2. 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

3. 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

4. The AND Gate Input A- Promoter which controls the Transcription of T7 RNA Polymerase (mod - 2 amber STOP codons) Input B- Promoter which controls the amber suppressor tRNA supD Output - T7 RNA is synthesized and activates T7 promoter

5. AND Gate to Translate T7 RNA Polymerase Resulting in GFP Expression

6. Common Genetic Circuit Problem - Impedance Matching Construct 1: GFP was produced in the absence of salicate. Rbs for P BAD was strong enough to produce T7 poly Input T7 Poly outputGFP

7. Saturation Mutation Library for P BAD Parental GGAGGAATTAACCATG LibrarybNNNGGAATTAACCRTG B9CTAGGAATTAACCGTG F11AAAGGAATTAACCGTG MgrBAAAGGAATTAACCATG SalcTCAGGAGTCATCATTATTTATG

8. B9 Characterization through Fluorimetry and Flow Cytommetry A.Visualised 64 combinations of inducers through Fluorimetry B.1000 fold induction between on and off states.

9. 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 P BAD and P sal

10. 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

11. B9 Transfer Function Data Fit to determine a and b a = 50 b = 3000

12. Range of Output Possibilities B9 and F11 visualization of input strength to output Demonstrates the entire range of possibilities. –Movement of the white box determined via rbs strength

13. AND Gate Modularity

14. 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

15. 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.

16. 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

17. Going Farther. Beijing iGEM 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