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 • 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: INPUT 1 INPUT 2 OUTPUT 0 0 1 1 1 • Identifies environment with high specificity by integrating multiple input

A Modular Genetic AND Logic Gate

The Ribosome Binding Site Problem • High basal expression of T 7 ptag (under control PBAD) and leaky promoter (Psal) o Compensated by weakening the ribosome binding site upstream of PBAD

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

Circuit Output Characterization for B 9

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 2 D Transfer Function

Individual input promoter transfer functions Parent rbs F 11 rbs B 9 rbs

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

Models yields greater insight into AND gate requirements • For any range of I 1 , it is possible to generate a functional AND gate

Circuit Modularity: Demonstration 1 Original B 9 rbs (weaker) No fluorescence 15 Fold Induction Functional AND Gate Stronger rbs Functional AND Gate

Circuit Modularity: Demonstration 2

Summary • Pitfalls – – – Lacks Scalability (T 7 polymerase or Sup. D 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

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

Circuit influence on cell growth Wildtype MC 1061 Cells with circuit, OFF state Cells with circuit, ON state

Construct Maps

(A) Wildtype MC 1061 (B) MC 1061 with circuit, OFF state (C) MC 1061 with circuit, ON state