Team:Peking/Modeling/Luminesensor/Orthogonality

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Orthogonal Test in silico

To modularize the genetic system, our Luminesensor is expected to be bio-orthogonal with the origin system in bacteria. 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).

By adding several nodes into the network, we constructed modeling for orthogonality test:

Fig 5. 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

Tab 2. Reaction Parameters for Orthogonal Test

Fig 6. 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).

Conclusion

The modeling above points out a way to optimize our Luminesensor -- the two critical mutation. It also shows the system still works well even if considering noise and endogerous competition.

Referrence

  • [1] Light Activation of the LOV Protein Vivid Generates a Rapidly Exchanging Dimer. B. D. Zoltowski etc. Biochemistry
  • [2] LexA Repressor Forms Stable Dimers in Solution. R. Mohana-Borges etc. THE JOURNAL OF BIOLOGICAL CHEMISTRY
  • [3] Mechanism-based tuning of a LOV domain photoreceptor, Brian D. Zoltowski, etc. NATURE CHEMICAL BIOLOGY
  • [4] Protein Vivid Generates a Rapidly Exchanging Dimer, Brian D. Zoltowski, etc. BIOCHEMISTRY
  • [5] A new LexA-based genetic system for monitoring and analyzing protein heterodimerization in Escherichia coli, M. Dmitrova. etc. Springer-Verlag Mol Gen Genet
  • Totop Totop