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Synthetic Biology1October 8, 2003These slides and notes were produced and written by Drew Endy(MIT). They were not reviewed and thus do not necessarily reflectthe individual or consensus opinions of study members or workshopparticipants.2003 Synthetic Biology Study1

Synthetic BiologyDrew EndyFellow of Biology & Biological Engineering, MITPatrick LincolnDirector of Computer Science, SRI, Inc.Richard MurrayDivision Chair, Engineering & Applied Science, Caltech2October 8, 2003Table of ContentsExecutive Summary3Participants4Engineering WorkflowCurrent5Proposed6Standard Biological PartsSynthetic Systems7Parts Standardization & Libraries8Abstraction9Decoupling Design & FabricationDNA Synthesis10Workflow11Registries of Standard Biological Parts12Biological RiskBackground13Current Tactics14Future Strategy15Suite of Solutions16ConclusionSummary17Technology Roadmap182003 Synthetic Biology Study2

Executive Summary Biology is a powerful technology––––Processing informationFabricating materialsConverting energyMaintaining & enhancing healthDesignParts &FabricationSystemsApplicationsApplication Biological technology poses a danger on par with any past experience– Existing threats– Emerging threats– Engineered threats Synthetic biology advances science & technology while mitigating danger– General capability to engineer biological systems– Increased speed and scope of response to threatsOctober 8, 20033Biology is a technology for processing information, materials, andenergy. As a technology platform, biological systems provide access to artifactsand processes across a range of scales (e.g., the ribosome is a programmablenanoassembler, a bamboo shoot can grow 12” per day). Biology also forms thebasis for human welfare (e.g., modest amounts of memory and logic,implemented as genetically encoded systems,would directly impact biologicalresearch and medicine). However, our ability to deploy biology as atechnology and to interact intentionally with the living world is nowlimited; the charge to our study was to begin to specify enabling technologiesthat, if developed, would provide a general foundation for the engineering ofbiology and make routine the creation of synthetic biological systems that behaveas predicted.We focused on improvements to the process of engineering biological systems.Three specific process improvements that should be pursued now are:(i) component standardization, (ii) substrate and componentabstraction, and (iii) design and fabrication decoupling.The development of technologies that enable the systematic engineering ofbiology must take place within the context of current and future risks due tonatural and engineered biological agents. While the development of technologiesfor engineering biology appears inevitable, and their distribution uncontrollable,the net impact such technologies will have on the creation of biological risk is notknown. However, any technology-based increase in risk creation seems likely to atleast be offset by a concomitant increase in the speed and scope of response torisks. Consequently, any meaningful strategy for minimizing futurebiological risk requires that the development of technologies forengineering biology proceeds alongside the development of nontechnical approaches to risk management; new training programs andprofessional societies will serve an important role in creating a cadre of engineerswho can work in biology and who will serve as a strategic resource for respondingto natural and engineered biological threats.2003 Synthetic Biology Study3

Study Participants Drew Endy (chair)Patrick Lincoln (co-chair)Richard Murray (co-chair)Frances Arnold (Caltech)Ralph Baric (UNC)Roger Brent (TMSI)Rob Carlson (U.Washington)Jim Collins (BU)Lynn Conway (Michigan)Ron Davis (Stanford)Mita Desai (NSF)Eric Eisenstadt (DARPA)Stephanie Forrest (U.New Mexico)Seth Goldstein (CMU)Homme Hellinga (Duke)Tom Kalil (UC Berkeley)Jay Keasling (UC Berkeley)October 8, 2003 Doug Kirkpatrick (DARPA)Tom Knight (MIT)Bill Mark (SRI)John Mulligan (Blue Heron)Radhika Nagpal (MIT/Harvard)Carl Pabo (Sangamo)Randy Rettberg (MIT)Pam Silver (Harvard)Brad Smith (Johns Hopkins)Christina Smolke (Caltech)Gerry Sussman (MIT)Jack Thorpe (ISAT)Claire Tomlin (Stanford)Jeff Way (Lexigen)Chris Webb (Stanford)Ron Weiss (Princeton)Erik Winfree (Caltech)4Study participants included representatives from universities, industry, andgovernment. Participants provided expertise in basic biological research,biological systems modeling, DNA synthesis, device analysis & design, selfassembly, systems analysis & design, computer science, electricalengineering, engineering theory, and biological security. Rich Entlich andother staff provided expert organizational support throughout the study.The study held three workshops and four executive meetings:1. October 23-24th (2002) at the Beckman Center in Irvine, CA2. March 3-4th (2003) at SRI, Inc. in San Mateo, CA (workshop)3. March 24-25th at Norton’s Woods in Cambridge, MA (workshop)4. April 10-11th at IDA in Alexandria, VA5. May 29-30th at Caltech in Pasadena, CA (workshop)6. August 18-22nd at Johnson House in Woods Hole, MA7. October 8th in Alexandria, VAThe following related events occurred while the study was underway:1. IBEA contracted by DOE to synthesize a bacterial genome (11/02)[see http://www.bioenergyalts.org/news.html]2. MIT conducts Synthetic Biology Lab (1/03)[see l]3. Caltech announces Center for Biological Circuit Design (3/03)[see f]4. EU NEST proposes Synthetic Biology research program (8/03)[see ftp://ftp.cordis.lu/pub/nest/docs/synthetic biology.pdf]5. Lawrence Berkeley Lab creates Dept. of Synthetic Biology (8/03)[see r/]2003 Synthetic Biology Study4

