iGEM 2010 Parasite detection with a rapid response 200m Making the - - PowerPoint PPT Presentation

Imperial College London iGEM 2010

“Parasite detection with a rapid response”

200µm

Making the Invisible Visible

Rapid detection for the real world Schistosomiasis: 200 million infected Consulted experts Specifications Human Practices Panel Discussion

Synthetic biology: Solving problems in the developing world

Schistosomiasis is a major global health problem

Caribbean area Schistosomiasis-Endemic Areas

Urinary Hepatic/Intestinal Very Low Risk Both Types

Rapid detection for the real world Schistosomiasis: 200 million infected Consulted experts Specifications Human Practices Panel Discussion

Schistosoma: Complex lifecycle, problematic detection

Rapid detection for the real world Schistosomiasis: 200 million infected Consulted experts Specifications Human Practices Panel Discussion Professor Alan Fenwick & Dr Wendy Harrison, Schistosomiasis Control Initiative (SCI) at Imperial College London Dr Julie Balen London School of Hygiene and Tropical Medicine Dr Martha Betson Natural History Museum, London

Schistosoma: Complex lifecycle, problematic detection

Rapid detection for the real world Schistosomiasis: 200 million infected Consulted experts Specifications Human Practices Panel Discussion

Multidisciplinary panel consulted to inform our design

Rapid detection for the real world Schistosomiasis: 200 million infected Consulted experts Specifications Human Practices Panel Discussion

Human practices defined our initial specifications

Specifications for a detection kit for water-borne parasites:

Fast (minutes) Inexpensive

• Production
• Testing equipment

Easy to use, store and transport Safe

Rapid detection for the real world Schistosomiasis: 200 million infected Consulted experts Specifications Human Practices Panel Discussion

Signal Transduction

Input – Prarasitic protease Output – Autoinducing peptide Input – Autoinducing peptide Input – TEV protease Output – TEV protease Output – Coloured compound

Output Signal transduction Detection

Our three stage synthetic biology device

The Engineering Design Cycle

Human practices

Optimization

S D M A S D

M

A T T

Overview

Output Time P

Output Signal transduction Detection

Bacillus subtilis can be used safely

AmyE Starch catabolism PyrD Pyrimidine synthesis Dif-sites no spread of resistance

CMR dif dif

Int Int

3 novel genomic integration vectors

GAAATTTC

Bacillus subtilis

Non-pathogenic Sporulation

dif

LytC CWB domain Surface protein TESTING AmyE final vector AmyE test vector

GFP-XylE

ComE and ComD

ComE and ComD TESTING

XylE TESTING for ComE-ComD

TEV and GFP-XylE TESTING AmyE FINAL construct

LacI repressed XylE

D e s i g n a n d A s s e m b l y

Detection Signal transduction Output Complete Assembly

Detection

Output Signal transduction Detection

Module I

Specific

single protease

Robust

reduced noise

Fast

efficient signalling

Reliable Detection

S D M A T

Signalling peptide

The surface protein contains an Auto Inducing Peptide

Efficient

Linear peptide

No

• S. pneumoniae

crosstalk

AIP

CWB

Linker

AIP M A T

S D

The cleavable linker confers specificity

S W P L

CWB

AIP

Linker

Recognition motive

Specific

M A T

S D

Cell-wall binding domain sequesters ComC

Cell Wall Binding Domain for attachment

Cell Wall Binding Domain

Isolated from LytC- protein

Linker

CWB

AIP

Sequestration

• n cell wall

M A T

S D

The detection module is highly efficient

Optimal [AIP] = 4.4x10-9 M Surface [AIP] = 1.3×10-3 M

D A T S

M

Threshold levels are easily reached

Using standard TEV-protease kinetics

[protease]

Testing with TEV-protease

0.1 1 10 100 1000 0.01 0.1 1 10 100 1000 10000 40 1.7

1.7 min

D A T S

M

Assembly of the Detection Module

Linker

AIP

CWB

Linker

AIP

CWB

His

Surface protein Surface protein testing construct with 6 alternative linkers Cell wall binding domain

