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Environmental signal integration by a modular AND gate

Environmental signal integration by a modular AND gate. J Christopher Anderson, Christopher A Voigt and Adam P Arkin. Presented by: Pei-Ann Lin and Jessica Perez. Genetic Circuit Design. Coupling environmental signal(s) to new functions

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Environmental signal integration by a modular AND gate

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  1. Environmental signal integration by a modular AND gate J Christopher Anderson, Christopher A Voigt and Adam P Arkin Presented by: Pei-Ann Lin and Jessica Perez

  2. Genetic Circuit Design • Coupling environmental signal(s) to new functions • Harnesses the amazing power of cells to integrate complex signals into reliable output • Transcription factors and other regulatory elements • Modularity has been a common challenge in these systems • AND Logic Gate: •  Identifies environment with high specificity by integrating multiple input

  3. A Modular Genetic AND Logic Gate

  4. High basal expression of T7ptag  (under control PBAD) and leaky promoter (Psal) •  Compensated by weakening the ribosome binding site upstream of PBAD The Ribosome Binding Site Problem

  5. Arabinose-/Salicylate- Arabinose-/Salicylate+ Arabinose+/Salicylate- Arabinose+/Salicylate+ Mutagenized rbs clone selection

  6. Circuit Output Characterization for B9

  7. The Transfer Function Model • Mathematical relation between system’s input and output • Helps us understand how input affects function of circuit • Assumes steady state in deterministic model, linear relationship between fluorescence and y, a, b Input 1 Output Input 2 2D Transfer Function

  8. Individual input promoter transfer functions Parent rbs F11 rbs B9 rbs

  9. Fitting experimental data to model a=50±20 and b=3000±1000

  10. Models yields greater insight into AND gate requirements • For any range of I1 , it is possible to generate a functional AND gate

  11. Circuit Modularity: Demonstration 1 Stronger rbs Functional AND Gate Original B9 rbs (weaker) No fluorescence 15 Fold Induction Functional AND Gate

  12. Circuit Modularity: Demonstration 2

  13. Summary • Pitfalls • Lacks Scalability (T7 polymerase or SupD cannot be reused) • Not totally orthogonal design • Logic gate behavior is density dependent • High DNA load for each gate • Parametizing modular examples • True modularity? • Significance • Integrating multiple signal to achieve sensing specificity • AND gate critical for creating genetic programs • Parametizing logic gate transfer function can be used to predict different promoters that can be connected to the AND gate

  14. TAG codon codes for Release Factor 1 Steitz. (2008) Nature Reviews Molecular Cell Biology 9, 242-253

  15. Circuit influence on cell growth Wildtype MC1061 Cells with circuit, OFF state Cells with circuit, ON state

  16. Construct Maps

  17. (A) Wildtype MC1061 (B) MC1061 with circuit, OFF state (C) MC1061 with circuit, ON state

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