1. Addressable Conjugation in Bacterial Networks High School Matt Fleming Kaitlin A. Davis Undergrads Bryan HernandezFreshman Jennifer LuFreshman Samantha LiangFreshman Will BosworthSophomore Daniel KluesingJunior Postdocs John E. Dueber J. Christopher Anderson Faculty Advisors Adam P. Arkin Jay D. Keasling The University of California, Berkeley

3. Every Cell is assigned a Unique RNA Address UGAUCUAAGUGGAGGACGAA GUUCAAGACAAUCACUCACG UCAAUAUAAAACUACAAACU GAUGAUAGAGGUUUCUUUUA Cell 1 Cell 2 Cell 3 Cell 4

4. Implementation Need: To transfer DNA messages from one bacterial cell to another Means: Bacterial Conjugation Need: To specifically control who can read the DNA message Means: Riboregulation

5. Our specific goals: Construct high-performance riboregulators Harness bacterial conjugation Transmit a coded message Construct a bacterial learning network

6. The Riboregulator: DNA Promoter RBS RFP Double T RNA Polymerase

7. Locked Riboregulator: RNA Ribosome Locked RBS RFP

8. Unlocked Riboregulator Ribosome Unlocked Revealed RFP Lock RBS + Key

9. The Locks

10. Mismatches are not necessary for Unlocking Secondary structure is critical for activity

11. Shorter loops unlock better Transcriptional terminators but not open reading frames improve activity

12. Bacterial Conjugation

13. R System x OriTr Transfer of Message Plasmids F System

14. Cell Sends a Coded Message to Recipient Cell

15. Conjugation Does Not Impede Riboregulator Function Conjugation and Riboregulators Riboregulators Don't Effect Conjugation Efficiency

16. NAND Logic Gates

17. Building Computational Networks Graded Response Inhibitory Signals Stimulatory Signals Parallel Signals

18. Concentration in Culture gives Graded Responses enrichment

19. Trainable Bacterial Networks

20. Acknowledgements Arkin and Keasling Labs Funding from Microsoft LBL EHS Jonathon Goler, Melissa Li

21. Nodes are Built from Multiple Cell Types

22. Addressable Networks