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