from the lactin-SOS system in bacteria may cause unexpected crosstalk. In order to remove this obstacle on the application prospects of our Luminesensor, we use LexA408 instead of the wild-type LexA. LexA408 and LexA are bio-orthogonal with each other since the sequence of the binding sites have variations (See Characterization).
Figure 10. Moecular Modeling for Luminesensor LexA408-VVD.
By adding several nodes into the network, we constructed modeling for orthogonality test:
Figure 11. 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:
| Parameter | Value | Unit | Description |
| k6 | 1.x10-4 | s-1 | dimered LexA releasing rate constant from non-specific binding site |
| K6 | 1.x10-2 | (n mol/L)-1 | dimered non-specific binding equilibrium constant |
Tab 2. Reaction Parameters for Orthogonal Test
Figure 12. Orthogonal Test 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 endogenous 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
