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

Synthetic biology: Solving problems in the developing world Rapid detection for the real world Schistosomiasis: 200 million infected Consulted experts Human Practices Panel Discussion Specifications

Schistosomiasis is a major global health problem Rapid detection for the real world Schistosomiasis: 200 million infected Consulted experts Caribbean area Human Practices Schistosomiasis-Endemic Areas Panel Discussion Hepatic/Intestinal Urinary Both Types Very Low Risk Specifications

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

Schistosoma: Complex lifecycle, problematic detection Rapid detection for the real world Schistosomiasis: 200 million infected Consulted experts Human Practices Panel Discussion Specifications

Multidisciplinary panel consulted to inform our design Rapid detection for the real world Schistosomiasis: 200 million infected Consulted experts Human Practices Panel Discussion Specifications

Human practices defined our initial specifications Rapid detection for the real world Specifications for a detection kit for water-borne parasites: Schistosomiasis: 200 million infected  Fast (minutes)  Inexpensive Consulted experts  Production  Testing equipment Human Practices  Easy to use, store and transport Panel Discussion  Safe Specifications

Our three stage synthetic biology device Input – Input – Input – Autoinducing Prarasitic TEV peptide protease protease Detection Signal transduction Output Signal Transduction Output – Output – Output – Autoinducing TEV Coloured peptide protease compound

The Engineering Design Cycle Human practices S D T Optimization A M S D M A T

Overview Detection Signal transduction Output P Output Time

Bacillus subtilis can be used safely Non-pathogenic Bacillus subtilis Sporulation 3 novel genomic integration vectors dif dif dif CMR GAAATTTC Int Int AmyE Starch catabolism PyrD Pyrimidine synthesis Dif-sites no spread of resistance

D e s i g n a n d A s s e m b l y GFP-XylE AmyE test vector AmyE final vector LytC CWB domain Surface protein TESTING Detection TEV and GFP-XylE TESTING ComE and ComD TESTING XylE TESTING for ComE-ComD AmyE FINAL construct ComE and ComD LacI repressed XylE Output Signal transduction Complete Assembly

Module I Detection Output Detection Signal transduction

Reliable Detection Specific single protease Robust reduced noise Fast efficient signalling S D M A T

The surface protein contains an Auto Inducing Peptide CWB AIP Linker AIP Signalling peptide Efficient Linear peptide No S. pneumoniae crosstalk S D M A T

The cleavable linker confers specificity CWB AIP Linker S Recognition W motive P L Specific S D M A T

Cell Wall Binding Domain for Cell-wall binding domain sequesters attachment ComC CWB AIP Linker Sequestration on cell wall Isolated from LytC- protein Cell Wall Binding Domain S D M A T

The detection module is highly efficient Surface [AIP] = 1.3 × 10 -3 M Optimal [AIP] = 4.4x10 -9 M S D A T M

Threshold levels are easily reached [protease] Using standard TEV-protease kinetics 10000 Testing with 1.7 min 1000 TEV-protease 100 10 1.7 1 0.1 0.01 10 1 100 1000 0.1 40 S D A T M

Assembly of the Detection Module Cell wall binding domain CWB Surface protein CWB AIP Linker CWB AIP His Linker Surface protein testing Linker 1 Linker 1 construct with 6 Linker 2 Linker 2 alternative linkers Linker 3 Linker 3 S D M A T

The Modularity of our Fundamental Technology • Schistosomiasis • Chagas‘ disease • C3 Convertase • Leishmanolysin Lumbar puncture Indicate acute infection 350 million people S D M A T

Our Software Tool S D M A T

Testing allows determination of optimal linker-version His 1. Salt elution and protein Surface [AIP] purification 2. Protease exposure and Cleavage efficiency protein purification S D M A T

Summary: Detection module Detection Signal transduction Output Specific single protease Robust reduced noise Assembled and Fast 1.7 minutes ready for testing Modular wide applicability S D M A T

Module II Signal Transduction Detection Signal transduction Output

Reliable Signal Transduction Specific single output Robust reduced noise S D M A T

S. pneumonia Peptide Quorum Sensing in B. subtilis S. pneumonia Com-system reduced In B. subtilis cross-talk ComE P TEV S D M A T

ComE activation depends on [Auto Inducing Peptide] and [Receptor] AIP-ComD*- ComE ↔ AIP -ComD + ComE* Production rate of ComE-P x10 -11 5 Rate(ComE P) ≈ Concentration (M) 4 [Receptor] 0 + [AIP] 0 3 2 1 0 0 15 5 10 Time (min) S D A T M

Assembly of the Signal Transduction Module K316013 K316014 ComE, ComD ComD expression ComE ComE responsive promoter, optimized RBS, TEV-protease S D M A T

Summary: Signal transduction Detection Signal transduction Output Specific single output Robust reduced noise + Detection Amenable to optimisation Signal transduction S D M A T

Module III Output Detection Signal transduction Output

Specification • Fast • Simple • Visual S D M A T

Direct Transcription/Translation Output Molecule (e.g. GFP) DNA Output Visibility Threshold Time S D M A T

1 Step Enzymatic Amplification Pre-synthesised DNA Enzyme Substrate Output Visibility Threshold Time S D M A T

2 Step Enzymatic Amplification Enzyme Deactivated Pre-synthesised DNA Enzyme Substrate Output Visibility Threshold Time S D M A T

Output Modelling • Equations developed to describe system • 1, 2 and 3 step amplifications modelled S D A T M

Our Design Catechol DNA XylE TEV Substrate Deactivated Enzyme Enzyme XylE Enzyme S D M A T

Our Design XylE XylE TEV Cleavable GFP XylE Linker XylE S D M A T

Output Assembly BBa_K316010 LacI XylE BBa_K316009 LacI TEV GFP XylE S D M A T

Extensive XylE characterisation Existing part - BBa_J33204 Spectra of cultures after catechol addition Optical Density / Arbitrary Units Wavelength / nm S D M A T

New Method for Characterising Promoters at Low Copy Numbers Pveg 3C/J23101 3C X E XylE Pveg 3K3/J23101 3K3 S D M A T

Determination of visibility threshold S D M A T

XylE-GFP fusion successfully limits enzyme activity 7,0 6,0 Natural XylE 5,0 Absorbance at 380nm 4,0 3,0 2,0 Visibility threshold 1,0 GFP-XylE 0,0 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 Time / minutes Time/minutes S D M A T

TEV cleavage successfully activates GFP-XylE 7,0 6,0 Natural XylE 5,0 Absorbance at 380nm Cleaved GFP-XylE 4,0 3,0 2,0 Visibility threshold 1,0 GFP-XylE 0,0 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 Time / minutes Time/minutes S D M A T

With characterisation data, our model shows 50% improvement S D M A T

Breakdown product attenuates cell growth S D M A T

Output Summary Output Detection Signal transduction • New mechanism • Faster than traditional systems • Simple visual output S D M A T

Specifications Achieved Detection Signal transduction Output Modular Customisable inputs and outputs 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) Extra data on the Characterisation of existing XylE registry Extra data on the Characterisation of novel GFP-XylE registry Submitted application to Gates Foundation

Thank you Any questions? Our Team of 10 undergraduates

Imperial College London