Environmental Signal Integration by a Modular AND Gate
Environmental Signal Integration by a Modular AND Gate By J Christopher Anderson, Christopher A Voigt and Adam P Arkin Presented by Alexandra Doolittle for 20. 385 02. 16. 2010
Hypothesis • AND Gates can be used to integrate environmental information • The AND gate is modular since promoters comprise both the inputs and output of the system
The AND Gate Input A- Promoter which controls the Transcription of T 7 RNA Polymerase (mod - 2 amber STOP codons) Input B- Promoter which controls the amber suppressor t. RNA sup. D Output - T 7 RNA is synthesized and activates T 7 promoter
AND Gate to Translate T 7 RNA Polymerase Resulting in GFP Expression
Common Genetic Circuit Problem - Impedance Matching output GFP Input T 7 Poly Construct 1: GFP was produced in the absence of salicate. Rbs for PBAD was strong enough to produce T 7 poly
Saturation Mutation Library for PBAD • • • Parental Libraryb B 9 F 11 Mgr. B Salc GGAGGAATTAACCATG NNNGGAATTAACCRTG CTAGGAATTAACCGTG AAAGGAATTAACCATG TCAGGAGTCATCATTATG
B 9 Characterization through Fluorimetry and Flow Cytommetry A. Visualised 64 combinations of inducers through Fluorimetry B. 1000 fold induction between on and off states.
Transfer Function • steady state response of the system as a function of the activity of the input promoters – Can understand how the input promoters affect the function – Black Box Model: levels of input output • I 1 and I 2 represent PBAD and Psal
Transfer Function Characterization for Each Promoter • Currently - use mutation and screening to determine the best rbs strength • Now- use the transfer function to determine the strength of rbs needed
B 9 Transfer Function Data Fit to determine a and b • a = 50 • b = 3000
Range of Output Possibilities • B 9 and F 11 visualization of input strength to output • Demonstrates the entire range of possibilities. – Movement of the white box determined via rbs strength
AND Gate Modularity
Critique • Transfer Function - based on steady state. Fails to take into account dynamic system responses. – Black Box model to simple. • Cell characteristics could affect the response – Nutrient levels – Temperature – Phase of Growth
Why Do We Care? • Integrating multiple signals from the environment can increase sensing specificity. • Can identify new environments where there is not a strong recognizable single symbol. • The modularity allows the circuit to be attached to multiple inputs and outputs.
Why Do We Care? • Transfer function can be used to predict strength levels of the inputs to produce a functional system. • Other potential Regulators: – Nonsense, missense and frameshift regulators – riboregulators
Going Farther. • Beijing i. GEM 2009 - Used the and gate to create conditional memory in ecoli • Biosensors - respond to changes in the environment. • A stop growth switch in cells when certain nutrients are lacking
- Slides: 16