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

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

Flow Cytometer (BD FcsCalibur) Reporter: GFP

lacI -> lacR -> Ptrc -> lcI -> lcI -> PL

lacI -> lacR -> Ptrc -> lcI -> lcI -> PL

MMC – 15 h UV – 1-10 s

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

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

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

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

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

Something to think about…

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

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