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Workshop Overview
Pages 1-104

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From page 1...
... The realization of viral and microbial genomics, in the last few decades of the 20th century, coupled with the completion of the initial draft of the human genome sequence in 2001, reflect a fundamental shift in the way biology is stud ied, and has opened a portal to vast postgenomic possibilities. Because of the Human Genome Project, scientists have already identified more than 1,800 genes associated with particular diseases.
From page 2...
... By inserting the genetic machinery for metabolic pathways into Escherichia coli and other host organisms, scientists are attempting to create microbial bio-factories for the production of pharmaceutical ingredients, flavors, fragrants, and other chemical products (Ro et al., 2006)
From page 3...
... This workshop summary report is not intended to be an exhaustive exploration of the subject matter nor does it represent the findings, conclusions, or recommendations of a consensus committee process.
From page 4...
... What differentiates synthetic biology from genetic engineering is its goal of designing new genetic systems and organisms using standardized parts from the "ground up." Although the term "synthetic biology" has been used in various ways, it is generally understood to describe research that combines biology with the principles of engineering to design, construct, or adapt existing DNA, or other biological structures into standardized, interchangeable, building blocks for use in creating genetic systems that carry out desired functions. The vision behind this science is that these biological "parts" can be joined to create engineered cells, organisms, or biological systems that reliably behave in predictable ways to perform specific tasks (Khalil and Collins, 2010; NSABB, 2010; Presidential Com mission for the Study of Bioethical Issues, 2010; Royal Academy of Engineering, 2009)
From page 5...
... Don't believe the hype." (Dr. Collins' contribution to the workshop summary report can be found in Appendix A, pages 117-150.)
From page 6...
... Investigators have increasingly embraced systems approaches in their efforts to understand biological interactions, taking advantage of the power of mathematical and computer modeling to exam ine the complex interactions between components of a biological system (Royal Academy of Engineering, 2009)
From page 7...
... Synthetic biologists are making headway toward handling more complex structures in more efficient ways. Chris Voigt's work with code refactoring is an example of how advances in synthetic biology are paving the way for more re 5 Disease-perturbed proteins and gene regulatory networks differ from their healthy counterparts, because of genetic or environmental influences.
From page 8...
... (Dr. Ellington's contribution to the workshop summary report can be found in Appendix A, pages 150-159.)
From page 9...
... . Ellington reported that the reengineered viruses that he and his team built did not function once they were actually inserted into host cells.
From page 10...
... 10 FIGURE WO-2 Construction of bacteria that are capable of light-dark edge detection. SOURCE: Ellington (2011; adapted from Cell)
From page 11...
... (Dr. Sauro's contribution to the workshop summary report can be found in Appendix A, pages 394-417.)
From page 12...
... According to Collins, most practicing engineers use mathematical modeling as a guide only -- in much the same way that synthetic biologists do. When actually assembling components, engineers rely on intuition and "tinkering," often without even understanding how a system works.
From page 13...
... As discussed by Ellington and Sauro, evolution can, for example, change the dynamics of a synthetic biological system. As Sauro explained, the unpredict able nature of, and responses to, selection makes it very difficult for synthetic biologists to control the behavior of their engineered systems.
From page 14...
... (Dr. Keasling's contribution to the workshop summary report can be found in Appendix A, pages 243-254.)
From page 15...
... One of the key milestones for bottom-up synthetic biologists is the development of libraries of diverse, well-characterized biological components that can be assembled to form new systems -- analogous to how one assembles component parts on a computer motherboard. The aspirational goal of this effort is to one day be able to select these "parts" from a catalogue and use them to create completely synthetic "novel" self-replicating life forms that are purpose-built rather than derived from a preexisting organism.
From page 16...
... First, a design objective is identified. Next, a suitable synthetic biological system is designed given the known properties of well-characterized components (bottom-up)
From page 17...
