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Synchronized Chemotactic Oscillators Summer Synthetic Biology Competition Massachusetts Institute of Technology November 6, 2004 S.M.U.G.
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S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

May 04, 2018

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Page 1: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

SynchronizedChemotactic Oscillators

Summer Synthetic Biology CompetitionMassachusetts Institute of Technology

November 6, 2004

S.M.U.G.

Page 2: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Motivation

• Our goal: an interesting, complex system –something cool. But how to make it happen?

• We focused on implementing modularity– Breaking biological systems into modular pieces– At a low level, this is BioBricks

• Building a modular system allowed efficientdivision of labor – key for a team this large

Page 3: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

A Chemotactic Oscillator

Attractant Plug

Bacterial Swimming Pool

Chemoattractant Gradient

Page 4: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

A Chemotactic Oscillator

Bacteria are added to theswimming pool

Page 5: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

A Chemotactic Oscillator

Chemotaxis is enabled,indicated by green bacteria

Bacteria start swimming up thegradient towards the attractant plug

Page 6: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

A Chemotactic Oscillator

The bacteria congregate around theattractant plug

Page 7: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

A Chemotactic Oscillator

Each bacteria has an internaloscillator, driving switch between:

chemotaxis enabledchemotaxis disabled

Page 8: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

A Chemotactic Oscillator

The bacteria communicate theirinternal oscillator phase with eachother using cell-to-cell signaling …

Page 9: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

A Chemotactic Oscillator

… enabling the entire population tochange state synchronously

Page 10: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

A Chemotactic Oscillator

In red bacteria, chemotaxis is disabled

Bacteria start to randomly move awayfrom the attractant plug

Page 11: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

A Chemotactic Oscillator

Eventually the bacteria are dispersedaround the swimming pool

Page 12: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

A Chemotactic Oscillator

Again the bacteria communicate witheach other using cell-to-cell signaling

Page 13: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

A Chemotactic Oscillator

The inter-cellular signaling moleculediffuses throughout out the swimming

pool to the entire population

Page 14: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

A Chemotactic Oscillator

State changes: chemotaxis is enabled

Page 15: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

A Chemotactic Oscillator

... and onward they swim, doomed to afate worse than that of Sisyphus

Page 16: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

A Chemotactic Oscillator

“Futile, this wretched swimming!”

Page 17: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Top-Level System Diagram

OscillatorModule

ChemotaxisModule

Individual’sOsc Phase

Population’sOsc Phase

ChemotaxisEnable

Cell BoundaryCell-to-CellSignalingModule

Page 18: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Top-Level System Diagram

OscillatorModule

ChemotaxisModule

Individual’sOsc Phase

Population’sOsc Phase

ChemotaxisEnable

Cell BoundaryCell-to-CellSignalingModule

Page 19: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Outline

• Overview– Motivation– System Description– How we got there: Synopsis of Summer Activities

• Module Discussion– Cell-to-cell Signaling Module– Oscillator Module– Chemotaxis Module– Module Integration

• Final Remarks

Cell-to-CellSynchronization

Module

OscillatorModule

ChemotaxisModule

Cell Boundary

Page 20: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Learning about SynthBio

• Preliminary discussion and design work

• Previous Class Experiences at MIT– Much design, little implementation

June July August

Cool ideas, but we wondered: “Can we do this?”

Had to hit the lab …

Page 21: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Introduction to a Biology Lab

Objective during this period was parts characterization– Achieved useful work on RBS characterization– Attempted to build sets of linked inverters– Began work on cell-to-cell signalling

June July August

“So...you're saying that was supposed to be refrigerated?”

“Agarose gels, Agar gels...what's the difference?”

Page 22: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Finalizing Design

Brainstormed several comprehensiveand full-system designs

Choosing one design gave us purposeand focus in lab

June July August

Page 23: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Thereafter: Making it Happen

• Final element– Integrating modules is surely easier said than done– How to prepare the experimental setup for our work?

• Barry, Jason, Fred, and Vikki will now discussthese topics. Chris will offer final remarks.

Cell-to-CellSynchronization

Module

OscillatorModule

ChemotaxisModule

Cell Boundary

Page 24: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Cell-to-Cell Signaling Objectives

PoPSSignalReceiver

SignalPoPSSender

SignalSignal

• Decompose signaling into elements

• Design, build and test elements

• Explore how elements mightfunction as part of the full system

Cell-to-CellSynchronization

Module

OscillatorModule

ChemotaxisModule

Individual’sOsc Phase

Population’sOsc Phase

ChemotaxisEnable

Cell Boundary

Page 25: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

The Lux System

Lux box withleft and rightpromoters

luxR luxCDABEGluxI

L

H

R

Page 26: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

The Lux System

Lux box withleft and rightpromoters

luxR luxCDABEGluxI

LR

H

Luciferase

LuxRDimerization

HR

H HR

R

R

Page 27: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

The Lux System

Lux box withleft and rightpromoters

luxR luxCDABEGluxI

LR

H

Luciferase

H

LuxRDimerization

HR

H HR

R

R

R

RH

Page 28: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Utilizing Existing Components

