Synthetic Biology - Imperial College London

Synthetic Biology Synthetic Biology Engineering BiologicallyEngineering Biologically--based Devices and Systemsbased Devices and Systems

Professor Richard I Kitney

Imperial College London

Developments in Biology

The Molecular Biology The Molecular Biology Revolution 1953Revolution 1953--20032003

April 25April 25thth 19531953

The Double Helix Model

Scanning Tunnelling MicrographWatson and Crick – Nature 25th April 1953

Inventing the Information Age

Norbert Wiener

Cybernetics

Cybernetics (1948)

Claude Shannon

A Mathematical Theory of Communication in the Bell System Technical Journal (1948)

Information Theory

Sampling Theory

A sample

The Molecular Biology The Molecular Biology Revolution 1953Revolution 1953--20032003

April 25April 25thth 19531953

The Molecular Biology Revolution 1953-2003

1960 Sydney Brenner, et al prove the existence of mRNA

1961 Brenner and Crick determine how DNA instructs cells to make specific proteins

The Molecular Biology Revolution 1953-2003

1973 Genes are transferred to bacteria, which reproduce, generating multiple copies

1977 Fred Sanger and Walter Gilbert independently develop a technique to read the DNA chemical bases

We stand at the dawn of a new understanding of disease…

Nature 409, 860 - 921 (2001)Initial sequencing and analysis of the human genomeInternational Human Genome Sequencing Consortium The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

The dawn of molecular based medicine

The Tools and Techniques of The Tools and Techniques of Modern BiologyModern Biology

The Biological Continuum

• Systems• Viscera• Tissue• Cells• Proteins• Genes

• Systems• Visera• Tissue• Cells• Proteins• Genes

...

Interface

...Interface

Visualisation2

...

Interface

...Interface

Database

...Interface

User Input

DisplayVisualsation

1...

Display

... ...

Interface

...

Interface

User Input

Model 1

... ...

Interface

Model 2

Interface

Database

...

Interface

Advanced Web-based Information Systems

• Gel Electrophoresis • Western Blot• 1D Gel• 2D Gel

• Flow Cytometry / FACS• Microarray Experiments• Mass Spectrometry• Microscope Images

Techniques for Biological Data

Biology Levels

• Systems• Visera• Tissue• Cells• Proteins• Genes

Multi-scale Modelling

Network dynamicNetwork dynamicNetwork dynamic

Population

Cell-Agent

Petri Netsgene expression

modelling

within a diffusive environment

emergent properties due to interactive cell-agents

death

division

secretion

uptak

e

migration

differentiation

death

division

secretion

uptak

e

migration

differentiation

Population behaviourPopulation behaviourPopulation behaviour

So, what is Synthetic Biology?

Taking an engineering approach to design and applying it to Biology

The Engineering Approach to Design

• Abstraction• Decoupling• Standardisation

Implementation Modelling

Design

Specifications

Testing/Validation

The Engineering Approach

Standard

Engineering

PracticeCertified

The Engineering Approach to Design

• Engineering systems are built from a hierarchy PartsDevicesSystem

• At each level the characteristics of the Part, Device or System are well defined and reproducible

• In engineering the aim is to build a system on the basis of devices which comprise standard parts

Synthetic Biology: aims to build applications from Biobricks

• Parts – encode biological functions• Devices – made from a collection of

parts and encode human-defined functions (eg logic gates)

• Systems – perform tasks, eg counting

Programmed pattern formation (Basu, 2005)Image recording multi-cellular system (Levskaya, 2005)Terpenoids production system (Martin, 2003)Cancer-fighting drugs synthesis (Pfeifer, 2001)Biofilm formation (Kobayashi, 2004)Programmed cell population control (You, 2004)Controlled invasion of cancer cells (Anderson, 2006) …

Multicellular Systems

Inverters (Yolobayashi, 2002; Karig, 2004)Biophasic switch (Michalowski, 2004)Toggle switch (Gardner, 2000) Logic gates: AND and OR gates (Rackham, 2005) Oscillators (Elowitz, 2000; Fung, 2005)Pulse generator (Basu, 2004) …

Biological Devices

The MIT Registry of Standard Biological Parts (700+)PromotersTranscriptional regulators/terminatorsProtein coding regions Ribosome binding sites …

Biological Parts

ExamplesCategories

Genetic Circuits – Switches and Logic

After Christopher A Voigt 2006

Synthetic Biology - Building Parts, Devices and Systems

Modelling Underpinning Technology

Quality Assurance

Engineering Design Applications

Modelling (examples)– Part behaviour– Engineering modelling– Flux modelling– Stability (of living devices)– Metabolic engineering– Protein networks

The Key Components

Underpinning Technology– Part categorisation– DNA synthesis– Micro fluidics– Parts into single cells– Minimal organisms (synthetic host organisms)– Imaging

Quality Assurance– Adaptation evaluation– Engineering Design

» Circuits» Fabrication

Carlson R (2003). The Pace and Proliferation of Biological Technologies. Biosecurity and Bioterrorism: Biodefense Strategy, Practice, and Science, Vol. 1, No. 3, pp. 1-12.

