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Synthetic killer circuits in bacteria. Lingchong You Duke University www.duke.edu/~you. DAC 44@ San Diego2007.06.07. Better living through bacteria. Signals. drug production targeted cell killing. actuator. sensor. containment module. bacterium.

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Synthetic killer circuits in bacteria l.jpg

Synthetic killer circuits in bacteria

Lingchong You

Duke University

www.duke.edu/~you

DAC 44@ San Diego2007.06.07


Better living through bacteria l.jpg

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 l.jpg

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 l.jpg

United we shine: quorum sensing in bacterium Vibrio fischeri

V. fischeri

Function: light production at high density


A population controller l.jpg

A population controller

AHL

I

R

R

luxR

luxI

ccdB

PluxI

CcdB

You, Cox, Weiss, & Arnold. Nature (2004)


A population level negative feedback l.jpg

A population-level negative feedback

Cells (N)

E

A

  • Steady-state density control

  • Sustained oscillations


Slide8 l.jpg

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 l.jpg

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 l.jpg

Monitoring dynamics with single-cell resolution


Long term monitoring of circuit dynamics l.jpg

Long term monitoring of circuit dynamics

Top10F’ cells; buffered LBK (pH=7.0); 32C


From autonomous population control to synthetic ecosystems l.jpg

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 l.jpg

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 l.jpg

Bifurcation analysis

Maximum of oscillation

Stable steady state

Unstable steady

Hopf bifurcation point

Oscillations!

Predator

Predator

Minimum of oscillation

Prey

Prey


Slide15 l.jpg

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 l.jpg

Predator-prey oscillations

prey

predator

Cells per screen

hours

Frederick Balagadde


Summary i l.jpg

Summary I

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


Epop a bacterial popping circuit l.jpg

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


Slide19 l.jpg

Synchronized killing in small populations


Slide20 l.jpg

Sustained oscillations

in macroscopic batch cultures

induced

Un-induced

induced

induced


Why oscillations l.jpg

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 l.jpg

…oscillates even without the QS module!!

PluxI

E

?

E

Thus cells were unable to produce or sense AHL


Oscillations via hidden interactions l.jpg

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


Slide24 l.jpg

rpoS

sdiA

cAMP

CRP

indole

E

E

PluxI

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


Lessons l.jpg

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


Slide26 l.jpg

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


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