Current WorkflowDesign &FabricationDevicesSystemApplication5October 8, 2003At present, the design and fabrication of any specific engineeredbiological system is an ad hoc process. The process often involvesfundamental scientific research, making impossible accurate prediction of finalsystem behavior and time-to-delivery. Furthermore, building any one systemdoes not directly enable the construction of other engineered biologicalsystems. For a given application, work begins with the coupled design andfabrication of unique, application-specific components that, given further work,can sometimes be assembled into a functioning system. By contrast, “mature”engineering disciplines (e.g., mechanical, electrical, civil, and software) canroutinely integrate large numbers of well-characterized components to producemany functioning systems.2003 Synthetic Biology Study5

Scaleable WorkflowDesignParts cationsApplicationSynthesisOctober 8, 20036Biology presents a new medium for engineering; we expect to encounter manymedium-specific challenges (e.g., evolution). Still, a scaleable developmentpath for biological systems engineering can make use of past successfulexperience in other engineering disciplines. The approach that emerged overthe course of the study is informed by three past lessons:(1) Standardization of components (mechanical engineering, 1800s).Libraries of standard parts that allow a combination of systems to be designedand assembled.(2) Component abstraction (from physics to electrical engineering, 1900s).Standard parts can be defined prior to absolute scientific knowledge; manyknowable facts are unnecessary. Simpler representations of many-componentdevices help to manage complexity and increase attainable system scale.(3) Decoupling of design & fabrication (VLSI electronics, 1970s).Engineered systems can be designed by experts to exploit every detail of aparticular fabrication process. However, too much attention to such detailslimits both the rate of design and the complexity of designable systems.In addition to the above, improvements in four technical areas would help toenable the engineering of biological systems:(1) Registries of standard biological parts that coordinate parts synthesisand system assembly, and help to develop and promulgate standards ofpractice (e.g., design, fabrication, and characterization).(2) Long polymer nucleic acid synthesis enabling rapid delivery of newcomponents and systems.(3) Design, simulation, and analysis tools that make use of standard partsand methods of assembly.(4) Methods and standards for measurement that enable rapid systemcharacterization and refinement.2003 Synthetic Biology Study6

Synthetic SystemscILacIOLacRBSλ cI-857TcIMichael Elowitz c.1999LacI7October 8, 2003Synthetic biological systems are currently created from naturalbiological “parts” via an expert-driven design and fabricationprocess.For example, a genetic “inverter” based on well-known natural biological partsregulating gene expression in bacteria is shown (top left). The inverter is builtfrom a “repressor” protein (LacI) that acts on an operator (O Lac) to inhibittranscription of the DNA-encoded elements to the right of O Lac. Absentinhibition at O Lac, RNA molecules encoding both a ribosome binding site(RBS) and the gene cI-857 are produced. The resulting RNA are translated tocreate a new protein (cI).The inverter’s transfer function (bottom left) depicts the relationship betweeninverter input (concentration of LacI protein) and output (concentration of cIprotein). A ring oscillator can be created by linking three such inverters in acyclic system (A inverts B inverts C inverts A).The actual behavior of bacterial cells containing a genetically-encoded ringoscillator is shown (right); cells blink over time as a function of oscillator state- see Elowitz et al. in Nature v403 p335, “A synthetic oscillatory network oftranscriptional regulators.”2003 Synthetic Biology Study7