Lux box withleft and rightpromoters

luxR luxCDABEGluxI

LR

H

Luciferase

H

LuxRDimerization

HR

H HR

R

R

R

RH

SignalPoPSSender

Page 29: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Utilizing Existing Components

Lux box withleft and rightpromoters

luxR luxCDABEGluxI

LR

H

Luciferase

H

LuxRDimerization

HR

H HR

R

R

R

RH

PoPSSignalReceiver

SignalPoPSSender

Page 30: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Utilizing Existing Components

Lux box withleft and rightpromoters

luxR luxCDABEGluxI

LR

H

Luciferase

H

LuxRDimerization

HR

H HR

R

R

R

RH

PoPSSignalReceiver

SignalPoPSSender

SignalSignal

Page 31: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Receiver Design

BBa_I13270

PoPSSignalReceiver

R

H

R

H HR

R

R

R

RH

Page 32: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Receiver Building

• Varied Upstream Promoter - Ptet, luxPL

• High (100-200) and Low Copy (10-20) Plasmid

• Used YFP Output Device as a PoPS Reporter

• Built in DH5alpha using standardized assembly,Transformed into MC4100 and HCB1103

BBa_I13273

PoPSSignalReceiver

Page 33: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Receiver Testing PoPSSignalReceiver

1. Hi/Lo ratio

2. Switch Point

3. Response Time

Page 34: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Receiver Testing

I13273 - pSB1A2

PoPSSignalReceiver

Hi/Lo ratio ≈ 17

Switch Point ≈ 2nM

Page 35: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Receiver Testing

I13273 - pSB1A2 - Growth Defects

PoPSSignalReceiver

Page 36: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Receiver Testing

I13273 - pSB3K3 - Growth Restored

PoPSSignalReceiver

Page 37: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Receiver Testing

I13273 - pSB3K3 - Response Time

PoPSSignalReceiver

Page 38: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Senders SignalPoPSSender

10mL

H

luxI

20m

30m

15m

25m

40m

Page 39: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Transmission

• Diffusion rate

• Degradation due to dilution (e.g. in chemostat)

• Degradation due to raised pH

• Active enzymatic degradation - aiiA

SignalSignal

Page 40: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

pH Dependent Degradation

Transmission

60 minute incubation of HSL at various pH

Use that HSL to activate receivers at neutral pH

SignalSignal

Page 41: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

TransmissionaiiA

Enzymatic intracellular Degradation of HSL

SignalSignal

Page 42: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Future Work

• Develop the ability to adjust receiver transferfunction parameters at will

• Complete characterization of existing senderdevice using the receiver device

• Build and test the sender device used in thesynchronized oscillator

• Continue to test the aiiA degradationmechanism

• Test under different operating conditions -microfluidic chemostat, microscope slides etc.

Page 43: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Oscillator Module

• Stand-alone Oscillator– Relaxation Oscillator– Ring Oscillator

• Synchronized Oscillator– Synchronators– Synchronized Ring

Oscillator

• Future Work Cell-to-CellSynchronization

Module

ChemotaxisModule

Individual’sOsc Phase

Population’sOsc Phase

ChemotaxisEnable

Cell Boundary

OscillatorModule

Page 44: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Input/Output

cell boundary

OscillatorDevice

PoPS

Page 45: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Lux/aiiA Relaxation Oscillator

P LuxR

R L A

ConstitutivePromoter

LuxR LuxI aiiA

LuxR is constitutively expressed, while LuxI andaiiA are regulated by a LuxR activated promoter

Page 46: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Lux/aiiA Relaxation Oscillator

R L

H

A

LuxRDimerization

HR

ConstitutivePromoter

R

LuxR LuxI aiiA

LuxR forms a dimer while LuxI synthesizes HSL

P LuxR

Page 47: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Lux/aiiA Relaxation Oscillator

R

H

L

H

A

LuxRDimerization

HR

H HR

RConstitutivePromoter

R

R

RH

LuxR LuxI aiiA

LuxR and HSL bind to form the transcriptionalactivator providing positive feedback

P LuxR

Page 48: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Lux/aiiA Relaxation Oscillator

R

H

L

H

A

LuxRDimerization

HR

H HR

RConstitutivePromoter

R

R

RH

LuxR LuxI aiiA

LuxR and HSL bind to form the transcriptionalactivator providing positive feedback

P LuxR

Page 49: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Lux/aiiA Relaxation Oscillator

R

H

L A

LuxRDimerization

Degradationof HSL by aiiA

HR

H HR

RConstitutivePromoter

R

R

RH

LuxR LuxI aiiA

aiiA degrades HSL providing negative feedback

P LuxR

Page 50: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Lux/aiiA Relaxation Oscillator

R

H

L A

LuxRDimerization

Degradationof HSL by aiiA

HR

H HR

RConstitutivePromoter

R

R

RH

LuxR LuxI aiiA

aiiA degrades HSL providing negative feedback

P LuxR

Page 51: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Simplified Relaxation Oscillator

