Cell-Cell Communications Ron Weiss Department of Electrical - - PDF document

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Cell-Cell Communications

Ron Weiss Department of Electrical Engineering Princeton University

Computing Beyond Silicon Summer School, Caltech, Summer 2002

• E. coli

Diffusing signal

Programming Cell Communities

proteins

Program cells to perform various tasks using:

• Intra-cellular circuits

– Digital & analog components

• Inter-cellular communication

– Control outgoing signals, process incoming signals

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Intercellular Communications

• Certain inducers useful for communications:
• 1. A cell produces inducer
• 2. Inducer diffuses outside the cell
• 3. Inducer enters another cell
• 4. Inducer interacts with repressor/activator change signal

(1) (2) (3) (4)

main metabolism

The Intercellular AND Gate

• Inducers can activate activators:

– VAI (3-N-oxohexanoyl

• L-Homoserine lacton) luxR
• Use as a logical AND gate:
• perator

promoter gene RNAP inactive activator

• perator

promoter gene RNAP active activator inducer no transcription transcription Output Activator Inducer Output 1 1 1 1 1 Activator Inducer

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Communications Simulator

agar cells

Ai,j,k Ai+1,j,k Ai-1,j,k Ai,j+1,k Ai,j-1,k

3D diffusion: dAi,j,k/dt = kdiff (Ai,j-1,k + Ai,j+1,k + Ai-1,j,k + Ai+1,j,k + Ai,j,k+1+ Ai,j,k-1 - 6Ai,j-1,k) Cells sit on 3D agar grid Model genetic networks in cells (ODE, stochastic) ODE diffusion model with reflective boundaries

Two Cell Simulation

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Light organ

Eupryma scolopes Quorum Sensing

• Cell density dependent gene expression

Example: Vibrio fischeri [density dependent bioluminscence]

The lux Operon LuxI metabolism autoinducer (VAI)

luxR luxI luxC luxD luxA luxB luxE luxG LuxR LuxI

(Light) hv (Light) hv Luciferase Luciferase

P P Regulatory Genes Structural Genes

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The lux box Low and High Cell Densities

free living, 10 cells/liter <0.8 photons/second/cell symbiotic, 1010 cells/liter 800 photons/second/cell

luxR luxI luxC luxD luxA luxB luxE luxG LuxR LuxI P P

Low Cell Density Low Cell Density

luxR luxI luxC luxD luxA luxB luxE luxG LuxR LuxI

(Light) hv (Light) hv

Luciferase Luciferase

P P

High Cell Density High Cell Density

LuxR

O O O O N H O O O O N H O O O O N H O O O O N H

LuxR (+)

O O O O N H O O O O N H O O O O N H O O O O N H O O O O N H O O O O N H O O O O N H O O O O N H O O O O N H O O O O N H O O O O N H O O O O N H

Acyl-HSL

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• P. Aeruginosa
• P. Aeruginosa
• Two autoinducer

systems regulate virulence/biofilm formation

• Secrete virulence

factors when population high enough to

• vercome host defenses

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Sources for a Library of Signals

N-acyl-L-Homoserine Lactone Autoinducers in Bacteria

Species Relation to Host Regulate Production of I Gene R Gene Vibrio fischeri marine symbiont Bioluminescence luxI luxR Vibrio harveyi marine symbiont Bioluminescence luxL,M luxN,P,Q Pseudomonas aeruginosa Human pathogen Virulence factors lasI lasR Rhamnolipids rhlI rhlR Yersinia enterocolitica Human pathogen ? yenI yenR Chromobacterium violaceum Human pathogen Violaceumproduction Hemolysin Exoprotease cviI cviR Enterobacter agglomerans Human pathogen ? eagI ? Agrobacterium tumefaciens Plant pathogen Ti plasmid conjugation traI traR Erwinia caratovora Plant pathogen Virulence factors Carbapenem production expI expR Erwinia stewartii Plant pathogen Extracellular Capsule esaI esaR Rhizobium leguminosarum Plant symbiont Rhizome interactions rhiI rhiR Pseudomonas aureofaciens Plant beneficial Phenazine production phzI phzR

