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Synthetic Biology I
The Programming of Cells
Nawwaf Kharma & Luc VarinArtificial Life GroupElec. & Comp. Engineering and Biology DepartmentsConcordia University, Montréal (QC), [email protected]
Artificial Life Group The Programming of Cells 2
Synthetic Biology- What & Why
• It is an emerging area of research that combines science and engineering to envision, design and realize novel structures and functions (mainly in cells) by (primarily) modifying the genome (within the cells)
• Cells have advantages:• They have/can have built-in
interfaces, to sense and produce many substances
• They are easy to mass produce, store and distribute
• They are generally more robust than man-made systems
• They are optimizable via directed evolution
Artificial Life Group The Programming of Cells 3
Biological Refresher:Flow of genetic information
DNA
Transcription
RNA
RNA
Translation
Protein
Proteins
Can interact with each other
Artificial Life Group The Programming of Cells 4
Basic Paradigm:From the ground up
• First: Basic Components
Combinatorial & Sequential
• Second: Larger Circuits
With more/larger components and/or more functionality
• Third: Communications & Pattern Formation
Leading to even greater functionality
>> Finally, future Challenges that must be met if the field is to advance >>>
Artificial Life Group The Programming of Cells 5
Combinatorial Components I:Promoter-based Regulation
Kaern et al. (2003)
Combinatorial Components II:siRNA-based Regulation
Artificial Life Group The Programming of Cells 6Deans et al. (2007)
Artificial Life Group The Programming of Cells 7
Combinatorial Components III:Ribozyme-based Regulation
Win et al. (2008)
Artificial Life Group The Programming of Cells 8
Sequential Components I: First Toggle
Gardner et al. (2000)
Sequential Components II:Various Oscillators
Artificial Life Group The Programming of Cells 9Elowitz et al. (2000)Wong et al. (2006)
Artificial Life Group The Programming of Cells 10
Sequential Components III:Mammalian Memory
Ajo-Franklin et al. (2007)
Sequential Components IV: Pulsers & Delays
Artificial Life Group The Programming of Cells 11Kaesling et al. (2006)
Delays using repressors only- how?
Combinatorial Components include: Switches Inverters Logic Gates
Sequential Components include: Oscillators (ON-OFF-ON-OFF-…)
Toggles (ON or OFF, depending on Input & State)
1-bit Memory (ON or OFF, depending on Input)
Pulse-rs (ON for a short while, then auto-OFF)
Delays (Output follows Input after a Delay)
Next: Scale-up & combine components to build circuits
Artificial Life Group The Programming of Cells 12
Summary 1 of 3
Artificial Life Group The Programming of Cells 13
Larger Circuits I: Combinatorial Logic (4 inputs/1 output)
Rinaudo et al. (2007)
Artificial Life Group The Programming of Cells 14Rinaudo et al. (2007)
Larger Circuits II:Sequential Logic (multi-bit memory)
Artificial Life Group The Programming of Cells 15Ham et al. (2008)
Artificial Life Group The Programming of Cells 16
Larger Circuits III: Modular Design (- also involving intercellular communication)
Kobayashi et al. (2004)
Ex1: Larger Combinatorial Circuit with: Multi-input Logic Function with 4 (and 5) inputs and 1 output
Ex2: Multi-bit Memory that: Set precisely using a series of inputs Grows exponentially with no. of invertases
Ex3: Modular Circuit with: 3 intra-cellular modules that Sense, Process, and Output information It also communicates with other cells
Artificial Life Group The Programming of Cells 17
Summary 2 of 3
Artificial Life Group The Programming of Cells 18
Advanced Topics I: Pattern Formation (using inter-cellular communications)
Basu et al. (2005)Bernhardt et al. (2007)
Advanced Topics II: Inter-kingdom Communications (using another kind of inter-cellular communications)
Artificial Life Group The Programming of Cells 19Weber et al. (2007)
Advanced Topics II continued
Inter-kingdom Communications
Artificial Life Group The Programming of Cells 20Weber et al. (2007)
Advanced Topics III:Applications
Artificial Life Group The Programming of Cells 21Rodrigo et al. (2007)
Ex1: Pattern Formation Is necessary for autonomous
differentiation of large collections of cells
Ex2: Inter-cellular Communications Is necessary for coordinated action by
larger collections of cells
Ex3: Applications Necessary to justify the significant cost
of investment in “blue-sky” research
Artificial Life Group The Programming of Cells 22
Summary 3 of 3
Future Challenges I: Reliable Well-Characterized Modules
Artificial Life Group The Programming of Cells 23Canton et al. (2008)
Future Challenges II: Synchronization & Communications
Artificial Life Group The Programming of Cells 24
How can a large number of components communicate and work synchronously across large distances? Possible answers include:
A.A large number of freely-diffusing signalsB.A much smaller number of short-distance signalsC.Isolated channels (e.g. neurons) that pass signals
As to operating synchronously: simulations have been made but no implementations yet!
Garcia-Ojalvo et al. (2004)
Future Challenges III: Interfacing with Electro/Optical Components
Artificial Life Group The Programming of Cells 25Weber et al. (2009)
How can electronic devices be interfaced to the new cellular devices? Possibilities include:
A.Light at different frequenciesB.Electric fieldC.Chemical reactions, such as:
A mammalian cell-based frequency generator: DC power converts ethanol into acetaldehyde, which dose-dependently triggers expression of the BMP-2 in engineered rat cardiomyocytes (AIRNRC-BMP-2) and increases the contraction frequency
Artificial Life Group The Programming of Cells 26
Final Summary
Cells are not like human-engineered machinesCells, if fully understood, may be treated as machinesCells are not fully understood or characterized
Researchers are starting to see potential in manipulating the genome of cells
And they are continuing to expand the frontier while simultaneously improving the reliability of existing components
There has been great progress, but still more needs to be done to:A. Build a basic repertoire of reliable well-characterized combinatorial &
sequential componentsB. Communicate and synchronize across large distances and in different
mediumsC. Establish controllable methods for growing large patterns of inter-acting
cells
Artificial Life Group The Programming of Cells 27
References
Kaern et al. (2003): Mads Kærn,William J Blake, and J J Collins. “The Engineering of Gene Regulatory Networks.” Annual Review of Biomedical Engineering 5:179–206.