• Initial modeling work used a system of continuousdifferential equations to examine a simplified oscillator

• Folds the positive feedback into a single Protein Aignoring the details of LuxI, HSL, and LuxR

• Even with these simplifications, the model can giveinsight into what experimental constructs would beuseful when building the actual Lux/aiiA oscillator

Promoter Protein A Protein B

Page 52: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

State Space Analysis

Intersection of nullclines yieldssystem equilibrium point

Equlibrium point changes withProtein B degradation rate

Page 53: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Preliminary Modeling Results

A vs B State Space Concentration of A vs Time

Page 54: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Experimental Work

• Modeling work suggested possible test constructs

• Experimental work on the Lux/aiiA relaxationoscillator was put on hold– Initial results on aiiA were discouraging– Not enough degradation tags were available to

effectively tune the aiiA degradation rate

Page 55: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Input/Output

cell boundary

OscillatorDevice

PoPS

Page 56: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Ring Oscillator

Page 57: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Input/Output

OscillatorDevice

Averageoscillationphase of

population

Individualoscillation

phase

cell boundary

PoPs

senderreceiver

PoPs

AHL

Page 58: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Synchronized Oscillator Options

• Repressilator & Synchronization Device– Functional oscillator– Need to design synchronization device

• Synchronator– 4 designs available from the MIT 2003 Synthetic

Biology course– Designed to synchronize, completely built, but

untested and uncharacterized

• See-ya-lator– Modeled after Yankees ALCS performance

Page 59: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

gfp

Synchronator Designs

gfp

Design 1

Design 3gf

p

Design 2

Design 4

gfp

Page 60: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Synchronator Design 2

gfp

Page 61: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

gfp

Synchronator Designs

gfp

Design 1

Design 3gf

p

Design 2

Design 4

gfp

Page 62: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Oscillator Lockdown Experiment

IPTG

ATC

gfp

Add IPTG = GFP high

Add ATC = GFP high

Add HSL = GFP low

Page 63: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Synchronator Lock-Down

0

500

1000

1500

2000

2500

3000

synch 1 synch 2 synch 3 synch 4

Flu

ore

scen

ce U

nit

s

Control

IPTG

ATC

HSL

Page 64: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Synchronator 2 Movie

Page 65: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Synchronized Ring Oscillator

• Add a synchronization element to the Repressilator(Garcia-Ojalvo,Elowitz,Strogatz, PNAS 2004)

receiver sender

Page 66: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Synchronized Ring Oscillator

• Add a synchronization element to the Repressilator(Garcia-Ojalvo,Elowitz,Strogatz, PNAS 2004)

receiver sender

Page 67: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Construction of SynchronizationDevice

I13905

I13974 I13975

I13973 S3511 R0062 Q04121

S03167

I0461

Page 68: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Future Work

• Synchronized Ring Oscillator– Lock-down experiments– Agarose Pad Time Lapse Movie– Continuous Culture (chemostat)– Plate Reader Time Course

• Relaxation Oscillator– Explore aiiA further to determine why it isn’t

functioning as expected– Build test constructs and characterize

Page 69: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Chemotaxis Module

• Chemotaxis biology

• Chemotaxis devices– Restoring motility

– Deactivating motility

• Chemotaxis assay

• Results

• Future work

Cell-to-CellSynchronization

Module

ChemotaxisModule

Individual’sOsc Phase

Population’sOsc Phase

ChemotaxisEnable

Cell Boundary

OscillatorModule

Page 70: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Chemotaxis in Escherichia Coli

• Four intracellular signaling proteins– CheB, CheR, CheY, and CheZ– Maintained at specific levels and ratios

Page 71: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Motile Behavior

• Net movement toward oraway from chemicalsresult from the combinedeffect of smooth runs andtumbles

• The expression andactivity of signalingproteins (CheB, CheR,CheY and CheZ)determines the frequencyof tumbles and runs

http://www.jameshallsweb.co.uk/dissertation/about_chemotaxis.htm

Page 72: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Too Much or Too Little?

smooth runs+ΔCheZ

none-ΔCheY

smooth runs+ΔCheR

tumbles+ΔCheB

smooth runs and tumbles+wt

PhenotypeMotilityGenotype

smooth runsCheZwt

tumblesCheYwt

tumblesCheRwt

smooth runsCheBwt

PhenotypeOverexpressionGenotype

Absence of anysignaling protein

affects motilebehavior

Overexpressionof any signalingmolecule affectsmotile behavior

Page 73: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Too Much or Too Little?

smooth runs+ΔCheZ

none-ΔCheY

smooth runs+ΔCheR

tumbles+ΔCheB

smooth runs and tumbles+wt

PhenotypeMotilityGenotype

smooth runsCheZwt

tumblesCheYwt

tumblesCheRwt

smooth runsCheBwt

PhenotypeOverexpressionGenotype

Absence of anysignaling protein

affects motilebehavior

Overexpressionof any signalingmolecule affectsmotile behavior

CheY concentration in RP437: 8,200 ± 310 per cell in richmedia and 6,300 ± 70 per cell in minimal media