... . Despite the fact that this is still a young science -- arguably in need of a "clear methodology" -- scientists are relying on the tools and approaches of systems bi ology to make tremendous leaps in their understanding of various physiological phenomena, such as the mammalian immune response, and creating the potential for synthetic biologists to translate that knowledge into practice (Westerhoff et al., 2009)
From page 18...
... Sequencing also allows synthetic biologists to verify whether their designed DNA circuits and parts have been correctly fabricated (Royal Academy of Engineering, 2009)
From page 19...
... FIGURE WO-6 Cost per base of DNA synthesis and sequencing. Figure WO-6.eps SOURCE: Carlson (2011)
From page 20...
... coli host cells in a combinatorial fashion and then uses accelerated directed evolution to select for cells with the desired properties (Mosberg et al., 2010)
From page 21...
... . Synthetic biologists use directed evolution to rap idly modify preexisting proteins and chemical pathways so that they perform new functions, and to develop new parts without needing to understand the mechanistic properties of a pathway or system at the level that would be necessary to design the parts from scratch (Dougherty and Arnold, 2009; Forster and Church, 2010)
From page 22...
... 22 FIGURE WO-7 Schematic presentation of directed evolution studies. In place of screening, one can exploit selection using conditions that favor the growth of mutants with desired properties.
From page 23...
... 1998. DNA shuffling of a family of genes from diverse species accelerates directed evolution.
From page 24...
... (Dr. Joyce's contribution to the workshop summary report can be found in Appendix A, pages 236-243.)
From page 25...
... . Arguably, the ultimate bottom-up synthetic biology achievement would be to build life from scratch.
From page 26...
... None of the known directed evolution technologies, at the moment, meet generally agreed-upon criteria for a working definition of life: "a self-sustained chemical system capable of undergoing Darwinian evolution." As Joyce observed, the pioneering Miller-Urey experiments of the 1950s did not meet the criteria of "life" either (Miller, 1953)
From page 27...
... . The only informational macromolecules in the system are the enzymes and their components, which themselves are subject to Darwinian evolution within the system.
From page 28...
... ( A) Beginning with 12 pairs of cross-replicating RNA enzymes, amplification was sustained for 20 successive rounds of ~20-fold amplification and 20-fold dilution.
From page 29...
... 29 WORKSHOP OVERVIEW along the diagonal. The number of clones containing each combination of components is shown on the vertical axis.
From page 30...
... The ultimate application, Joyce said, would be a replicator that invents its own function by evolving over time in response to the constraints of its envi ronment -- a feat that would require a significant level of genetic complexity. As Joyce observed, life on Earth, although vulnerable to extreme changes of environ mental conditions, has demonstrated extraordinary resiliency and inventiveness in adapting to highly disparate niches.10 Perhaps the most significant invention of life is a genetic system that has an extensible capacity for inventiveness, something that likely will not be achieved soon for synthetic biological systems (Joyce, 2011)
From page 31...
... Metagenomic Mining In addition to screening DNA databases, scientists are screening the natural world for potentially useful DNA sequences. Metagenomic mining involves the extraction of microbial genes from environmental samples without having cultivated the organisms (Rondon et al., 2000)
From page 32...
... . Synthetic biologists now can use the genetic and enzymatic machinery of this newly discovered pathway to engineer cells to convert renewable raw materials into biofuels (Sommer et al., 2010)
From page 33...
... According to Johnston, the ability to synthesize genes and other molecules, like synbodies, and then screen those compounds for their bioactivity holds great promise for vaccine discovery, antibody production, drug discovery, diagnostics, and other tools for managing emerging infectious diseases. Rebuilding Complex Functions Encoded by Multiple Genes Speaker Christopher Voigt's group, at the University of California, San Francisco, has been working with several of these gene clusters as part of an effort to develop a methodology for reengineering entire gene clusters.
From page 34...
... When software manufacturers experience a problem with their software, they may fix that problem by rewriting the code in such a way that the underlying software continues to function unchanged. In synthetic biology, refactoring involves rewriting the DNA sequence so that it is easier to engineer but in such a way that the fundamental functionality of that sequence remains the same.
