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  1. CARD Computer-Aided Rational Design of the phosphotransferase system for enhanced glucose uptake in Escherichia coli. http://www.cadlive.jp

  2. Objectives Propose CAD-based rational design of a biochemical network for an engineering purpose cell Substrate Product Substrate

  3. Computer simulation Biological experiment Network design CADLIVE System Concept of CAD

  4. Robustness Perturbation Parameter Time Biological systems maintain their homeostasis against environmental stress, genetic changes and noises. Feedback Feedforward Pathway redundancy How do you change such a robust system?

  5. Methods

  6. Architecture check Mathematical check Design strategy for rational design of biochemical networks

  7. Modular decomposition Define an engineering purpose. Function of sub-networks is assigned in analogous to control engineering architecture Check our paper: Heat shock response PLoS Comp Biol, 2006 PNAS, 2005

  8. Perturbation analysis for finding critical genes Cell substrate Product vs vp E: enzyme

  9. Results and Discussion

  10. Plant FB Sensor Accelerator Actuator Computer Brake Actuator Glucose PTS network map

  11. Plant FB Sensor Accelerator Actuator Computer Brake Actuator Accelerator Flux Module Brake Flux Module

  12. cAMP IIA-P IIA ptsI ptsH ptsG Dynamic simulation reproduces the experimental behaviors Glucose depletion on 500 min

  13. Model validation by experiments cAMP Experimental data are reproduced by our dynamic model IIA-P Mlc knockout ptsG

  14. Plant FB Sensor Accelerator Actuator Computer Brake Actuator Critical genes are explored for enhanced glucose uptake PTS proteins Accelerator Flux Module crp mlc Brake Flux Module

  15. Recombinant strategy • Brake flux module Negative feedback DELETE • Accelerator flux module Positive feedbackENHANCE

  16. PERTURBATION ANALYSIS (SIMULATION) Prediction of changes in the specific glucose uptake rate for mathematical mutants. The values are the ratios of the specific glucose uptake rate for a mutant to that for wild type.

  17. Dynamic simulations PtsI overexpression is effective for enhanced PLANT and increases cAMP

  18. EXPERIMENT Enhanced specific glucose uptake by ptsI overexpression in an mlc knockout mutant as predicted.

  19. PERTURBATION EXPERIMENT Experimental results of growth, glucose uptake, specific glucose uptake, and cAMP concentration in growing cells. 11.08 (prediction)

  20. MODEL IMPROVEMENT FB Sensor Plant Accelerator Actuator Computer Brake Actuator

  21. PERTURBATION ANALYSIS (SIMULATION) In the improved model

  22. Conclusion A computer-aided rational design approach was successfully applied to the Escherichia coli glucose PTS to increase the specific glucose uptake rate. The proposed method predicted that the mlc knockout mutant with ptsI gene overexpression greatly increases the specific glucose uptake rate and verified it by biological experiments.