Parts: Standardization & LibrariesM1752, Western Electric c. 1951Bardeen & Brattain c. 1947Many sorts, most places, c. 2003 Zif268, Paveltich & Pabo c. 1991Random Zif268s, Greisman & Pabo c. 1997TATAZF-6 & TATAZF-2, Wolfe et al. c. 20018October 8, 2003The ring oscillator on the preceding page took an expert over a year to design andbuild; the system makes use of three of the best-characterized natural proteins thatregulate gene expression in bacteria. Today, an electrical engineer could build a ringoscillator in 5 minutes by, for example, taking a N7404A hex-inverter “off the shelf”and connecting three of the inverters in a cycle using standard gauge wire.To enable the routine production of many-component integrated biologicalsystems we should develop libraries of standard biological parts. Initially,most parts will be “harvested” from natural systems. Development of domainspecific parts would enable the engineering of systems responsive to differentapplications of biological technology. Parts specific to the following application areaswould find immediate use: biological information processing and control, materialfabrication, metabolism and energy production, and human health.Also, it is worth noting that the continued improvement and successful application ofcomputation-based design of ligand-domain reactions will help to enable theengineering of synthetic parts libraries - e.g., see Looger et al. in Nature v423 p185,“Computational design of receptor and sensor proteins with novel function.” Weshould foster the transition from the collection and characterization ofnatural parts, to the design and fabrication of libraries of synthetic parts.One early example of the transition from natural to synthetic parts is likely to be thedevelopment of a library of synthetic DNA binding proteins for use in the regulationof gene expression; “back of the envelope” estimates suggest that a standard libraryof 1,000 zinc-finger protein:DNA binding site pairs can be created with 1% intralibrary component “crosstalk.” Crosstalk refers to the fact that biological systems areoften composed of self-mixing molecules. Specificity of interaction is determined bynon-covalent interactions between molecule surfaces (in contrast to wires thatdetermine component interactions in electrical circuits); non-specific or unintendedmolecule-molecule interactions can create undesirable side-effects in biologicalsystems.2003 Synthetic Biology Study8

Parts: AbstractionRBScILacIcIrate inλ cI-857Trate inOLacRBSλ cI-857Oλrate outTBBa QPI 05.29.70rate outcIOctober 8, 2003rate outrate inLacI9We also need to promote the characterization and representation ofstandard biological parts in ways that insulate relevant physicalcharacteristics from overwhelming physical detail. For example, thefour DNA elements defining a typical genetic inverter (above left) are organizedsuch that the concentration of the LacI and cI protein are the inverter inputand output, respectively; the result is a unique device with characteristicsspecific to LacI and cI (transfer function, bottom left). If LacI protein wereused to regulate a different output (e.g., TetR protein) then a new device,requiring additional characterization, would be created. Instead, byreorganizing the genetic components, we can create an inverter that isindependent of specific input and output chemicals (top right); devicecharacteristics are now defined as a function of input and output rates (bottomright). The resulting inverter can be used in combination with any other sostructured device. Also, for the purpose of integrated system design, the fourcomponent inverter (top right) can be replaced with a simpler representation ofa new part, a “quad-part inverter” (middle right).Again, representations of biological systems should (1) provide simpledescriptions of complex, oftentimes poorly understood, biologicalcomponents and processes and (2) allow the creation of parts that canbe used in combination with other parts (e.g., parts whose inputs andoutputs are not specific to other parts).2003 Synthetic Biology Study9

