paper-based synthetic gene networks
Post on 05-Aug-2015
128 Views
Preview:
TRANSCRIPT
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
Synthetic Biology: Reprogramming Life
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
Potential for Synthetic Gene Networks
Medicine and Global Health
Food Supply
Ecological Monitoring
Synthetic Gene Networks
Harness the Power and Diversity of Biology
Potential for Synthetic Gene Networks
Medicine and Global Health
Food Supply
Ecological Monitoring
Synthetic Gene Networks
Living cells
In vitro Transcription and Translation
Cytoplasmic extracts: cell-free transcription and translation
E. coli
• Handling of solution phaseCytoplasmic extracts: cell-free transcription and translation
Challenges
E. coli
In vitro Transcription and Translation
2 mm paper disc
Paper-Based Synthetic Biology
E. coli
Cell-free extracts
Synthetic gene network
K Pardee et al., Cell, 2014
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
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
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.
Freeze-dried system maintains activity > 225 days
Fresh
Freeze Dried
Reconstitution of transcription and translation activity from freeze-dried cell extracts.
Distribution without Refrigeration
Toehold Switches: De Novo Designed Regulators
A Green et al., Cell, 2014
Switch RNA
Trigger RNA
Activated Complex
Paper-Based: Inducible RNA Toehold Switches
Paper-Based: Inducible RNA Toehold Switches
10–100 fold induction
Measurable signal in 25-30
minutes
5 uM RNA trigger
2 mm paper discs
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
Paper-Based SGN: Colorimetric Output
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
Electronic Optical Reader: Quantification & Automation
Chip with printed paper-based array is loaded into the device
Device cost < $35
Full-length mRNA Sensors
Full-length mRNA Sensors: Antibiotic Resistance
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
Rapid Prototyping: Ebola Sensors
4 to 77 fold induction in presence of
36-nt RNA trigger
Sudan strain RNA sensors
Zaire strain RNA sensors
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
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
Paper-based platform for synthetic gene networks
Moving Forward
Synthetic biology applications
Portable molecular manufacturing
Diagnostics
Research and education
Acknowledgements
Collins Labhttp:// www.bu.edu/abl
Keith Pardee
AlexGreen
EwenCameron
Tom Ferrante
Peng Yin
AjayKeyser
top related