From page 35...
... Synthetic parts 7. Synthetic "Controller" Sensors Circuits Wires Inducer Growth Stage Oxygen Membrane Stress DNA Synthesis FIGURE WO-13 "Refactoring" gene clusters.
From page 36...
... . Voigt's team demonstrated that they could modify the type III secretion system of Salmonella using synthetically designed genes to, in effect, turn the Salmonella into small spider silk factories (Widmaier and Voigt, 2010; Widmaier et al., 2009)
From page 37...
... (Dr. Freemont's contribution to the workshop summary report can be found in Appendix A, pages 159-178.)
From page 38...
... Moreover, accord ing to Palsson, microbial metabolic systems biology is being applied more and more to infectious disease and speaks to the interesting coupling that takes place between the host and the pathogen, and the many different microenvironmental niches that pathogens find in the human body. A microbial cell is a very crowded and interconnected space, placing severe constraints on biological functioning.
From page 39...
... SOURCE: Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Microbiology, copyright 2009.
From page 40...
... . The major objectives of speaker Bali Pulendran's research program at Emory University are to take a systems-level approach toward understanding how some of the many existing successful vaccines mediate immune responses.
From page 41...
... The first clues to how the vaccine "works" were reported in 2006, and suggest that the vaccine activates multiple Toll-like receptors (TLRs) via multiple subsets of dendritic cells,14 eliciting a broad spectrum of immune responses (Querec et al., 2006)
From page 42...
... This observation is illustrated in Figure WO-15. Pulendran expressed the hope that the growing effort to identify gene profiles that correlate with different types of immune system responses -- both the innate and adaptive immune responses -- may eventually lead to development of a generic vaccine chip containing 50 to 100 genes that can be used to predict the immune responsiveness to a broad range of vaccines.
From page 43...
... FIGURE WO-15 XBP-1 target genes correlated to the maximum HAI response. SOURCE: Image by Nakaya, provided by Pulendran (2011)
From page 44...
... Pulendran observed that the yellow fever vaccine's activation of four different TLRs, for example, seemed to be associated with the longevity of the effectiveness of the vaccine. If a synthetic system could be engi neered with the same TLR ligands, it may be possible to invoke a similar longlasting immune response in other vaccines.
From page 45...
... The insights gained from experimentation can then guide the design and development of new vaccines. A Systems-Level Approach to Understanding the Immune Response and Managing Immunotherapy A systems approach to disease is predicated on the idea that the analysis of dynamic, disease perturbed, networks and the detailed mechanistic understand ing of disease that it provides can transform every aspect of how we practice medicine -- better diagnostics, effective new approaches to therapy and even prevention.
From page 46...
... For example, immune responses to vaccination in clinical trials can be profiled in exquisite depth with technologies such as microarrays, deep sequencing, and proteomics. The high-throughput data generated can be mined using bioinformatics tools and can be used to create hypotheses about the biological mechanisms underlying vaccine-induced immunity.
From page 47...
... Heath mentioned one patient -- Patient 5 -- whose flow cytometry results suggested that the patient was responding well to therapy when in fact the cancer was returning; the patient ultimately died. Heath and his col leagues hypothesized that "a comprehensive functional analysis of defined T-cell populations, assayed over time, can reveal not just when and how the therapy is working but when and how it fails." Figure WO-20 summarizes the clinical trial timeline and results for "Patient 5." Heath described the engineered T-cells that are used in adoptive T-cell immunotherapy as "tremendously complicated drugs" -- that need continuous monitoring.
From page 48...
... The engineered cells are expanded to ~10 9 cells and then reintroduced back into the patient. The data shown are flow cytometry data of CD3+ T-cells, from patient F5-1, before and after the genetic engineering process that makes them MART-1 antigen–specific.The MART-1 antigen is associated with the heavy pigmentation of melanoma tumors.
From page 49...
... technology for the T-cell functional analysis (Ma et al., 2011; Shin et al., 2010)
From page 50...