Decouple: DNA Synthesis100000001000000Bases of DNAPer Person earFree-living cellSystemPart10October 8, 2003The above plot gives the bulk number of nucleotides that can be assembled intoshort chain oligomers by an individual during an 8-hour work day - see Carlson,Biosecurity & Biotechnology v1(3), “Pace & Proliferation of BiologicalTechnologies.” The increase in DNA synthesis capacity has been driven largelyby process parallelization; anecdotal reports suggest that equivalenttechnology is available worldwide. For scale, the length of a typical part is 1,000 bases, the length of a small integrated system is 10,000 bases, andthe length of a genome encoding a “simple” free-living cell is 1,000,000 bases.One critical factor not represented on the above plot is the time todelivery of a fully-assembled long chain oligomer. Commercial deliverytimes for an assembled 10,000 base DNA oligomer are now 10 weeks. Otherlimiting factors are specific to various fabrication processes. For example,methods based on short chain oligomer-assembly followed by cloning andligation allow for exact synthesis but require extra time for the processing ofassembly intermediates (e.g., cloning and sequencing); such methods are alsolimited by the potential genetic instability of assembly intermediates. Synthesismethods based on in-vitro assembly (e.g., PCR) can be faster but often accrueerrors during synthesis and assembly that carry forward into the final product.DNA sequencing technology underwent a similar increase in capacity beginning 1980; the science of biology changed in response (e.g., the genome projects,bioinformatics, and systems biology). Today, researchers in an average biologylab might spend half their time manipulating DNA molecules. Continuedimprovements in DNA synthesis technology should, in addition topromoting biological engineering, help change biology from a“discovery” science to a “synthetic” science (a lá the development ofsynthetic chemistry) and help to enable a more rapid response tofuture biological risks.2003 Synthetic Biology Study10

Decouple: Design, Parts & Fab., Systems– Model-based design using standard biological parts– Parts fabrication via commercial suppliersSynchronization ofan oscillator, IAP ‘038 Jan15 Jan22 Jan29 Jan8/0311October 8, 2003Just as being able to read DNA via genome-scale sequencing efforts has notimmediately translated into a perfect understanding of nature’s designs, ourincreasing ability to write DNA via de novo synthesis will not, byitself, result in useful engineered biological systems.During January 2003, MIT conducted a four-week long experimental course inwhich 16 students were asked to design genetically-encoded oscillators usingan early version of what future biological engineers might call “protein-DNAlogic” (or PDL). Students were given a 20,000 base pair DNA synthesisbudget. In addition, students were asked to design their systems usingstandard biological parts such that the resulting parts could be used in morethan one system (i.e., parts were shared across the class). The courseworkflow was: (i) model-based system design, (ii) model-driven simulation,(iii) layout, documentation, and plan of characterization, (iv) parts ordering viacommercial suppliers, and (v) parts return and system assembly. Design,simulation, layout and documentation took one month. Editing the studentspecified parts and placing the parts synthesis order took two months. Partssynthesis required another one to five months. System assembly fromstandard parts is taking an additional four months, for a total elapsed time ofone year. Current estimates are that the 2004 course will run to completion infive months and that, given current technology, three months start-finish willbe realized. The students who participated in the course did notperform laboratory experiments, instead they worked as standardbiological parts, device, and system engineers.By decoupling design and fabrication it will become possible to build morecomplex systems. The decoupling of system design and fabrication willsimultaneously enable a new cadre of engineers to participate in theanalysis and design of biological systems.2003 Synthetic Biology Study11

Decouple: Registries of Standard Parts Maintain & promulgatestandards of s & ExchangeApplications Coordinate partssynthesis Coordinate systemassembly12October 8, 2003The usefulness of standard biological parts depends on parts characterizationvia common standards of description and also parts availability via commonprotocols of exchange. In addition, system assembly requires coordination ofde novo DNA synthesis and final system assembly with bulk-service providers;as with DNA sequencing, DNA synthesis will benefit from economies of scale.Registries of standard biological parts should be created to best meetthese requirements.A standard biological parts registry is similar in concept to the MOSIS serviceprovided to the VLSI electronics community [see http://www.mosis.org/]. Byserving as a focal point for community organization registries willprovide a mechanism for community-wide organization and thedevelopment and propagation of standards of practice. Furthermore,such community-wide organization will help launch the future organizations(private and public) that will support the engineering of many-componentintegrated biological systems.At present, DNA synthesis and system assembly is slow and expensive (i.e.,fabrication is currently a limiting technology). As a result, early registries willneed to provide both the physical DNA itself and the information specifyingDNA sequence and encoded genetic function. In the future, if and when DNAsequence information becomes fungible with DNA molecules, knowledge ofwhat to synthesize will be limiting. At such time, the role of registries shouldshift to serve as maintainers and providers of the inf

Synthetic biology advances science & technology while mitigating danger –General capability to engineer biological systems –Increased speed and scope of response to threats Systems ApplicationsApplication Parts & Fabrication Design Biology is a technology for processing information, materials, and energy.

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