The Synthetic Biology Pipeline ?

SoftwareOligio Micro

ArraysAssemble DNA

ConstructsDNA Error Correction

Large Scale Assembly

Data Devices Molecules

The MIT Registry

Examples of Parts

To see the catalogue of standard parts from the diagram above, hover over the part at the website below

http://parts2.mit.edu

Implementation Modelling

Design

Specifications

Testing/Validation

The Engineering Approach

Standard

Engineering

PracticeCertified

A Case Study

Engineering a Molecular Predation OscillatorEngineering a Molecular Predation Oscillator

BiomedicalEngineers

Biochemists

iGEM 2006 @ Imperial

Biologists

Biochemist

BiomedicalEngineers

ElectricalEngineer

Dr Mann

• Sustained Oscillations

• High Signal to Noise Ratio

• Controllable Oscillations

• Standardized Device forEasy Connectivity

Requirement for a typical engineering oscillatorOur Specifications:

• Stability: >10 periods• SNR: High• Flexibility: ControllableAmplitude and Frequency• Modular Design• Easy Connectivity

The Main Challenges Main challenges of past oscillators:

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

• Unstable

• Noisy

• Inflexible

Time (min.)

Fluo

resc

ence

Repressilator

Initial Design Ideas

• Large populations of molecules to reduce influence of noise

• Oscillations due to population dynamics• A well characterized model

Molecular Predator Molecular Predator -- Prey Prey

Based on

X

Y

bXY

The Lotka-Volterra Modelddt

ddt

TimeTime

Population Size

aX

cXY dY

Prey Killingby PredatorPrey Growth

Predator Growth Predator Death

: Prey

: Predator

X

Y

Typical LV Simulations

Small

Amplitude

Graph of Prey vs. Time

Large

Amplitude

Low Frequency High Frequency

prey prey

prey prey

time time

timetime

Prey Killingby PredatorPrey Growth

A

Required Biochemical Properties

TimeTime

Population Size

A

B

B

Predator Growth Predator Death

A A

AB B

B

ddtddt

Self promoted expression of A

Degradation of A by B

Expression of B promoted by AB interaction

Degradation of B

Molecular System

A

B

A

A

B

B

Self promotedexpression of A

Expression of B promoted by AB

interaction

Degradationof A by B

Degradation of B

A

• Cell-cell communication• Constraint: Chemostat• Flexibility: Ratio of populations

Prey Generator Prey Generator CellCell

Predator Predator Generator CellGenerator Cell

Designing the Prey Generator

RequiredDynamic

UsefulBioBricks

FinalConstruct

LuxILuxRtetR pLux

Self promoted expression of A

LuxIC0061

LuxRtetR pLuxF2620

LuxILuxR

LuxR AHL AHL

AHL

Degradation of B

Degradation of A by B

Expression of B promoted by AB interaction

AHLAHL

Lactonase

AHL

Killing

LuxRLuxR

AHL

AHLAHL

Sensing

Designing the Predator Generator

RequiredDynamic

pLuxLuxR aiiA

NaturaldegradationLuxR

C0062pLux

R0062

aiiAC0060

UsefulBioBricks

FinalConstruct

Prey Generator Cell

System Overview

LuxI

pLux

LuxR

pTet

LuxILuxR

LuxR AHL

AHL

AHL

LuxR

pLux

LuxR aiiA

LuxR AHL Lactonase

AHL

AHLAHL

Pool of AHL will Pool of AHL will oscillateoscillate

Predator Generator Cell

Full System set-up

Well mixed cultureIn chemostat

LuxILuxR

tetR pLux

pLuxLuxR aiiA

Prey molecule generator

Predator molecule generator

Wash-out

reservoir

In-flow

time

Change in population ratio

[AHL] Output signal

Cell population

time

Input signal

Full System set-up

Well mixed cultureIn chemostat

LuxILuxR

tetR pLux

pLuxLuxR aiiA

Prey molecule generator

Predator molecule generator

Wash-out

reservoir

In-flow

[AHL]

Cell population

time

time

Output signal

Change in population ratio

ddt

Modelling the Full System

Expression of aiiA

Degradation of AHL

Self promoted expression of

AHL

Degradation of AHL by

aiiA

AHL

LuxR

aiiA

ddt

ddt

Expression of LuxR

Degradation of aiiA

Degradation of LuxR

Modelling the Full System

aiiA Expression of aiiA

Degradation of AHL

Self promoted expression of

AHL

Degradation of AHL by

aiiAAHL

LuxR

dtAHLd ][

dtLuxRd ][

dtaiiAd ][

Expression of LuxR

Degradation of aiiA

Degradation of LuxR

Modelling the Full System

aiiA Production of aiiA

Degradation of AHL

Degradation of AHL by

aiiA

Production of LuxR

AHL

LuxR

dtAHLd ][

dtLuxRd ][

dtaiiAd ][

[ ][ ][ ][ ]LuxRAHLc

LuxRAHLc+0

[ ][ ][ ][ ]LuxRAHLc

LuxRAHLc+0

Gene Expression

[ ][ ]AHLa

AHLa+0

Degradation of aiiA

Degradation of LuxR

Modelling the Full System

aiiA Production of aiiA

Degradation of AHL

Production of LuxR

AHL

LuxR

dtAHLd ][

dtLuxRd ][

dtaiiAd ][ [ ][ ]