... Within the SCBC, individual cells are isolated within ~2 nanoliter volume chambers, and each of these chambers is equipped with a full barcode structured antibody array. As an initial demonstration of the potential effectiveness and value of this approach, Heath reported that he and his team initially conducted this functional analysis on a single melanoma cancer patient, at a single time point.
From page 51...
... The technologies that Heath's research team developed while building a chip for use in immunotherapy monitoring can also be used to examine and describe immune cell function in terms of functional protein signaling networks. Heath emphasized an important distinction between functional protein signaling
From page 52...
... FIGURE WO-22 Data showing 52 the persistence of the population of engineered MART-1 antigen– specific CD8+ T-cells and the evolving functional performance of those cells. The graph at top illustrates the population of the engineered cells, as a function of time, over a 90-day period following infusion of the engineered cells into the melanoma cancer patient.
From page 53...
... (Dr. Westerhoff's contribution to the workshop summary report can be found in Appendix A, pages 480-494.)
From page 54...
... . In the past, quinolones were generally thought to kill bacterial cells by inhibiting bacterial DNA gyrase (topoisomerase II or topoisomerase IV)
From page 55...
... . On a more practical level these and subsequent studies led Collins and col leagues to wonder if it was possible to enhance the potency of certain antibiotics by blocking the pathways that bacterial cells use to protect themselves against antibiotic-induced oxidative damage (Kohanski et al., 2008, 2010a)
From page 56...
... Lawrence, James J Collins, A Common Mechanism of Cellular Death Induced by Bactericidal Antibiotics, 797-810, Copyright (2007)
From page 57...
... . PROGRESS IN SYNTHETIC BIOLOGY: FROM THE TOGGLE SWITCH TO THE SYNTHETIC CELL Unlike systems biologists, who adopt a big-picture approach to biology by analyzing troves of data on the simultaneous activity of thousands of genes and proteins, synthetic biologists reduce the very same systems to their simplest unique component parts.
From page 58...
... As alluded to earlier, because the molecular nature of many cellular reactions is only partially understood, most synthetic genetic circuits require considerable empirical refinement following the initial computational work. This process has been referred to as the "iterative process of modeling and experi ment" that is required to build synthetic genetic systems with desired characteristics (Atkinson et al., 2003)
From page 59...
... Unlike the many synthetic biology devices that "count" or turn things on and off -- such as counters, oscillators, and switches -- that essentially duplicate what computers do, French stated that a practical argument could be made that synthetic biologists "should concentrate on letting the biology do what biology is good at, which is biocatalysis and molecular recognition, and develop good interfaces that allow cells and machines to talk to each other." Biosensors have been developed that function at a variety of levels within cells, allowing researchers to alter the transcription of particular genes (transcriptional biosensors) , change how expressed genes are translated into proteins
From page 60...
... . These early biosensors have served primarily as "proofs-of-concept" dem onstrating their potential for widespread applications in medicine, environmental protection, and environmental remediation (van der Meer and Belkin, 2010)
From page 61...
... . French identified several ways that synthetic biology approaches can be used to improve whole-cell environmental biosensors.
From page 62...
... Craig Venter Institute's "synthetic cell." In May 2010, the J Craig Venter Institute (JCVI)
From page 63...
... (Dr. Hutchison's contribution to the workshop summary report can be found in Appendix A, pages 222-235.)
From page 64...
... SOURCE: J Craig Venter Institute (2011)
From page 65...
... Speaker Peter Greenberg of the University of Washington defined a biofilm as a "structured community of bacterial cells enclosed in a self-produced poly meric matrix." (Dr. Greenberg's contribution to the workshop summary report can be found in Appendix A, pages 213-222.)
From page 66...
... (Dr. Lu's contribution to the workshop summary report can be found in Appendix A, pages 278-324.)
From page 67...
... (Dr. Lewis' contribution to the workshop summary report can be found in Appendix A, pages 254-278.)
From page 68...