Li M, Hazelbauer G “Cellular Stoichiometery of the Components of the Chemotaxis Signaling Complex”Journal of Bacteriology, 2004, 186(12) 3687-3694

Page 74: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Building Chemotaxis Output• CheY quadparts: High Copy (150-200) and Low Copy (15-20)

• Two methods for coupling to Chemotaxis– Restoration of normal chemotaxis in ΔCheY mutant strain

– Deactivation of normal chemotaxis in wild type strain

• Characterizing quadpart expression– Time frame of protein expression

• Observing the inactivation and deactivation of bacterial chemotaxis– Swarm plate characterization– Drop assay and bacterial clustering on glass slide– Capillary Assay

lacI+pLR0011 BOO34

cheYC0020 B0015

Page 75: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Swarm Plate Assay

• Amino acids are bacterialchemoattractants

• Nutrient consumptionproduces gradient

• Ring formation on agarcorresponding to particularamino acid/chemoattractantconsumed by motile bacteria

• Movement away from thecenter (point of inoculation)

10ul of bacterial suspension

30ºC for duration of swarming

Page 76: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Deactivation: Expected Results

Page 77: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Deactivation: High Copy Results

High copy CheY expression in wild type

Page 78: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Deactivation: High Copy Results

High copy CheY expression in LacI- strain(wild type background)

Page 79: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Deactivation: Low Copy Results25µM IPTG induction for 2 hours, OD660 = 0.1

10ul spot, 16 hrs

LacI-

LacI-

(transformed)

wild type

wild type(transformed)

Expected:Swarm

Observed:Swarm

Expected:Swarm

Observed:Swarm

Expected:Swarm/No rings

Observed:Swarm/No rings

Expected:Swarm

Observed:No Swarm

Page 80: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Future Work

• More low copy construct (15-25 per cell) experiments

• Experimentation with ΔcheY mutants of E. coli RP437for the restoration of motility– Requires the tuned expression of CheY

• Drop assay or coverslip assay to observe bacterialaggregation– Characterization of immediate chemotaxis response under

varying levels of induction

• Coupling to population oscillator module and cell-cellsignaling module

Page 81: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Module Integration

• Operating Conditions– Strain

– pH

– Temperature

– Test setup

• Inter-ModuleCommunication– Signal Interpretation

– Timing

Cell-to-CellSynchronization

Module

OscillatorModule

ChemotaxisModule

Cell Boundary

Page 82: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Op Conditions: Strain

Chemotactic

LacI-

Known Requirements

RP437 (HCB33)Chemotaxis

MC4100Oscillator

MC4100, DH5alphaCell-Cell Signaling

Tested ConditionsModule

Future Plans– Testing Cell-Cell signaling module in RP437– Create LacI- version of RP437– Test combined construct in LacI- version of RP437

Page 83: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Op Conditions: pH and Temp

30o Celsius, pH 6.5-7.5

37o Celsius, pH 7

37o Celsius, pH 7

Tested Conditions

pH around 7, temperaturearound 30o Celsius

HSL stability is pHdependent

Known Requirements

Chemotaxis

Oscillator

Cell Cell Signaling

Component

Future Plans– Test combined system in pH7, 32o Celsius

Page 84: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Op Conditions: Test Setup

Steady gradient ofchemoattractants

Known Requirements

Swarm plates

ChemostatAgarose pads

Culture tubes

Tested conditions

Chemotaxis

Oscillator

Cell Cell Signaling

Component

Future Plans– Plan I: Swimming Pool for continuous observation– Plan II: Time Course Sampling with time course test for chemotaxis

Page 85: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Inter-Module: Signal Interpretation

Signals given as a logical “1” or “0” output from onemodule must be interpreted as a logical “1” or “0’

input by the other modules

Increasing Signal Strength

Module 1 ValidOutput Ranges Low High

Low High

Future Plans– Do signal strength characterization tests for modules– If strengths don’t match, fine tune by swapping promoter/RBS

Module 2 ValidInput Ranges

Page 86: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Inter-Module: Signal Interpretation

Signals given as a logical “1” or “0” output from onemodule must be interpreted as a logical “1” or “0’

input by the other modules

Increasing Signal Strength

Module 1 ValidOutput Ranges Low High

Low High

Future Plans– Do signal strength characterization tests for modules– If strengths don’t match, fine tune by swapping promoter/RBS

Module 2 ValidInput Ranges

Page 87: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Inter-Module: Signal Interpretation

Signals given as a logical “1” or “0” output from onemodule must be interpreted as a logical “1” or “0’

input by the other modules

Increasing Signal Strength

Module 1 ValidOutput Ranges Low High

Low High

Future Plans– Do signal strength characterization tests for modules– If strengths don’t match, fine tune by swapping promoter/RBS

Module 2 ValidInput Ranges

Page 88: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Inter-Module: Timing

Timing of oscillator signals must match up with timing of cell-cell signaling and chemotaxis modules

Sig

nal 2

Sig

n al 1

HighThreshold

LowThreshold

HighThreshold

LowThreshold

Increasing Time

Signal 1: (Oscillator) Rise Time=0; Fall time=0; Period =2

Signal 2: (Follower) Rise Time=1; Fall time=2

Page 89: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Inter-Module: Timing

• Cell-to-Cell Signaling and Chemotaxis must be able toturn on and off in at most the amount of time it takes theOscillator to turn on and off.

