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Building Biological Systems from Standard Parts Tom Knight MIT Computer Science and Artificial Intelligence Laboratory IGEM Headquarters Ginkgo Bioworks Inc. A Scientist discovers that which exists; an Engineer creates that which never Maxwell / Darwin Physics / Biology 1900’s / 2000’s Science ~ 1870 Science ~ 1960 Electrical engr. ~ 1905 Synthetic biology ~ 2000 Major ideas: modularity, hierarchy, information, black box behavior, feedback, design & synthesis, control of materials, technological substrate Major ideas: modularity, hierarchy, information, black box behavior, feedback, design & synthesis, control of materials, technological substrate Perfect devices Perfect behavior Major societal problems • Energy & raw materials • Environmental protection and cleanup • Health & aging • Defense against natural and unnatural events Science and Engineering Knowledge & understanding Excellent models Science Systems Biology Natural organisms Engineering Synthetic Biology Engineered organisms Science and Engineering Science & Systems Biology of natural organisms Knowledge & understanding Excellent models Parts Repository De novo DNA synthesis Engineered organisms Revised knowledge and new techniques Engineering & Synthetic Biology using standard parts Systems Biology vs. Synthetic Biology Based on Standard Parts Systems Biology Synthetic Biology Based on Parts - Models of natural systems - New discoveries from data analysis and fusion - Understanding of noise and other effects in natural systems - Success measured in match of the model to nature - Embrace natural complexity - Parts designed for use by others - Engineering design tools - Simulators - Industrial development of good parts and devices - Simple organisms to hold designs - iGEM team success is based on parts - Registry is the primary catalog of parts - Success measured in generality and utility of parts, systems and protocols -Remove natural complexity Powerful tools of engineering design • • • • • • abstraction hierarchy modularity standardization isolation, separation of concerns flexibility Abstraction model catabolism anabolism Real world complexity Constructed complexity Small core of standard parts Design information Abstraction model catabolism anabolism Living systems, waste Food Metabolic intermediates AAs, NTPs, core metabolites genome Abstraction model Abstraction barrier Requirements Implementations Abstraction layers Part Standard interfaces Contracts Abstractions Abstraction layer Abstractions in electronics User Application software Operating system, user interface Programming language Instruction set architecture Virtual machine Computer hardware design Functional computing units Logic synthesis Logic gates Circuit design Transistors Mask geometry Fabrication technologies Semiconductor physics Quantum physics State change, abstract behavior 1E9 components Differential equations: KCL, KVL, device models, network theory Types of designers User Application software Operating system, user interface Tall, thin designer Programming language Instruction set architecture Virtual machine Computer hardware design Functional computing units Broad, deep designer Logic synthesis Logic gates Circuit design Transistors Mask geometry Fabrication technologies Semiconductor physics Quantum physics Carver Mead, 1980 Mead & Conway, Introduction to VLSI Design Standards & Design Rules User Application software Operating system, user interface Programming language Instruction set architecture Virtual machine Computer hardware design Functional computing units Logic synthesis Logic gates Circuit design Transistors Mask geometry Fabrication technologies Semiconductor physics Quantum physics Run Microsoft software Fanout rules Signal restoration rules Spacing rules Carver Mead, 1980 Mead & Conway, Introduction to VLSI Design Complexity Reduction User Application software Operating system, user interface Programming language Instruction set architecture Virtual machine Computer hardware design Functional computing units Logic synthesis Logic gates Circuit design Transistors Mask geometry Fabrication technologies Semiconductor physics Quantum physics 100’s of OS calls 100 statements 100’s of instructions 10’s of units 10’s of gate types 4 types of transistors 15 mask layers 6 materials Complexity Reduction • Good News: Biology is modular and abstract Evolution needs modular design as much as we do We can discover the modular designs, modify them, and use them Learn New Engineering Principles from Biology Coping with errors Design with unreliable components Design with evolution Self organization Self repair Molecular scale construction Biology is the