Receiver cells

Cell-Cell Communication Circuits

pLuxI-Tet-8 pRCV-3

aTc

luxI◊VAI

VAI LuxR GFP

tetR aTc Sender cells

VAI VAI

Receiver cells Sender cells

tetR

P(tet)

luxI

P(Ltet-O1)

aTc

GFP(LVA)

Lux P(R)

luxR

Lux P(L)

+

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Time-Series Response to Signal

Fluorescence response of receiver (pRCV-3)

500 1000 1500 2000 2500 0:00 0:30 1:00 1:30 2:00 Time (hrs) Fluorescence pRCV-3 + pUC19 pRCV3 + pSND-1 pRCV-3 pRCV-3 + pRW-LPR-2 pRCV-3 + pTK-1 AI

p

• s

i t i v e c

• n

t r

• l

10X VAI extract d i r e c t s i g n a l l i n g negative controls

Characterizing the Receiver

Response of receiver to different levels of VAI extract

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25,000 50,000 75,000 10x AI Null 2 20 200 2,000 20,000 200,000 aTc (ng / ml) Receiver Fluorescence LuxTet4B9 RCV Only

Controlling the Sender’s Signal Strength

Dose response of receiver cells to aTc induction of senders receivers senders

• verlay

0.1mm

10 receivers senders

• verlay

20 µm

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Bi-Directional Communication

[Karig, Weiss]

• Explore substrate properties

– Crosstalk – Time scale/delay – Signal strength

• Create constructs useful in later systems

Construct A Construct B

lacI rhlI P(lac) luxR gfp rhlR luxI hcred PL(lux) qsc IPTG P(lacIq) PL(rhl)

3OC6HSL C4HSL

PR(lux)

Demonstrating rhlI Communications

senders receivers

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Testing Crosstalk

Does 3OC6HSL bind RhlR to activate transcription?

Signal Processing / Analog Circuits

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[HSL] GF P

Detecting Chemical Gradients

Analyte source detection analyte source reporter rings

signal

Circuit Components

[HSL] GFP

Components:

• 1. Acyl-HSL detect
• 2. Low threshold
• 3. High threshold
• 4. Negating combiner

L u xR

L u xR

P(lux)

X Y Z

P(W)

GFP

P(Z)

Z

P(X)

W

P(Y)

luxR

P(R)

14 Acyl-HSL Detection

L u xR

L u xR

P(lux)

X Y Z

P(W)

GFP

P(Z)

Z

P(X)

W

P(Y)

luxR

P(R)

Y high threshold X low threshold

Low Threshold Detection

L u xR

L u xR

P(lux)

X Y Z2

P(W)

GFP

P(Z)

Z1

P(X)

W

P(Y)

luxR

P(R)

15 High Threshold Detection

L u xR

L u xR

P(lux)

X Y Z2

P(W)

GFP

P(Z)

Z1

P(X)

W

P(Y)

luxR

P(R)

Protein Z Determines Range

L u xR

L u xR

P(lux)

X Y Z2

P(W)

GFP

P(Z)

Z1

P(X)

W

P(Y)

luxR

P(R)

16 Negating Combiner

L u xR

L u xR

P(lux)

X Y Z2

P(W)

GFP

P(Z)

Z1

P(X)

W

P(Y)

luxR

P(R)

Engineering Circuit Characteristics

HSL-mid: the midpoint where GFP has the highest concentration HSL-width: the range where GFP is above 0.3uM HSL-width HSL-mid

0.3

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Tuning the Range:

Repressor/Operator Affinities

1 2 3 4 1 2 3 4 0.5 1 1.5 2 2.5 3 k-bind-Y2-P-Y k-bind-X2-P-X hsl-width 1 2 3 4 1 2 3 4 0.5 1 1.5 k-bind-Y2-P-Y k-bind-X2-P-X hsl-mid

range width versus X & Y repressor efficiencies range mid-point versus X & Y repressor efficiencies rep/op affinity increases transfer-curve shifts left

