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 INPUT 1INPUT 2OUTPUT 000 010 100 111

3. A Modular Genetic AND Logic Gate

4. The Ribosome Binding Site Problem High basal expression of T7ptag (under control P BAD ) and leaky promoter (P sal ) o Compensated by weakening the ribosome binding site upstream of P BAD

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

6. Circuit Output Characterization for B9

7. The Transfer Function Model Mathematical relation between systems 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 Input 2 Output 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. For any range of I 1, it is possible to generate a functional AND gate Models yields greater insight into AND gate requirements

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