Transcript Input/output circuits

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
R
R
PluxI
luxR
ccdB
I
luxI
CcdB
You, Cox, Weiss, & Arnold. Nature (2004)
A population-level negative feedback
dN
 kN (1  N / N m )  dEN
dt
Cells (N)
E
dE
 kE A  d E E
dt
A
dA
 vAN  d A A
dt
•Steady-state density control
•Sustained oscillations
OFF
Typical circuit
dynamics
ON
1. Population behavior
2. Stable regulation
3. Captured by
simulation
4. Mutants arose after
~100 hrs
Cell density by serial dilution + plating
ON
OFF
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
N
Goals
1) Developing the strategy for
program sophisticated
dynamics
2) Fundamental insights into
complex ecological
dynamics
N2
Engineered
communications
coupled with survival
• Predation
• Synergism
• Competition
•…
A predator-prey system
predator
A
B
plac
lasI luxR
ccdA
ccdB
LasI
LuxR
3OC12HSL
3OC6HSL
prey
LasR
LuxI
ccdB
B
lasR
luxI
Bifurcation analysis
Maximum of oscillation
1.E+02
Stable steady state
1.E+01
Oscillations!
Predator
Predator [x107 CFU]
Unstable steady
Hopf bifurcation point
1.E+00
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.5
Predator
1.E-01
Minimum of oscillation
Prey
1.4
1.E-02
Growth rate of the prey [hr-1]
Prey
Microchemostat
• Reduce population size
to stabilize the circuits
• New version
– 14 reactors
– fluorescence
Total - Prey
 Predator
culture
Phase channel
 total
Green channel
 prey
F. Balagadde
Predator-prey oscillations
Cells per screen
140
prey
120
100
predator
80
60
40
20
0
0
50
100
150
200
250
hours
Frederick Balagadde
Summary I
Communication coupled with
cell killing enables robust control
of bacterial population dynamics
ePop: a bacterial popping circuit
AHL
PluxI
I
R
R
luxR
e
• At high levels, E
protein blocks cell
wall synthesis 
cell popping
luxI
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
E
cells
AHL
…oscillates even without the QS module!!
0.20
PluxI
E
?
Abs
0.15
0.10
E
0.05
10
20
30
hours
Thus cells were unable to produce or
sense AHL
40
Oscillations via hidden interactions
?
E
PluxI
X X
X X X
X
E
X: a diffusible factor that accumulates at high density
and induces expression of the E protein
rpoS
sdiA
CRP
indole
cAMP
E
PluxI
E
sdiA, rpoS, and CRP have been shown to interact with PluxI
Lessons
• Hidden interactions
complicates gene circuit
engineering.
Plux
activity
density
• Matching gates
– Only observed for E
protein
– pluxGFP doesn’t
generate response
without active luxR
• Gene circuits as probes
of cell physiology
Strong
response
E
Plux
activity
No
response
GFP
Plux
activity
You Lab
Postdoc
Hao Song
Thanks!
Graduate students
Tae Jun Lee
Philippe Marguet
Anand Pai
Chee Meng Tan
Yu Tanouchi
Elsewhere
Frances Arnold (Caltech)
Mat Barnet
Cynthia Collins
Sidney Cox
Frederick Blattner (Scarab/UW)
Stephen Quake (Stanford)
Frederick Balagadde
Ron Weiss (Princeton)
Undergraduate students
Meagan Gray
Maher Salahi
Cameron Smith
Eric Spitz
David Wang
Former members
Dennis Tu
Faisal Reza
Peter Blais
Jun Ozaki
Collaborators
Duke
Kam Leong
Joseph Nevins
Guang Yao
George Truskey
Mike West
Jarad Niemi
Fan Yuan
Funding
KECK Futures Initiative
NIH
NSF
Packard Foundation