• Current Knowledge– Repressilator : Period ~2 hours– Cell-to-Cell and Chemotaxis On : transcription+protein action is fast– Cell-to-Cell and Chemotaxis Off : protein degradation/dilution is slow

• Future Plans– Try getting the HSL signals to degrade faster by operating

at a higher pH (10) or in a chemostat– Characterize off times for chemotaxis module

Page 90: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Integration Summary

• Integration is HARD!– Operating Conditions– Inter-module Communication

• Still a lot of work to be done

Page 91: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Final Remarks

• Overview of summer accomplishments

• Advice for future summer competitions

• Key enablers for the field of synthetic biology– Assembly process– Device characterization– Standard operating conditions

Page 92: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Summer Accomplishments

• Over 200 new BioBrick parts added to the registry

• Device characterization– RBS measurements– Preliminary copy number measurements– Basic chemostat constructed and tested– Many inverter measurement constructs ready to be tested

• Cell-to-cell signaling module– Working Lux sender/receiver constructed with BioBrick parts– Characterizing receiver transfer curves– Verified importance of low-copy constructs– Characterization of cell-to-cell signaling channel

Page 93: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Summer Accomplishments

• Oscillator module– Modeling work on Lux/aiiA relaxation oscillator– Refined techniques for creating time-lapse movies– Verified repressilator ring oscillator– Tested previously designed Synchronators– New synchronized repressilator is built and ready for testing

• Chemotaxis module– Working swarm plate chemotaxis assay– Results on possibility of transcriptional control of chemotaxis

• Twelve synthetic biology students who are excitedabout the potential of this new field

• Five extremely frustrated advisors who are lookingforward to a long winter vacation