... Lewis' team has been using HTS in order to identify prodrug compounds that might show promise in targeting persister cells. How a Systems-Level Understanding of Biofilms Is Influencing Antibiotic Drug Discovery Part of why antbiotic discovery is still in a "Dark Age" is that most envi ronmental microorganisms -- more than 99 percent -- are unculturable.
From page 69...
... Synthetic Biology Approaches to Managing Biofilms The easiest way to remove biofilms from surfaces is mechanically by, for example, brushing one's teeth or surgically removing a biofilm infection. Unfortunately, mechanical removal of biofilms may not be practical in many situations, such as in industrial settings, where the current practice is to use biocides like chlorine bleach and quaternary ammonia.
From page 70...
... . As a proof-of-concept that phages can be used not just to kill but also to deliver biofilm matrix-removing enzymatic machinery into bacterial cells, much like a Trojan horse, the researchers re-engineered bacteriophage by incorporating the genetic circuitry for DspB.22 The goal was to create a phage that attacked not only the bacterial cells themselves but also the biofilm matrix.
From page 71...
... . These investigators also reported that the use of antibiotic-potentiating phage with antibiotics decreased the number of resistant bacterial cells that emerged later on (Lu and Collins, 2009)
From page 72...
... (Dr. Berry's contribution to the workshop summary report can be found in Appendix A, pages 105-117.)
From page 73...
... . Jay Keasling and colleagues engineered a Saccharomyces cerevisiae yeast to produce artemisinic acid by modifying an existing metabolic pathway in the yeast and adding in a gene from A
From page 74...
... In order to identify the genes involved in the final step in the pathway, Keasling and his team constructed a yeast -- Saccharomyces cerevisiae -- that produces amorphadiene with the goal of using the yeast system as a probe to screen a li brary of Artemisia annua genes and identify those involved in the hydroxylation of amorphadiene into artemisinic acid and then inserting these genes into E coli (Paradise et al., 2008; Ro et al., 2006)
From page 75...
... (Dr. Stephanopoulos' contribution to the workshop summary report can be found in Appendix A, pages 417-429.)
From page 76...
... . Stephanapoulos observed that metabolic engineering can also be used for compound discovery by using metabolic pathway scaffolds to screen libraries of synthetic molecules and identify those compounds that yield the greatest amount of end product.
From page 77...
... (Dr. Georgiou's contribution to the workshop summary report can be found in Appendix A, pages 202-213.)
From page 78...
... , are developing polyclonal recombinant antibodies for use as broad-spectrum anti-infective therapeutics. What Synthetic Ecosystems Are Teaching Biologists About Antibiotic Resistance and Antibiotic Drug Discovery The study of how organisms interact with each other -- and with their environment -- falls under the purview of ecosystems analysis.
From page 79...
... "Third Wave" Antibody Discovery - Experimental analysis and mining of the polyclonal immune response NextGen Sequencing Responding Patient/ of Ig Repertoire Immunized Individual Molecular Serology Serum Ig Deconvolution FIGURE WO-29B Isolation of monoclonal antibodies from responding patients. Figure WO-29B.eps SOURCE: Georgiou (2011)
From page 80...
... Freemont and his research group have been working with genetically coded biosensors to see if they could design a biosensor capable of detecting the acylHSL quorum-sensing signals -- discussed by Greenberg -- that diffuse in and out of bacterial cells as biofilms develop. Freemont agreed with Greenberg that regulation of the gene circuit activated by acyl-HSL quorum sensing is complex, with other factors at play.
From page 81...
... A cell-free design is essentially a biochemical mixture of the contents of the cell that has the ability to transcribe and translate a genetically encoded de sign into a device. Through modeling and in silico simulations, combined with in vivo testing of various parts -- such as the various promoters controlled by LasR -- Freemont's team has developed two devices ("V" and "L")
From page 82...
... Future applications in clude engineering biosensors to detect heavy metals and other common persistent toxins in soil and water (Royal Academy of Engineering, 2009)
From page 83...
