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:

• Identifies environment with high specificity by integrating multiple input

INPUT 1 INPUT 2 OUTPUT

0 0 0

0 1 0

1 0 0

1 1 1

A Modular Genetic AND Logic Gate

The Ribosome Binding Site Problem

• High basal expression of T7ptag  (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+ Arabinose+/Salicylate-

Arabinose+/Salicylate+

Circuit Output Characterization for B9

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

Input 2 Output

2D Transfer Function

Individual input promoter transfer functions

Parent rbsF11 rbsB9 rbs

Fitting experimental data to model

a=50±20 and b=3000±1000

• For any range of I1 , it is possible to generate a functional AND gate

Models yields greater insight into AND gate requirements

Circuit Modularity: Demonstration 1

Original B9 rbs (weaker) No fluorescence

Stronger rbs Functional AND Gate

15 Fold InductionFunctional AND Gate

Circuit Modularity: Demonstration 2

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

TAG codon codes for Release Factor 1

Steitz. (2008) Nature Reviews Molecular Cell Biology 9, 242-253

Circuit influence on cell growth

0.0

0.5

1.0

1.5

2.0

0 1 2 3 4Time (hrs)

OD

600

0.0

0.5

1.0

1.5

2.0

0 1 2 3 4Time (hrs)

OD

600

Wildtype MC1061Cells with circuit, OFF stateCells with circuit, ON state

Construct Maps

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