Page 94: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

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I13607

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E0669

E0130

I13016I13633I13105I0465I13312I13262I0468

C0056I13631I13104I0464I13311I13261I0467

I13990

S04010

S04003

S04002

S04001

S04000

Q04740

Q02400

Q02121

Q00400

Q00121

P0474

I13006

I13013

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I13011

I13010

I13009

I13008

I13007

I13667

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I13645

I13644

I13637

I13634

I13627I13103I0463I13310I13220I0466

I13626I13102C0261I13309I13213I13005

I13625I13101C0260I13308I13212I13004

I13624I13100C0163I13307I13211I13002

I13623I13033C0063I13306I13208I13002

I13621I13032I13207I13305I13206I13001

I13617I13031I534160I13304I13205G00701

I13614I13030I534060I13303I13203G00700

I13613I13029P34160I13283I13202S10000

I13611I13028P34060I13280I13201I13730

I13115I13027I1468I13279I13200I13721

I13114I13026I1466I13277I13053I13712

I13113I13025I13915I13274I13038I13711

I13112I13024I13914I13273I13037I13710

I13111I13023I13913I13271I13036I13702

I13110I13022I13912I13270I13035I13701

I13109I13021I13911I13266I13034I13700

I13108I13020I13910I13265I13018C0028

I13107I13019I13314I13264I13015C0024

I13106I13014I13313I13263I13000C0020

BioBrick Parts

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I13971

I13851

I13850

I13801

I13800

I13072

I13062

I13017

I6402

I6401

I13607

I13606

I13605

I13604

I13603

I13602

I13601

I13600

E0669

E0130

I13016I13633I13105I0465I13312I13262I0468

C0056I13631I13104I0464I13311I13261I0467

I13990

S04010

S04003

S04002

S04001

S04000

Q04740

Q02400

Q02121

Q00400

Q00121

P0474

I13006

I13013

I13012

I13011

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I13009

I13008

I13007

I13667

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I13653

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I13644

I13637

I13634

I13627I13103I0463I13310I13220I0466

I13626I13102C0261I13309I13213I13005

I13625I13101C0260I13308I13212I13004

I13624I13100C0163I13307I13211I13002

I13623I13033C0063I13306I13208I13002

I13621I13032I13207I13305I13206I13001

I13617I13031I534160I13304I13205G00701

I13614I13030I534060I13303I13203G00700

I13613I13029P34160I13283I13202S10000

I13611I13028P34060I13280I13201I13730

I13115I13027I1468I13279I13200I13721

I13114I13026I1466I13277I13053I13712

I13113I13025I13915I13274I13038I13711

I13112I13024I13914I13273I13037I13710

I13111I13023I13913I13271I13036I13702

I13110I13022I13912I13270I13035I13701

I13109I13021I13911I13266I13034I13700

I13108I13020I13910I13265I13018C0028

I13107I13019I13314I13264I13015C0024

I13106I13014I13313I13263I13000C0020

BioBrick Parts

Over 200 PartsAdded to Registry

Page 96: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

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I13906

I13905

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I13973

I13972

I13971

I13851

I13850

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I13072

I13062

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I6402

I6401

I13607

I13606

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I13604

I13603

I13602

I13601

I13600

E0669

E0130

I13016I13633I13105I0465I13312I13262I0468

C0056I13631I13104I0464I13311I13261I0467

I13990

S04010

S04003

S04002

S04001

S04000

Q04740

Q02400

Q02121

Q00400

Q00121

P0474

I13006

I13013

I13012

I13011

I13010

I13009

I13008

I13007

I13667

I13665

I13664

I13657

I13654

I13653

I13651

I13647

I13645

I13644

I13637

I13634

I13627I13103I0463I13310I13220I0466

I13626I13102C0261I13309I13213I13005

I13625I13101C0260I13308I13212I13004

I13624I13100C0163I13307I13211I13002

I13623I13033C0063I13306I13208I13002

I13621I13032I13207I13305I13206I13001

I13617I13031I534160I13304I13205G00701

I13614I13030I534060I13303I13203G00700

I13613I13029P34160I13283I13202S10000

I13611I13028P34060I13280I13201I13730

I13115I13027I1468I13279I13200I13721

I13114I13026I1466I13277I13053I13712

I13113I13025I13915I13274I13038I13711

I13112I13024I13914I13273I13037I13710

I13111I13023I13913I13271I13036I13702

I13110I13022I13912I13270I13035I13701

I13109I13021I13911I13266I13034I13700

I13108I13020I13910I13265I13018C0028

I13107I13019I13314I13264I13015C0024

I13106I13014I13313I13263I13000C0020

BioBrick Parts

166 Parts BeganAssembly Process

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I13016I13633I13105I0465I13312I13262I0468

C0056I13631I13104I0464I13311I13261I0467

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S04000

Q04740

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Q02121

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Q00121

P0474

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I13644

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I13634

I13627I13103I0463I13310I13220I0466

I13626I13102C0261I13309I13213I13005

I13625I13101C0260I13308I13212I13004

I13624I13100C0163I13307I13211I13002

I13623I13033C0063I13306I13208I13002

I13621I13032I13207I13305I13206I13001

I13617I13031I534160I13304I13205G00701

I13614I13030I534060I13303I13203G00700

I13613I13029P34160I13283I13202S10000

I13611I13028P34060I13280I13201I13730

I13115I13027I1468I13279I13200I13721

I13114I13026I1466I13277I13053I13712

I13113I13025I13915I13274I13038I13711

I13112I13024I13914I13273I13037I13710

I13111I13023I13913I13271I13036I13702

I13110I13022I13912I13270I13035I13701

I13109I13021I13911I13266I13034I13700

I13108I13020I13910I13265I13018C0028

I13107I13019I13314I13264I13015C0024

I13106I13014I13313I13263I13000C0020

BioBrick Parts

68 PartsCompletelyAssembled

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E0669

E0130

I13016I13633I13105I0465I13312I13262I0468

C0056I13631I13104I0464I13311I13261I0467

I13990

S04010

S04003

S04002

S04001

S04000

Q04740

Q02400

Q02121

Q00400

Q00121

P0474

I13006

I13013

I13012

I13011

I13010

I13009

I13008

I13007

I13667

I13665

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I13627I13103I0463I13310I13220I0466

I13626I13102C0261I13309I13213I13005

I13625I13101C0260I13308I13212I13004

I13624I13100C0163I13307I13211I13002

I13623I13033C0063I13306I13208I13002

I13621I13032I13207I13305I13206I13001

I13617I13031I534160I13304I13205G00701

I13614I13030I534060I13303I13203G00700

I13613I13029P34160I13283I13202S10000

I13611I13028P34060I13280I13201I13730

I13115I13027I1468I13279I13200I13721

I13114I13026I1466I13277I13053I13712

I13113I13025I13915I13274I13038I13711

I13112I13024I13914I13273I13037I13710

I13111I13023I13913I13271I13036I13702

I13110I13022I13912I13270I13035I13701

I13109I13021I13911I13266I13034I13700

I13108I13020I13910I13265I13018C0028

I13107I13019I13314I13264I13015C0024

I13106I13014I13313I13263I13000C0020

BioBrick Parts

29 PartsCompletely

Working

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C0056I13631I13104I0464I13311I13261I0467

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I13626I13102C0261I13309I13213I13005

