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 was. -- Theodore von Karman

Maxwell / Darwin Physics / Biology 1900’s / 2000’s Science ~ 1870 Electrical engr. ~ 1905 Major ideas: modularity, hierarchy, information, black box behavior, feedback, design & synthesis, control of materials, technological substrate Perfect devices Science ~ 1960 Synthetic biology ~ 2000 Major ideas: modularity, hierarchy, information, black box behavior, feedback, design & synthesis, control of materials, technological substrate Perfect behavior

Major societal problems Energy & raw materials Environmental protection and cleanup Health & aging Defense against natural and unnatural events

Science and Engineering Natural organisms Engineered organisms Knowledge & understanding Excellent models Science Systems Biology Engineering Synthetic Biology

Science and Engineering Engineered organisms Knowledge & understanding Excellent models Science & Systems Biology of natural organisms Engineering & Synthetic Biology using standard parts Parts Repository De novo DNA synthesis Revised knowledge and new techniques

Systems Biology vs. Synthetic Biology Based on Standard Parts Systems Biology - 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 Synthetic Biology Based on Parts - 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 Small core of standard parts Real world complexity Constructed complexity catabolismanabolism Design information

Abstraction model Metabolic intermediates AAs, NTPs, core metabolites Food Living systems, waste catabolismanabolism genome

Abstraction model RequirementsImplementations Abstraction barrier

Abstraction layers Standard interfaces Contracts Abstractions Part 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 Differential equations: KCL, KVL, device models, network theory State change, abstract behavior 1E9 components

Types of designers 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 Tall, thin designer Broad, deep designer 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 Carver Mead, 1980 Mead & Conway, Introduction to VLSI Design Spacing rules Fanout rules Signal restoration rules Run Microsoft software

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 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 Actuators 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 cgt CTTAAG cgccggcg a AGATCT c EcoRI XbaI t ACTAGT a GCGGCCG CTGCAG gct a TGATCA t cgccggc GACGTC cga SpeI PstI E X S P No internal sequences of the form EcoRI: GAATTC XbaI: TCTAGA SpeI: ACTAGT PstI: CTGCAG

Assembly 3-Way E P E X S t A CTAGA a a TGATC T t SpeI XbaI t ACTAGA a a TGATCT t mixed E X S P X S P vector origin antibiotic resistance

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 – ECFP Plac – EYFP Plac – ECFP – EYFP Plac – EYFP – ECFP Plac – ECFP – T1 – EYFP Plac – EYFP – T1 – ECFP Need standardized measurement techniques Need good modeling tools

No prerequisites, no credit, consumes most of January… 13 waitlisted students Laura Wulf, MIT News Office c.2003 MIT Synthetic Biology, IAP Class 2003 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 Me:

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 -- Leonardo da Vinci