[ ][ ]LuxRAHLcLuxRAHLc

+0

Gene Expression

[ ][ ][ ]AHLb

AHLaiiAb+0

Enzymatic Reaction

[ ][ ][ ][ ]LuxRAHLc

LuxRAHLc+0

[ ][ ]AHLa

AHLa+0

Degradation of aiiA

Degradation of LuxR

Modelling the Full System

aiiA Production of aiiA

Degradation of AHL

Production of LuxR

AHL

LuxRdt

LuxRd ][

dtaiiAd ][ [ ][ ]

[ ][ ]LuxRAHLcLuxRAHLc

+0

Gene Expression Enzymatic Reaction

[ ]AHLe

[ ]LuxRd1

[ ]aiiAd2

Degradation

dtAHLd ][

[ ][ ][ ][ ]LuxRAHLc

LuxRAHLc+0

[ ][ ][ ]AHLb

AHLaiiAb+0

[ ][ ]AHLa

AHLa+0

Full System Simulations

Small

Amplitude

Graph of Prey vs. Time

Large

Amplitude

Low Frequency High Frequency

prey prey

prey prey

time time

timetime

Typical System BehavioursOscillations with limit cycles No oscillations

Prey

Predator

Prey

Time

Predator

Prey

Prey

Time

[ ][ ]AHLa

AHLa+0

Modelling the Full System

aiiA Production of aiiA

Degradation of AHL

Production of LuxR

AHL

LuxRdt

LuxRd ][

dtaiiAd ][ [ ][ ]

[ ][ ]LuxRAHLcLuxRAHLc

+0

Gene Expression Enzymatic Reaction

[ ]AHLe

[ ]LuxRd1

[ ]aiiAd2

Degradation

dtAHLd ][

[ ][ ][ ][ ]LuxRAHLc

LuxRAHLc+0

[ ][ ][ ]AHLb

AHLaiiAb+0

Populationdependent

Constant

Wash-outrelated

Characterisation Predator Sensing

Test part

Predictive model transfer function

Experimental data

[AHL]

[GFP

]

Experimental Data

Average with variance and curve fit

Characterisation Predator Sensing

Test part

Predictive model transfer function

Experimental data

Fitting model to data

Parameter extractions

[AHL]

[GFP

]

Average with variance and curve fitting

Implementation

Prey

Prey with Riboswitch

Sensing predator

Sensing predator

Final predator

J37034

J37033J37019

J37031

J37032

++

++

++

+

+

+

++

RS+J37034

J37025

J37024AiiA Test

Construct

J37023

++

+

Registry Catalogue

PartsAssembly process

Implementation Modelling

Design

Specifications

Testing/Validation

The Project Cycle

The Wiki

• Documentation• Communication• Organization

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

A 3rd Industrial Revolution in the Making (?)

• Parts, Devices and Systems

• Biofuels • Biomaterials • Medicines/Drugs/Vaccines • Biosensors

Synthetic Biology promises a shift comparable in importance to the ICT revolution with the power to revolutionise many sectors of the economy including:

The Companies

John Mulligan – Genetics Engineer

“It is possible to design a protein with a specific configuration on a computer and hen to access Blue heron’s software system to construct the DNA sequence that would produce it inside the cell.The protein and DNA may not exist in nature”

http://www.raeng.org.uk/policy/engagement/pdf/Systems_Biology_Report.pdf

The Hierarchy of Synthetic Biology and Quantitative Systems Biology

Now

Next 10yrs and beyond

Level 1

Level 2

Synthetic BiologySynthesis of Engineering

Devices and Systems – and industrial applications

Synthetic BiologySystems, Devices, Parts -Engineering Specification

Systems Biology

Engineering Systems and Signal Theory

Biology and Basic Medical Science

Systems BiologyHealthcare Applications Level 3

Educational AspectsEducational Aspects

Some thoughts from the UK

1. BSc/BEng - three years, e.g. a first degree in engineering or physics

2. MSc - two years, a masters in biology or basic medical science

3. PhD - three years (minimum), a doctorate in Systems Biology

Systems biology training based on the Bologna model

Concluding Remarks• We are now at a similar point to the late

19th Century where the great industries of the 20th Century (automotive, aircraft, ICT etc) did not exist

• Synthetic Biology, coupled to Systems Biology, will produce a third industrial revolution

The EndThe End