Paper-based synthetic gene networks
- 1. Paper-based synthetic gene networks James J. Collins Howard Hughes Medical Institute Dept of Biomedical Engineering & Center of Synthetic Biology Boston University Wyss Institute for Biologically Inspired Engineering Harvard University
- 2. Synthetic Biology: Reprogramming Life
- 3. Transfer to cell Test network dynamics Encode into DNA plasmid Design & model network #ofCells Off On 1 2 43 Gene Expression Synthetic Biology: Engineered Gene Networks TS Gardner et al., Nature, 2000
- 4. Potential for Synthetic Gene Networks Medicine and Global Health Food Supply Ecological Monitoring Synthetic Gene Networks Harness the Power and Diversity of Biology
- 5. Potential for Synthetic Gene Networks Medicine and Global Health Food Supply Ecological Monitoring Synthetic Gene Networks Living cells
- 6. In vitro Transcription and Translation Cytoplasmic extracts: cell-free transcription and translation E. coli
- 7. • Handling of solution phase Cytoplasmic extracts: cell-free transcription and translation Challenges E. coli In vitro Transcription and Translation
- 8. 2 mm paper disc Paper-Based Synthetic Biology E. coli Cell-free extracts Synthetic gene network K Pardee et al., Cell, 2014
- 9. 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
- 10. 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
- 11. 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.
- 12. Freeze-dried system maintains activity > 225 days Fresh Freeze Dried Reconstitution of transcription and translation activity from freeze-dried cell extracts. Distribution without Refrigeration
- 13. Toehold Switches: De Novo Designed Regulators A Green et al., Cell, 2014 Switch RNA Trigger RNA Activated Complex
- 14. Paper-Based: Inducible RNA Toehold Switches
- 15. Paper-Based: Inducible RNA Toehold Switches 10–100 fold induction Measurable signal in 25-30 minutes 5 uM RNA trigger 2 mm paper discs
- 16. 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
- 17. Paper-Based SGN: Colorimetric Output
- 18. 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
- 19. Electronic Optical Reader: Quantification & Automation Chip with printed paper-based array is loaded into the device Device cost < $35
- 21. Full-length mRNA Sensors: Antibiotic Resistance
- 22. 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
- 23. Rapid Prototyping: Ebola Sensors 4 to 77 fold induction in presence of 36-nt RNA trigger Sudan strain RNA sensors Zaire strain RNA sensors
- 24. 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
- 25. 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
- 26. Paper-based platform for synthetic gene networks Moving Forward Synthetic biology applications Portable molecular manufacturing Diagnostics Research and education
Editor's Notes
- #5: For example, detection of antibiotic resistance gene is a matter of 20 mins in a hospital or heavy metals in water in 20 min. Of course there is an existing infrastructure that can do this, PCR or mass spectrometry. However, many of these options require expensive equipment, technical training and time. My goal is to use the strengths of SGNs to make this capabilities available on a mass scale and for little cost.
- #6: For example, detection of antibiotic resistance gene is a matter of 20 mins in a hospital or heavy metals in water in 20 min. Of course there is an existing infrastructure that can do this, PCR or mass spectrometry. However, many of these options require expensive equipment, technical training and time. My goal is to use the strengths of SGNs to make this capabilities available on a mass scale and for little cost.
- #7: In the lab, cell-free Ts/Tl systems have been run in test tubes for decades. In the real world however, these reactions are labile and difficult to handle.
- #8: In the lab, cell-free Ts/Tl systems have been run in test tubes for decades. In the real world however, these reactions are labile and difficult to handle.
- #9: Gene circuits run in cell extracts that contain the proteins for transcription and translation. Field is in early days, with simple sensors and switches.
- #10: Gene circuits run in cell extracts that contain the proteins for transcription and translation. Field is in early days, with simple sensors and switches.
- #11: Gene circuits run in cell extracts that contain the proteins for transcription and translation. Field is in early days, with simple sensors and switches.