nanotechnology which works Role of Standards in Engineering • Simplified thinking about interfaces: Design rules Composition: Structural / Functional • Reusable Parts • Contracts and commercial access • Independent evolution of components and technologies • Facile comparison of results “The good thing about standards is that there are so many to choose from” “In this country, no organized attempt has yet been made to establish any system, each manufacturer having adopted whatever his judgment may have dictated as best, or as most convenient for himself.” Williams Sellers “On a Uniform System of Screw Threads” Franklin Institute April 21, 1864 Several Standards • Standard components & interfaces • Standard composition • Standard function & interfaces • Standard measurements • Standard chassis Biobricks: Standard Biological Parts • Snap together Lego block assembly Mechanical compatibility • Output of one component suitable as input of next component Functional compatibility Input Sensors Computational Devices Output Actuators Naturally Occurring Sensor and Actuator Parts Catalog Actuators Sensors • • • • Light (various wavelengths) Magnetic and electric fields pH Molecules • Autoinducers H2S maltose serine ribose cAMP NO Internal State Cell Cycle Heat Shock • Chemical and ionic membrane potentials • Motors Flagellar Gliding motion • • • • • • • • • • Light (various wavelengths) Fluorescence Autoinducers (intercellular communications) Sporulation Cell Cycle control Membrane transport Exported protein product (enzymes) Exported small molecules Cell pressure / osmolarity Cell death Standard Component Form gca GAATTC gcggccgc t TCTAGA g t ACTAGT a GCGGCCG CTGCAG gct cgt CTTAAG cgccggcg a AGATCT c a TGATCA t cgccggc GACGTC cga EcoRI XbaI SpeI X S E No internal sequences of the form EcoRI: XbaI: SpeI: PstI: GAATTC TCTAGA ACTAGT CTGCAG PstI P Assembly 3-Way vector origin antibiotic resistance E P X E X S S P t A a TGATC SpeI CTAGA a T t XbaI t ACTAGA a a TGATCT t mixed E X S P DARPA Biocomp Plasmid Distribution 1.0 May 2002 • Standard vectors, components, protocols • Very limited coverage – Plac, ECFP, EYFP, lacZ, T1 Assembled compound structures • Enough to get started • More coming soon Lux systems from V. fischeri and P. luminescens cI, p22-C2, tetR, luxR Antibiotic resistance, pACYC & pSC101 ori Autoinducer systems from V. fischeri, P. aeruginosa Some toy experiments • • • • • • Plac Plac Plac Plac Plac Plac – – – – – – ECFP EYFP ECFP – EYFP EYFP – ECFP ECFP – T1 – EYFP EYFP – T1 – ECFP • Need standardized measurement techniques • Need good modeling tools MIT Synthetic Biology, IAP Class 2003 Laura Wulf, MIT News Office c.2003 Grace June-Wha Maia Connie Louis Alex Danny Jose Reshma Vinay Ty Samantha Neel Voichita Brian Peter Kenney Rhee Mahoney Tao Waldman Wissner-Gross Shen Pacheco Shetty Mahajan Thomson Sutton Varshney Marinescu Chow Carr 6/7 6/7 6 6 2/6 6/8/18 6 10/14 BE BE BE BE HST HST MAS MAS 2006 2006 2005 2004 2005 2003 2005 2003 G G G G G G G RS No prerequisites, no credit, consumes most of January… 13 waitlisted students Four project teams, shared components sixty fabricated components – Blue Heron Key Ideas • Build system out of standard parts Pre-optimized for assembly • Use standard techniques to assemble them No surprises Routine Robot assembly • Network effects on the size of the library 6 -> 5500 • Couple functional and physical designs Parts have a logical function, not random DNA fragments • Measured and characterized for modeling First time success • Part collections of similar interchangeable parts Standard Plasmids • pSB1A3 pSB “synthetic Biology” 1 -> high copy number origin (pUC19 e.g.) A -> Ampicillin resistant 3 -> Biobrick cloning site with up and downstream terminators • Available antibiotics A ampicillin (orange) 100 ug/ml C chloramphencol (green) 35 ug/ml K kanamycin (red) 50 ug/ml T tetracycline (yellow) 15 ug/ml • Available origins - pSC101, p15A, inducible • We need parts returned to the Registry in 1 series plasmids if possible • VF2, VR sequencing primer locations Resources • IGEM home pages: igem.org Past team project wikis, posters, presentations • Registry of standard biological parts: Partsregistry.org • Openwetware: openwetware.org Searching the literature • IGEM headquarters [email protected] • Me: [email protected] Synthetic Biology • An Engineering technology based on biology which complements rather than replaces standard approaches • Engineering synthetic constructs will Enable quicker and easier experiments Enable deeper understanding of the basic mechanisms Enable applications in nanotechnology, medicine and agriculture Become the foundational technology of the 21st century Simplicity is the ultimate sophistication