IGEM Jamboree MIT – Nov 4th 2006
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iGEM Jamboree MIT – Nov 4th 2006. Engineering a Molecular Predation Oscillator. iGEM 2006 @ Imperial. Electrical Engineer. Biochemist. Biologists. Biomedical Engineers. Biomedical Engineers. Biochemists. Dr Mann. Bio-Memory. Oscillator. Bio-Clock. Project Ideas. Feasibility

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Igem jamboree mit nov 4th 2006

iGEM Jamboree MIT – Nov 4th 2006


Igem jamboree mit nov 4th 2006

Engineering a Molecular Predation Oscillator


Igem jamboree mit nov 4th 2006

iGEM 2006 @ Imperial

Electrical Engineer

Biochemist

Biologists

Biomedical Engineers

Biomedical Engineers

Biochemists

Dr Mann


Project ideas

Bio-Memory

Oscillator

Bio-Clock

Project Ideas

  • Feasibility

  • Originality of Design

  • BioBrick Availability

  • Future Impact


What is an oscillator

Our Definition

Device producing a periodic variation in time

of a measurable quantity, e.g. amplitude.

What is an Oscillator?

Engineering

Biology


The engineering approach

Design

Specifications

Modelling

Implementation

Testing/Validation

The Engineering Approach

Standard

Engineering

Practice

Certified


The main challenges

Requirement for a typical engineering oscillator

Our Specifications:

  • Sustained Oscillations

  • High Signal to Noise Ratio

  • Controllable Oscillations

  • Standardized Device for Easy Connectivity

  • Stability: >10 periods

  • SNR:High

  • Flexibility: Controllable Amplitude and Frequency

  • Modular Design

  • Easy Connectivity

Fluorescence

Time (min.)

The Main Challenges

Main challenges of past oscillators:

  • Unstable

  • Noisy

  • Inflexible

Repressilator

Figure Reference : Michael B. Elowitz & Stanislas Leibler Nature 2000


Our initial design ideas

Large populations of molecules to reduce influence of noise

Oscillations due to population dynamics

A well characterized model

Our Initial Design Ideas

Based on

Molecular Predator - Prey


The lotka volterra model

Prey Growth

Prey Killing

by Predator

Predator Growth

Predator Death

Population

Size

d

d

dt

dt

Time

The Lotka-Volterra Model

X

aX

bXY

Y

cXY

dY

X

Y

:Prey

: Predator


Typical lv simulations

Typical LV Simulations

Graph of Prey vs. Time

Low Frequency

High Frequency

prey

prey

Small

Amplitude

time

time

prey

prey

Large

Amplitude

time

time


Required biochemical properties

A

B

A

B

A

B

d

d

dt

dt

Required Biochemical Properties

Prey Growth

Prey Killing

by Predator

Self promoted

expression of A

Degradation

of A by B

A

B

Predator Growth

Predator Death

Expression of B

promoted by

AB interaction

Degradation

of B

Population

Size

A

B

Time


Igem jamboree mit nov 4th 2006

A

B

A

B

Molecular System

Prey Generator Cell

A

Self promoted

expression of A

  • Cell-cell communication

  • Constraint: Chemostat

  • Flexibility: Ratio of populations

Degradation

of A by B

A

Predator Generator Cell

Degradation of B

Expression of B

promoted by AB

interaction

B


Quorum sensing quenching

AHL

+

LuxR

pTet

pLux

Quorum sensing/quenching

Vibrio fischeri

Bacillus

LuxR

LuxI

LuxI

LuxR

aiiA

aiiA

pLux

BioBricksavailable

Forms a complex with AHL to activate pLux

Makes AHL

BBa_C0062

BBa_C0061

Degrades AHL

pLux

pLux Promoter

BBa_R0062

BBa_C0160

AHL->Pops Receiver

BBa_F2620


Designing the prey generator

AHL

AHL

AHL

LuxI

C0061

F2620

LuxR

LuxR

pLux

tetR

pLux

tetR

LuxI

LuxR

LuxR

LuxI

Designing the Prey Generator

Required

Dynamic

Self promoted

expression of A

Useful

BioBricks

Final

Construct


Designing the predator generator

AHL

AHL

AHL

AHL

AHL

LuxR

aiiA

Natural

degradation

pLux

C0062

C0060

R0062

LuxR

LuxR

AHL Lactonase

pLux

LuxR

aiiA

Sensing

Killing

Designing the Predator Generator

Required

Dynamic

Expression of B

promoted by

AB interaction

Degradation

of A by B

Degradation

of B

Useful

BioBricks

Final

Construct


Igem jamboree mit nov 4th 2006

AHL

AHL

AHL

AHL

AHL

AHL

LuxR

LuxR

LuxR

LuxR

AHL Lactonase

System Overview

Prey Generator Cell

Pool of AHL will oscillate

LuxI

Predator Generator Cell

LuxR

LuxI

pTet

pLux

aiiA

LuxR

pLux


Full system set up

Cell population

Input signal

Change in population ratio

Prey molecule generator

time

[AHL]

Output signal

LuxR

LuxI

pLux

pLux

tetR

LuxR

aiiA

time

Predator molecule generator

Full System set-up

reservoir

In-flow

Wash-out

Well mixed culture

In chemostat


Path to our goal

Specifications

Design

Modelling

Implementation

Testing

Modelling

Full system

Path to Our Goal

Start!