Deans et al. (2007): Tara L Deans, Charles R Cantor & James J Collins. “A Tunable Genetic Switch Based on RNAi and Repressor Proteins for Regulating Gene Expression in Mammalian Cells.” Cell 130, 363–372.
Win et al. (2008): Maung Nyan Win & Christina D Smolke. “Higher-Order Cellular Information Processing with Synthetic RNA Devices.” Science vol. 322, 456-460.
Gardner et al. (2000): Timothy S Gardner, Charles R Cantor & James J Collin. “Construction of a genetic toggle switch in Escherichia coli.” Nature vol. 403, 339-342.
Elowitz et al. (2000): Michael B Elowitz & Stanislas Leibler. “A synthetic oscillatory network of transcriptional regulators.” Nature vol. 403, 335- 338.
Wong et al. (2006): W W Wong and J C Liao. “The design of intracellular oscillators that interact with metabolism.” Cell. Mol. Life Sci. vol. 63:1215–1220.
Ajo-Franklin et al. (2007): Caroline M Ajo-Franklin, David A Drubin, Julian A Eskin, Elaine P S Gee, Dirk Landgraf, Ira Phillips & Pamela A Silver. “Rational design of memory in eukaryotic cells.” Genes and Development 21:2271–2276.
Kaesling et al. (2006): http://keaslinglab.lbl.gov/wiki/index.php/Synthetic_Biology_-_Devices_-_Pulse_Generator
Rinaudo et al. (2007): Keller Rinaudo, Leonidas Bleris, Rohan Maddamsetti, Sairam Subramanian, Ron Weiss & Yaakov Benenson. “A universal RNAi-based logic evaluator that operates in mammalian cells.” Nature Biotechnology vol. 25, No. 7: 795-801.
Artificial Life Group The Programming of Cells 28
References continued
Ham et al. (2008): Timothy S Ham, Sung K Lee, Jay D Keasling & Adam P Arkin. “Design and Construction of a Double Inversion Recombination Switch for Heritable Sequential Genetic Memory.” PloS ONE, vol. 3, No. 7: 1-9.
Kobayashi et al. (2004): Hideki Kobayashi, Mads Kærn, Michihiro Araki, Kristy Chung, Timothy S Gardner, Charles R Cantor & James J Collins. “Programmable cells: Interfacing natural and engineered gene networks.” PNAS vol. 101, No. 22: 8414–8419.
Basu et al. (2005): Subhayu Basu, Yoram Gerchman, Cynthia H Collins, Frances H Arnold & Ron Weiss. “A synthetic multicellular system for programmed pattern formation.” Nature, vol. 434: 1130-1134. IET Synth. Biol., vol. 1, no. 1–2: 29–31.
Bernhardt et al. (2007): K Bernhardt, E J Carter, N S Chand, J Lee, Y Xu, X Zhu, J W Ajioka, J M Goncalves, J Haseloff, G Micklem and D Rowe. “New tools for self-organised pattern formation.”
Weber et al. (2007): Wilfried Weber, Marie Daoud-El Baba & Martin Fussenegger. “Synthetic ecosystems based on airborne inter- and intrakingdom communication.” PNAS, vol. 104, no. 25: 10435–10440.
Rodrigo et al. (2007): G Rodrigo, A Montagud, A Aparici, M C Aroca, M Baguena, J Carrera, C Edo, P Fernandez-de-Cordoba, A Ferrando, G Fuertes, D Gimenez, C Mata, J V Medrano, C Navarrete, E Navarro, J Salgado, P Tortosa, J Urchueguia and A Jaramillo. “Vanillin cell sensor.” IET Synth. Biol., vol. 1, no. 1–2: pp. 74–78.
Artificial Life Group The Programming of Cells 29
References continued
Alexic et al. (2007): J Aleksic, F Bizzari, Y Cai, B Davidson, K de Mora, S. Ivakhno, S L Seshasayee, J Nicholson, J Wilson, A Elfick, C French, L Kozma-Bognar, H Ma & A Millar. ” Development of a novel biosensor for the detection of arsenic in drinking water.” IET Synthetic Biolology, vol. 1, no. 1–2: 87–90.
Canton et al. (2008): Barry Canton, Anna Labno & Drew Endy. “Refinement and standardization of synthetic biological parts and devices.” Nature Biotechnology, vol. 26, no. 7: 787-793.
Garcia-Ojalvo et al. (2004): Jordi Garcia-Ojalvo, Michael B Elowitz, and Steven H Strogatz. “Modeling a synthetic multicellular clock: Repressilators coupled by quorum sensing.” PNAS, vol. 101, no. 30: 10955–10960.
Weber et al. (2009): Wilfried Weber, Stefan Luzi, Maria Karlsson, Carlota Diaz Sanchez-Bustamante, Urs Frey, Andreas Hierlemann & Martin Fussenegger. “A synthetic mammalian electro-genetic transcription circuit.” Nucleic Acids Research, vol. 37, no. 4.
Artificial Life Group The Programming of Cells 30