Synthetic killer circuits in bacteria

Synthetic killer circuits in bacteria Lingchong You Duke University www.duke.edu/~you DAC 44@ San Diego 2007.06.07

Better living through bacteria Signals • drug production • targeted cell killing actuator sensor containment module bacterium Baker, Nat. Biotech. 2005, 23:645

Design strategy:Coordinating cell killing by communication Prototypes • Population controller • Predator-prey system Goal: Precise control of bacterial dynamics – growth, death, and aggregation

United we shine: quorum sensing in bacterium Vibrio fischeri V. fischeri Function: light production at high density

A population controller AHL I R R luxR luxI ccdB PluxI CcdB You, Cox, Weiss, & Arnold. Nature (2004)

A population-level negative feedback Cells (N) E A • Steady-state density control • Sustained oscillations

OFF Typical circuit dynamics ON • Population behavior • Stable regulation • Captured by simulation • Mutants arose after ~100 hrs ON OFF Cell density by serial dilution + plating

Population control in a microchemostat • Miniaturized (102-104 cells) • Automated • Single-cell resolution Balagadde*, You*, Hansen, Arnold, & Quake, Science 2005

Monitoring dynamics with single-cell resolution

Long term monitoring of circuit dynamics Top10F’ cells; buffered LBK (pH=7.0); 32C

From autonomous population control to synthetic ecosystems N1 N2 N Engineered communications coupled with survival • Goals • Developing the strategy for program sophisticated dynamics • Fundamental insights into complex ecological dynamics • Predation • Synergism • Competition • …

A predator-prey system predator B A plac lasI luxR ccdA ccdB LasI LuxR 3OC12HSL 3OC6HSL prey LasR LuxI ccdB lasR luxI B

Bifurcation analysis Maximum of oscillation Stable steady state Unstable steady Hopf bifurcation point Oscillations! Predator Predator Minimum of oscillation Prey Prey

Microchemostat • Reduce population size to stabilize the circuits • New version • 14 reactors • fluorescence Total - Prey  Predator culture Green channel  prey Phase channel  total F. Balagadde

Predator-prey oscillations prey predator Cells per screen hours Frederick Balagadde

Summary I Communication coupled with cell killing enables robust control of bacterial population dynamics

ePop: a bacterial popping circuit • At high levels, E protein blocks cell wall synthesis  cell popping AHL I R R luxR luxI e PluxI E Lysis protein from phage X174 Philippe Marguet & Eric Spitz

Synchronized killing in small populations

Sustained oscillations in macroscopic batch cultures induced Un-induced induced induced

Why oscillations? We thought: • Cell-cell communication via LuxR/I leads to negative feedback control of cell density cells E AHL

…oscillates even without the QS module!! PluxI E ? E Thus cells were unable to produce or sense AHL

Oscillations via hidden interactions ? X X E E X X X PluxI X X: a diffusible factor that accumulates at high density and induces expression of the E protein

rpoS sdiA cAMP CRP indole E E PluxI sdiA, rpoS, and CRP have been shown to interact with PluxI

Lessons Plux activity • Hidden interactions complicates gene circuit engineering. • Matching gates • Only observed for E protein • pluxGFP doesn’t generate response without active luxR • Gene circuits as probes of cell physiology density Strong response No response E GFP Plux activity Plux activity

Thanks! Collaborators You Lab Postdoc Hao Song Graduate students Tae Jun Lee Philippe Marguet Anand Pai Chee Meng Tan Yu Tanouchi Undergraduate students Meagan Gray Maher Salahi Cameron Smith Eric Spitz David Wang Former members Dennis Tu Faisal Reza Peter Blais Jun Ozaki • Duke • Kam Leong • Joseph Nevins • Guang Yao • George Truskey • Mike West • Jarad Niemi • Fan Yuan • Elsewhere • Frances Arnold (Caltech) • Mat Barnet • Cynthia Collins • Sidney Cox • Frederick Blattner (Scarab/UW) • Stephen Quake (Stanford) • Frederick Balagadde • Ron Weiss (Princeton) • Funding • KECK Futures Initiative • NIH • NSF • Packard Foundation

Synthetic killer circuits in bacteria