Specifications

oscillator

Based on

Lotka-Volterra

Modelling

Lotka-Volterra

Based on

Quorum Sensing


Modelling the full system

LuxR

aiiA

Modelling the Full System

AHL

d

Degradation of AHL by aiiA

Self promoted expression of AHL

Degradation of AHL

dt

Expression of LuxR

Degradation of LuxR

d

dt

Expression of aiiA

Degradation of aiiA

d

dt


Modelling the full system1

Modelling the Full System

Degradation of AHL by aiiA

Self promoted expression of AHL

Degradation of AHL

AHL

Expression of LuxR

Degradation of LuxR

LuxR

Expression of aiiA

Degradation of aiiA

aiiA


Modelling the full system2

Modelling the Full System

Gene Expression

Degradation of AHL by aiiA

Degradation of AHL

AHL

Degradation of LuxR

Production of LuxR

LuxR

Production of aiiA

Degradation of aiiA

aiiA


Modelling the full system3

Modelling the Full System

Gene Expression

Enzymatic Reaction

Degradation of AHL

AHL

Degradation of LuxR

Production of LuxR

LuxR

Production of aiiA

Degradation of aiiA

aiiA


Modelling the full system4

Modelling the Full System

Gene Expression

Enzymatic Reaction

Degradation

Degradation of AHL

AHL

Production of LuxR

LuxR

Production of aiiA

aiiA


Full system simulations

Full System Simulations

Graph of Prey vs. Time

Low Frequency

High Frequency

prey

prey

Small

Amplitude

time

time

prey

prey

Large

Amplitude

time

time


Typical system behaviours

Typical System Behaviours

Oscillations with limit cycles

No oscillations

Predator

Predator

Prey

Prey

Prey

Prey

Time

Time


Modelling the full system5

Population

dependent

[

]

[

]

b

aiiA

AHL

[

]

+

b

AHL

0

Wash-out

related

Constant

Modelling the Full System

Gene Expression

Enzymatic Reaction

Degradation

Degradation of AHL

AHL

Production of LuxR

LuxR

Production of aiiA

aiiA


Path to our goal1

Specifications

Design

Modelling

Implementation

Testing

Specifications

test constructs

Design

test constructs

Modelling

Test construct

Build

test constructs

Characterized

test constructs

Path to Our Goal

Start!

Specifications

oscillator

Based on

Lotka-Volterra

Modelling

Lotka-Volterra

Based on

Quorum Sensing

Modelling

Full system


Breaking down the complexity

Prey Generator

Predator Generator

Predator Sensing

Predator Killing

c

b

PoPs

AHL

c0

AHL

time

b0

Breaking Down the Complexity

Prey Sensing

a

PoPs

AHL

a0


Characterization predator sensing

Characterization Predator Sensing

Test part

Experimental Data

Predictive model

transfer function

Average with variance and curve fit

[GFP]

Experimental data

[AHL]


Characterization predator sensing1

Characterization Predator Sensing

Test part

Average with variance and curve fitting

Predictive model

transfer function

[GFP]

Experimental data

Fitting model to data

[AHL]

Parameter extractions


Implementation

Implementation

Assembly process

Registry Catalogue Parts

+

Prey

+

J37034

RS+J37034

+

Prey with Riboswitch

+

J37023

+

J37024

AiiA Test Construct

+

+

J37025

Final predator

+

+

J37033

+

J37019

Sensing predator

+

+

J37031

+

Sensing predator

+

J37032


On our experience

On Our Experience

Specifications

Design

Testing/Validation

Implementation

Modelling


Path to our goal2

Specifications

Design

Modelling

Implementation

Testing

Path to Our Goal

Start!

Specifications

oscillator

Based on

Lotka-Volterra

Modelling

Lotka-Volterra

Based on

Quorum Sensing

Modelling

Full system

Specifications

test constructs

Design

test constructs

Modelling

Test construct

Build

test constructs

Characterized

test constructs

Modelling

characterized sys

Build full

system

Characterized

Full system

Our Goal!


Contributions to the registry

Contributions to the Registry

Functional Parts

Built

Sequenced

Tested

Characterized

Documented

Final PreyJ37015

Sensing PreyT9002

Sensing PredatorJ37016

Cre/Lox J37027

Intermediate Parts

Built

Sequenced

Built

Sequenced

J37019

J37033

J37032

J37034

J37023


Our wiki

Our Wiki

  • Documentation

  • Communication

  • Organization

http://openwetware.org/wiki/IGEM:IMPERIAL/2006


Thank you

Acknowledgements:

Prof. Tony Cass

Dr. Anna Radomska

Dr. Rupert Fray

Dr. David Leak

Dr. Mauricio Barahona

Dr. Danny O'Hare

Dr. Geoff Baldwin

Susan E. Wryter

David Featherbe

Ciaran Mckeown

James Mansfield

Thank You

Funding:

  • European Commission

  • Imperial College Deputy Rectors Fund

  • Faculty of Engineering

  • Faculty of Natural Sciences


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