I13625I13101C0260I13308I13212I13004

I13624I13100C0163I13307I13211I13002

I13623I13033C0063I13306I13208I13002

I13621I13032I13207I13305I13206I13001

I13617I13031I534160I13304I13205G00701

I13614I13030I534060I13303I13203G00700

I13613I13029P34160I13283I13202S10000

I13611I13028P34060I13280I13201I13730

I13115I13027I1468I13279I13200I13721

I13114I13026I1466I13277I13053I13712

I13113I13025I13915I13274I13038I13711

I13112I13024I13914I13273I13037I13710

I13111I13023I13913I13271I13036I13702

I13110I13022I13912I13270I13035I13701

I13109I13021I13911I13266I13034I13700

I13108I13020I13910I13265I13018C0028

I13107I13019I13314I13264I13015C0024

I13106I13014I13313I13263I13000C0020

BioBrick Parts

13 Parts Canceled

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I13634

I13627I13103I0463I13310I13220I0466

I13626I13102C0261I13309I13213I13005

I13625I13101C0260I13308I13212I13004

I13624I13100C0163I13307I13211I13002

I13623I13033C0063I13306I13208I13002

I13621I13032I13207I13305I13206I13001

I13617I13031I534160I13304I13205G00701

I13614I13030I534060I13303I13203G00700

I13613I13029P34160I13283I13202S10000

I13611I13028P34060I13280I13201I13730

I13115I13027I1468I13279I13200I13721

I13114I13026I1466I13277I13053I13712

I13113I13025I13915I13274I13038I13711

I13112I13024I13914I13273I13037I13710

I13111I13023I13913I13271I13036I13702

I13110I13022I13912I13270I13035I13701

I13109I13021I13911I13266I13034I13700

I13108I13020I13910I13265I13018C0028

I13107I13019I13314I13264I13015C0024

I13106I13014I13313I13263I13000C0020

BioBrick Parts

69 Parts forCell-to-Cell

Module

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I13627I13103I0463I13310I13220I0466

I13626I13102C0261I13309I13213I13005

I13625I13101C0260I13308I13212I13004

I13624I13100C0163I13307I13211I13002

I13623I13033C0063I13306I13208I13002

I13621I13032I13207I13305I13206I13001

I13617I13031I534160I13304I13205G00701

I13614I13030I534060I13303I13203G00700

I13613I13029P34160I13283I13202S10000

I13611I13028P34060I13280I13201I13730

I13115I13027I1468I13279I13200I13721

I13114I13026I1466I13277I13053I13712

I13113I13025I13915I13274I13038I13711

I13112I13024I13914I13273I13037I13710

I13111I13023I13913I13271I13036I13702

I13110I13022I13912I13270I13035I13701

I13109I13021I13911I13266I13034I13700

I13108I13020I13910I13265I13018C0028

I13107I13019I13314I13264I13015C0024

I13106I13014I13313I13263I13000C0020

BioBrick Parts

22 Parts forOscillator Module

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I13606

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I13602

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C0056I13631I13104I0464I13311I13261I0467

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I13634

I13627I13103I0463I13310I13220I0466

I13626I13102C0261I13309I13213I13005

I13625I13101C0260I13308I13212I13004

I13624I13100C0163I13307I13211I13002

I13623I13033C0063I13306I13208I13002

I13621I13032I13207I13305I13206I13001

I13617I13031I534160I13304I13205G00701

I13614I13030I534060I13303I13203G00700

I13613I13029P34160I13283I13202S10000

I13611I13028P34060I13280I13201I13730

I13115I13027I1468I13279I13200I13721

I13114I13026I1466I13277I13053I13712

I13113I13025I13915I13274I13038I13711

I13112I13024I13914I13273I13037I13710

I13111I13023I13913I13271I13036I13702

I13110I13022I13912I13270I13035I13701

I13109I13021I13911I13266I13034I13700

I13108I13020I13910I13265I13018C0028

I13107I13019I13314I13264I13015C0024

I13106I13014I13313I13263I13000C0020

BioBrick Parts

11 Parts forChemotaxis

Module

Page 103: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

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I13602

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C0056I13631I13104I0464I13311I13261I0467

