Programmable cells: Interfacing natural and engineered gene networks

1. Programmable cells: Interfacing natural and engineered gene networks Hideki Kobayashi, Mads Kærn, Michihiro Araki, Kristy Chung, Timothy S. Gardner, Charles R. Cantor, and James J. Collins

2. Scope • create novel cellular behaviors and characteristics by coupling engineered gene networks to the cell’s natural regulatory circuitry • Four examples • Detects and retains memory of DNA damage • Forms biofilm in response to DNA damage • Detects and retains memory of quorum sensing molecules • Density dependent protein synthesis

4. Reporter: GFP Flow Cytometer (BD FcsCalibur)

7. lacI -> lacR -> Ptrc -> lcI -> lcI-> PL

8. lacI -> lacR -> Ptrc -> lcI -> lcI-> PL

9. MMC – 15 h UV – 1-10 s

10. (With traA gene) (lacking the traA gene) Replace gfp with traA Biofilm was only observed if traA was expressed for > 4h

11. acylated-homoserine lactone (AHL) LuxI LuxR luxR luxICDABE LuxI-LuxR quorum-sensing systems

12. Quorum sensing molecules lacI -> lacR -> Ptrc -> lcI -> lcI-> PL

13. GFP Density-dependent gene activation Toggle lacI -> lacR -> Ptrc -> lcI -> lcI-> PL

15. Discussions • Programmable cells have been constructed by interfacing natural and engineered gene networks • Programmable cells have been demonstrated using four different constructs with the toggle gene network as a building block

16. Something to think about…

17. b) DNA damage sensing Original design Modified design Will the modified design work? If not, why not? If yes, how would it differ from (a)?

18. Q: What if we replace the lacI gene with gfp and forget about the regulatory circuit Density-dependent gene activation gfp