Linker 1 Linker 2 Linker 3

CWB

Linker 1 Linker 2 Linker 3

D T S

M A

Lumbar puncture

The Modularity of our Fundamental Technology

• Leishmanolysin
• C3 Convertase
• Chagas‘ disease
• Schistosomiasis

Indicate acute infection 350 million people

D T S

M A

Our Software Tool

D T S

M A

His

• 1. Salt elution and protein

purification

• 2. Protease exposure and

protein purification

Testing allows determination of

• ptimal linker-version

Surface [AIP] Cleavage efficiency

D S

M A T

Specific single protease Robust

reduced noise

Fast

1.7 minutes

Summary: Detection module

Modular

wide applicability

Output Signal transduction Detection

Assembled and ready for testing

S D M A T

Signal Transduction

Output Signal transduction Detection

Module II

Specific single output Robust reduced noise

Reliable Signal Transduction

S D M A T

In B. subtilis

ComE

P

• S. pneumonia Peptide Quorum

Sensing in B. subtilis

• S. pneumonia Com-system

reduced cross-talk

TEV

M A T

S D

ComE activation depends on [Auto Inducing Peptide] and [Receptor]

AIP-ComD*-ComE ↔ AIP-ComD + ComE*

Production rate of ComE-P

5 10 15 1 2 3 5 4 x10-11 Time (min) Concentration (M)

Rate(ComEP) ≈ [Receptor]0 + [AIP]0 D A T S

M

Assembly of the Signal Transduction Module

ComE

ComD

ComE, ComD expression

K316013 K316014

ComE responsive promoter,

• ptimized RBS, TEV-protease

D T S

M A

Specific single output Robust reduced noise

Summary: Signal transduction

Output Signal transduction Detection

Amenable to

• ptimisation

Signal transduction Detection

+

S D M A T

Output

Output Signal transduction Detection

Module III

• Fast
• Simple
• Visual

Specification

S D M A T

Output Molecule (e.g. GFP) DNA

Direct Transcription/Translation

Time Output Visibility Threshold

M A T

S D

1 Step Enzymatic Amplification

Pre-synthesised Substrate DNA Enzyme

Time Output Visibility Threshold

M A T

S D

2 Step Enzymatic Amplification

DNA Enzyme Pre-synthesised Substrate Deactivated Enzyme

Time Output Visibility Threshold

M A T

S D

Output Modelling

• Equations

developed to describe system

• 1, 2 and 3 step

amplifications modelled D A T S

M

Our Design

Catechol Substrate Deactivated XylE Enzyme DNA TEV Enzyme XylE Enzyme

M A T

S D

Our Design

GFP Cleavable Linker XylE TEV XylE XylE XylE

M A T

S D

Output Assembly

BBa_K316010 BBa_K316009

XylE

GFP TEV LacI XylE LacI

D T S

M A

Extensive XylE characterisation

Existing part - BBa_J33204

Spectra of cultures after catechol addition

Optical Density / Arbitrary Units

Wavelength / nm

D S

M A T

New Method for Characterising Promoters at Low Copy Numbers

Pveg 3K3/J23101 3K3 Pveg 3C/J23101 3C

XylE E X

D S

M A T

Determination of visibility threshold

D S

M A T

0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5

Absorbance at 380nm Time / minutes

XylE-GFP fusion successfully limits enzyme activity

Natural XylE GFP-XylE Visibility threshold

Time/minutes

D S

M A T

TEV cleavage successfully activates GFP-XylE

0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5

Absorbance at 380nm Time / minutes

Cleaved GFP-XylE Visibility threshold Natural XylE GFP-XylE

Time/minutes

D S

M A T

With characterisation data, our model shows 50% improvement

D S

M A T

Breakdown product attenuates cell growth

D S

M A T

Output Summary

• New mechanism
• Faster than

traditional systems

• Simple visual output

Output Signal transduction Detection

S D M A T

Specifications Achieved

Output Signal transduction Detection

Modular Customisable inputs and

• utputs

Fast response < 8 minutes Easy to use Clear visual output Easy to store Spore-forming chassis & transport Inexpensive Low cost testing kit Safe Non-pathogenic chassis, vectors & Dif excision

Building different prototypes allowed us to contextualise our detection kit

Synthetic Biology School Workshops

Synthetic Biology School Workshops

Parasight Achievements

Real world application - Schistosomiasis Human practices defined our project Fast visual output Modular (surface protein – software tool) Characterisation of existing XylE Characterisation of novel GFP-XylE Submitted application to Gates Foundation

Extra data on the registry Extra data on the registry

Thank you Any questions?

Our Team of 10 undergraduates

Imperial College London