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I13645

I13644

I13637

I13634

I13627I13103I0463I13310I13220I0466

I13626I13102C0261I13309I13213I13005

I13625I13101C0260I13308I13212I13004

I13624I13100C0163I13307I13211I13002

I13623I13033C0063I13306I13208I13002

I13621I13032I13207I13305I13206I13001

I13617I13031I534160I13304I13205G00701

I13614I13030I534060I13303I13203G00700

I13613I13029P34160I13283I13202S10000

I13611I13028P34060I13280I13201I13730

I13115I13027I1468I13279I13200I13721

I13114I13026I1466I13277I13053I13712

I13113I13025I13915I13274I13038I13711

I13112I13024I13914I13273I13037I13710

I13111I13023I13913I13271I13036I13702

I13110I13022I13912I13270I13035I13701

I13109I13021I13911I13266I13034I13700

I13108I13020I13910I13265I13018C0028

I13107I13019I13314I13264I13015C0024

I13106I13014I13313I13263I13000C0020

BioBrick Parts

63 Parts forDevice

Characterization

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I13921

I13920

I13906

I13905

I13904

I13902

I13901

I13900

I13210

I13209

I13973

I13972

I13971

I13851

I13850

I13801

I13800

I13072

I13062

I13017

I6402

I6401

I13607

I13606

I13605

I13604

I13603

I13602

I13601

I13600

E0669

E0130

I13016I13633I13105I0465I13312I13262I0468

C0056I13631I13104I0464I13311I13261I0467

I13990

S04010

S04003

S04002

S04001

S04000

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Q00121

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I13010

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I13008

I13007

I13667

I13665

I13664

I13657

I13654

I13653

I13651

I13647

I13645

I13644

I13637

I13634

I13627I13103I0463I13310I13220I0466

I13626I13102C0261I13309I13213I13005

I13625I13101C0260I13308I13212I13004

I13624I13100C0163I13307I13211I13002

I13623I13033C0063I13306I13208I13002

I13621I13032I13207I13305I13206I13001

I13617I13031I534160I13304I13205G00701

I13614I13030I534060I13303I13203G00700

I13613I13029P34160I13283I13202S10000

I13611I13028P34060I13280I13201I13730

I13115I13027I1468I13279I13200I13721

I13114I13026I1466I13277I13053I13712

I13113I13025I13915I13274I13038I13711

I13112I13024I13914I13273I13037I13710

I13111I13023I13913I13271I13036I13702

I13110I13022I13912I13270I13035I13701

I13109I13021I13911I13266I13034I13700

I13108I13020I13910I13265I13018C0028

I13107I13019I13314I13264I13015C0024

I13106I13014I13313I13263I13000C0020

BioBrick Parts

12 NewReporter Parts

Page 105: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Advice for Future Competitions

Structured introductory two-week curriculum

– Daily lectures in the mornings and specific lab tutorialsin the afternoons

– Students would model, assemble, and characterizea simple synthetic system such as a single OR gate

– Teaches synthetic biology basics and experimentallab technique as well as providing a solid foundationfor initial design work

June July August

Page 106: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Advice for Future Competitions

More milestones and incremental deliverables

– Report on simple synthetic system

June July August

Page 107: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Advice for Future Competitions

More milestones and incremental deliverables

– Report on simple synthetic system

– Preliminary design specification

June July August

Page 108: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Advice for Future Competitions

More milestones and incremental deliverables

– Report on simple synthetic system

– Preliminary design specification

– Interim progress report

June July August

Page 109: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Advice for Future Competitions

More milestones and incremental deliverables

– Report on simple synthetic system

– Preliminary design specification

– Interim progress report

– Periodic logs kept by each student and lab-group

June July August

Page 110: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Advice for Future Competitions

Inter-team collaboration

– Periodic conference calls

– Distribute designspecifications and interimreports to all teams

– Logs managed in onlineforum accessible by allteams

Page 111: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Key Enablers for SynthBio

Through our summer experiences we identifiedthree key enablers which will greatly help futurework in the field of synthetic biology

Standard Operating Conditions

Device Characterization

Assembly Process

Page 112: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Assembly Process

• Remarkable that a group of students withvery little biology background was able to buildworking biological parts relatively quickly

• Even so, current assembly process placedsignificant constraints on what was possible– Took on average one week per stage– When assembly failed very difficult to determine why

• Assembly is an important research topic– Optimize each stage– Characterize and model error rates– Develop more assembly tools

Page 113: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Device Characterization

• Modeling work is significantly hamperedby lack of useful device characterization

• Device characterization is challenging– What do we actually measure?– How do we measure it?– How do we make measurements repeatable?

• Accurate device characterization will enable– Effective parameterized models– More rational design– Easier reuse of previously developed parts

Page 114: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Operating Conditions

• Currently no standard operating conditions

– Strain, media, growth phase are usually documentedbut they still vary with each experiment

– Standard conditions enable easier result comparisons

– Standard operation conditions also make it easier topredict how future systems will behave

• Standard operating conditions is challenging

– Difficult to choose a single set of conditions sincedifferent experiments have different requirements

– Continuous culture using chemostat is an attractivepossibility but needs more work

Page 115: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Cell-to-CellSynchronization

Module

OscillatorModule

ChemotaxisModule

Cell Boundary

Conclusions

• We have designed and made strong progress towardsbuilding a synchronized chemotactic oscillator

• The assembly process, device characterization, andstandard operating conditions are key enablers whichwill greatly benefit the field of synthetic biology

Page 116: S.M.U.G. - Amazon S3 as part of the full system Cell-to-Cell Synchronization Module Oscillator Module Chemotaxis Module Individual’s Osc Phase Population’s Osc Phase Chemotaxis

Cell-to-CellSynchronization

Module

OscillatorModule

ChemotaxisModule

Cell Boundary

Conclusions

• We have designed and made strong progress towardsbuilding a synchronized chemotactic oscillator

• The assembly process, device characterization, andstandard operating conditions are key enablers whichwill greatly benefit the field of synthetic biology

Questions?


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