Tuning the Range:

Ribosome Binding Sites

0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.6 0.8 1 0.5 1 1.5 2 kxlate-Y kxlate-X hsl-mid 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.6 0.8 1 1 2 3 4 kxlate-Y kxlate-X hsl-width

range width versus X & Y RBS efficiencies range mid-point versus X & Y RBS efficiencies RBS efficiency increases transfer-curve shifts left

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HSL Detection

VAI VAI

Receiver cells Sender cells

tetR

P(tet)

luxI

P(Ltet-O1)

aTc

GFP(LVA)

Lux P(R)

luxR

Lux P(L)

+

Low Threshold Component

IPTG YFP cI CFP lacI

[high] (Off) P(tet) λP(R) P(lac)

measure TC

lacI

P(tet)

P(lac) IPTG

YFP

λP(R)

cI CFP

RBS #1: modify RBS #2: mutate operator

#1 #2

Weiss & Basu, NSC 2002

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tetR P(bla) P(tet) aTc cI P(lac) lacI CFP YFP λ

P(R)

Genetic Circuit for High Threshold

pCMB-2/pCMB-100 1 10 100 1000 10000 0.1 1 10 100 aTc Concentration Level Fluorescence pCMB-2/pCMB-100

Circuit Design Principles

• Separation of low threshold and high threshold

– RBS efficiency of X must be higher than that of Y – Binding affinity of X to its respective promoter has to be higher than that of Y

• Constants associated with Y have more impact on range-

width and range-midpoint

– Y passes through an additional gain stage

• Leakiness and sensitivity of lux promoter determines the

lower bound of detection of acyl-HSL

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Amorphous Computing Programming Cell Aggregates

• Amorphous Computing:

“How does one engineer prespecified, coherent behavior from the cooperation of vast numbers of unreliable parts that are interconnected in unknown, irregular, and time-varying ways.”

• An aggregate of cells is an example of an

amorphous computing substrate

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MCL [Weiss, 1998] GPL [Coore, 1997] Origami [Nagpal, 2001]

Engineering Coordinated Behavior

• High-level specifications for pattern formations
• Translate programs to genetic circuits

Another Example: Differentiation

Cells differentiate into bands of alternating C and D type segments.

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A program for creating segments:

(start Crest ((send (make-segC 1) 3))) ((make-seg seg-type seg-index) (and Tube (not C) (not D)) ((set seg-type) (set seg-index) (send created 3))) (((make-seg) (= 0)) Tube ((set Bottom))) (((make-seg) (> 0)) Tube ((unset Bottom))) (created (or C D) ((set Waiting 10))) (* (and Bottom C 1 (Waiting (= 0))) ((send (make-seg D 1) 3))) (* (and Bottom D 1 (Waiting (= 0))) ((send (make-segC 2) 3))) (* (and Bottom C 2 (Waiting (= 0))) ((send (make-seg D 2) 3))) (* (and Bottom D 2 (Waiting (= 0))) ((send (make-segC 3) 3)))

Microbial Colony Language (MCL)

message condition actions

The Microbial Colony Language

• Language primitives:

– asynrchronous rules – boolean state variables – boolean logic – local communications with chemical diffusion

• These primitives can be mapped to

engineered biochemical processes

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Reaction/Diffusion Pattern Formation

[Millonas/Rauch]

Kinetic rates determine emergence of patterns

Reaction/Diffusion Simulation

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Reaction/Diffusion Simulation II Future Work

• Quantitative prediction of engineered cell behavior
• Self-perfecting genetic circuits
• Intercellular communication architectures
• Signal processing circuits
• Additional CAD tools
• Bio-fab

– Large scale circuit design, production, and testing

• Simpler & more complex organisms:

– Eukaryotes – Mycoplasmas

• Biologically inspired logic gates
• Molecular scale fabrication

vs.