paper-based synthetic gene networks
TRANSCRIPT
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Paper-based synthetic gene networks
James J. Collins
Howard Hughes Medical InstituteDept of Biomedical Engineering & Center of Synthetic BiologyBoston University
Wyss Institute for Biologically Inspired EngineeringHarvard University
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Synthetic Biology: Reprogramming Life
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Transfer to cell Test networkdynamics
Encode into DNAplasmid
Design & model network
# o
f C
ells
Off On
1 2 43Gene Expression
Synthetic Biology: Engineered Gene Networks
TS Gardner et al., Nature, 2000
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Potential for Synthetic Gene Networks
Medicine and Global Health
Food Supply
Ecological Monitoring
Synthetic Gene Networks
Harness the Power and Diversity of Biology
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Potential for Synthetic Gene Networks
Medicine and Global Health
Food Supply
Ecological Monitoring
Synthetic Gene Networks
Living cells
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In vitro Transcription and Translation
Cytoplasmic extracts: cell-free transcription and translation
E. coli
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• Handling of solution phaseCytoplasmic extracts: cell-free transcription and translation
Challenges
E. coli
In vitro Transcription and Translation
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2 mm paper disc
Paper-Based Synthetic Biology
E. coli
Cell-free extracts
Synthetic gene network
K Pardee et al., Cell, 2014
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Paper-based transcription and translation reactions
Paper-Based Synthetic Biology
Bright Field
2 mm paper disc2 mm paper disc
E. coli
Cell-free extracts
Fluorescence
GFP Plasmid
- +
Synthetic gene network
K Pardee et al., Cell, 2014
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Paper-based transcription and translation reactions
Paper-Based Synthetic Biology
2 mm paper disc2 mm paper disc
E. coli
Cell-free extracts
Synthetic gene network
GFP Plasmid
- +
Bright Field
Fluorescence
K Pardee et al., Cell, 2014
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Freeze dried-cell extracts
Freeze-dried system maintains activity
Fresh
Freeze Dried
Distribution without Refrigeration
Reconstitution of transcription and translation activity from freeze-dried cell extracts.
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Freeze-dried system maintains activity > 225 days
Fresh
Freeze Dried
Reconstitution of transcription and translation activity from freeze-dried cell extracts.
Distribution without Refrigeration
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Toehold Switches: De Novo Designed Regulators
A Green et al., Cell, 2014
Switch RNA
Trigger RNA
Activated Complex
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Paper-Based: Inducible RNA Toehold Switches
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Paper-Based: Inducible RNA Toehold Switches
10–100 fold induction
Measurable signal in 25-30
minutes
5 uM RNA trigger
2 mm paper discs
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Paper-Based SGN: Colorimetric Output
Chlorophenol Red-β-D-galactopyranoside (CPRG)
Enzyme-mediated color change of CPRG substrate to phenol red.
Phenol red Abs 575 nm
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Paper-Based SGN: Colorimetric Output
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2. Print reaction chambers on chromatography paper
Printing Paper-Based Arrays for Reactions
3. Spot reactions onto arrays for freeze drying
1. Wax-based printer
4. Rehydrate and incubate at room temperature
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Electronic Optical Reader: Quantification & Automation
Chip with printed paper-based array is loaded into the device
Device cost < $35
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Full-length mRNA Sensors
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Full-length mRNA Sensors: Antibiotic Resistance
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Rapid Prototyping: Ebola Sensors
• Constructed and tested 24 sensors in 12 hours
• Sudan and Zaire-strain specific sensors
• Input DNA cost $21/sensor
• Compares very favorably to antibody-based technologies
• Current outbreak, took > 80 days for first molecular confirmation
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Rapid Prototyping: Ebola Sensors
4 to 77 fold induction in presence of
36-nt RNA trigger
Sudan strain RNA sensors
Zaire strain RNA sensors
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Rapid Prototyping: Ebola Sensors
240 reactions tested in parallel
Colorimetric output for direct deployment to low technology environments
Good strain discrimination Sensitivity to 30 nM for both strains
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Key Features of Our Fieldable Paper-Based System
Freeze-dried reactions
•Room temperature storage (many months)
•4¢ – 65¢ per sensor
Electronic optical reader
•Quantification and automation
•< $35/device
Colorimetric output
•Read with the naked eye
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Paper-based platform for synthetic gene networks
Moving Forward
Synthetic biology applications
Portable molecular manufacturing
Diagnostics
Research and education
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Acknowledgements
Collins Labhttp:// www.bu.edu/abl
Keith Pardee
AlexGreen
EwenCameron
Tom Ferrante
Peng Yin
AjayKeyser