Building Biological Systems from Standard Parts

1. 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

2. Maxwell / DarwinPhysics / Biology1900’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

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

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

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

7. Systems Biologyvs.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

8. Powerful tools of engineering design • abstraction • hierarchy • modularity • standardization • isolation, separation of concerns • flexibility

9. Abstraction model catabolism anabolism Real world complexity Constructed complexity Small core of standard parts Design information

10. Abstraction model catabolism anabolism Living systems, waste Food Metabolic intermediates AAs, NTPs, core metabolites genome

11. Abstraction model Abstraction barrier Requirements Implementations

12. Abstraction layers Part Standard interfaces Contracts Abstractions Abstraction layer

13. 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

14. 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 Broad, deep designer Tall, thin designer Carver Mead, 1980 Mead & Conway, Introduction to VLSI Design

15. 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

16. 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

17. 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

18. 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

19. 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”

20. “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

21. Several Standards • Standard components & interfaces • Standard composition • Standard function & interfaces • Standard measurements • Standard chassis

22. 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

23. 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

24. 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

25. Assembly 3-Way vector origin antibiotic resistance E P X S 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

26. DARPA Biocomp PlasmidDistribution 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

27. 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

28. MIT Synthetic Biology, IAP Class 2003 Laura Wulf, MIT News Office c.2003 No prerequisites, no credit, consumes most of January… 13 waitlisted students Four project teams, shared components sixty fabricated components – Blue Heron

29. 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

31. 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

32. 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 hq@igem.org • Me: tk@mit.edu

33. 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