... Indeed, synthetic biology is providing a novel means to help address energy challenges through the design of organisms that can more efficiently manufacture biofuels and are less wasteful than the current processes used to make ethanol-based fuels (Fortman et al., 2008; French, 2009; Royal Academy of Engineering, 2009)
From page 84...
... Spider silk has a similar tensile strength as steel, yet it is much lighter. By modifying the type III secretion system of Salmonella using synthetically designed genes, these investigators have essentially turned the bacterial cell into a small spider silk production factory (Widmaier et al., 2009)
From page 85...
... While most systems and synthetic biologists who work with biofilms are seeking novel ways to treat biofilms, Lu noted that there are also some potential applications of biofilms. One of those applications is in materials engineering.
From page 86...
... Cells that have been engineered to deliver a drug could be programmed to deliver doses directly to the target sites. The basic biological switches and oscillators developed over the past decade and previously discussed represent the first steps in this direction (Khalil and Collins, 2010; Royal Academy of Engineering, 2009)
From page 87...
... . Bottom-up approaches in particular are far from reaching a point at which synthetic biologists will be able to build, from scratch, systems capable of doing novel things, let alone systems capable of self-replication and otherwise functioning as living organisms.
From page 88...
... are poorly characterized, as the tradition of characterizing biological components has been losing ground to the use of high-throughput methods for collecting large quantities of data. Part of the problem, Sauro observed, is that synthetic biologists rarely re ported exactly what they did in their experiments, making the experimental results difficult for others to replicate.
From page 89...
... . BOX WO-2 The International Genetically Engineered Machine Competition The iGEM competition resembles a giant science fair for budding synthetic biologists.
From page 90...
... . Synthetic biologists typically insert their engineered circuits into Escherichia coli, but they also use Bacillus subtilis, Saccharomyces spp., and other microbial species.
From page 91...
... The iGEM competition, described earlier, is one way that the scientific com munity has responded to this challenge. By providing a curated community in which budding synthetic biologists from a variety of backgrounds can work to
From page 92...
... . Indeed, iGEM, the Registry of Standard Biological Parts and the BioBricks® Foundation are at the forefront of the kind of open-source science that many synthetic biologists argue is the best way to encourage innovation and progress in the field.
From page 93...
... In the case of synthetic biology, the call for collaboration is not just a matter of combining and coordinating efforts among scientists in order to accelerate the generation of knowledge; it is also a matter of safety. WORKSHOP OVERVIEW REFERENCES Agarwal, K
From page 94...
... Paper read at Meeting of the Presidential Commission for the Study of Bioethical Issues, July 8-9, 2010, Washington, DC. Cathcart, R
From page 95...
... 2007. Gyrase inhibitors induce an oxidative damage cellular death pathway in Escherichia coli.
From page 96...
... Nature Reviews Molecular Cell Biology 3:685-695. Fuqua, W
From page 97...
... Presentation given at the March 14-15, 2011, public workshop, "Synthetic and Systems Biology," Forum on Microbial Threats, Institute of Medicine, Washington, DC. iGEM (International Genetically Engineered Machine competition)
From page 98...
... 2011. Synthetic biology "from scratch." Presentation given at the March 14-15, 2011, public workshop, "Synthetic and Systems Biology," Forum on Microbial Threats, Institute of Medicine, Washington, DC.
From page 99...
... 2005. Synthetic biology: Engineering Escherichia coli to see light.
From page 100...
... 2004. Structural in sights into the assembly of the type III secretion needle complex.
From page 101...
... Nature Reviews Drug Discovery 8:442. Nimmerjahn, F., S
From page 102...
... 2006. Production of the antimalarial drug precursor artemisinic acid in engineered yeast.
From page 103...
... 2003. Synthesis and localization of the Salmonella SPI-1 type III secretion needle complex proteins PrgI and PrgJ.
From page 104...
... 2009. Engineering the Salmonella type III secretion system to export spider silk mono mers.


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