MEETING REPORT

Programming cellular function

Christopher A Voigt & Jay D Keasling

The process of cellular engineering is rapidly accelerating owing to advances in technologies to manipulate DNA and other biomolecules, giving rise to the field of synthetic biology. A meeting was held in August 2005 to present progress in the field and to discuss topics in ethics, safety and security.

An aim of synthetic biology is to program new cellular functions using well-characterized genetic components. This requires the construc- tion of synthetic sensors that receive environ- mental information, new circuits to integrate and interpret the signals and mechanisms to link the circuit output to control cellular pro- cesses. The time required to design, construct and test a synthetic organism is declining owing to several central technologies. Large-scale genome sequencing projects have provided a toolbox of microbial genetic components that can be swapped between organisms and com- bined. In addition, plummeting DNA synthesis costs are rendering the slow processes of clon- ing and molecular biology obsolete. Finally, there is a new effort to standardize genetic parts to improve the predictability of designs and facilitate the exchange of materials between The Life Engineering symposium was held at the California Institute for Quantitative Biomedical © 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology research groups. These technologies will drive Research on the UCSF Mission Bay Campus. new applications and will require rethinking the ethical, safety and security underpinnings of genetic engineering. A two-day Life Engineering Symposium was Institute for Quantitative Biomedical Research Miniature manipulators of the human body held on August 19–20, 2005 to address recent Institute (QB3), the Lawrence Berkeley already exist. Pathogenic bacteria and viruses advances in synthetic biology. The sympo- National Laboratory (LBNL) Department of are experts at identifying specific cell types sium was held at the University of California, Synthetic Biology and the University of Oxford. and organs, can evade immune responses San Francisco’s (UCSF) Mission Bay Campus Here, we highlight the applications and themes and can manipulate individual cells. John and was cosponsored by the Keck/National that emerged at this meeting. Pawelek (Yale University) demonstrated Academies Futures Initiative, the California that wild-type Salmonella typhimurium and Live therapeutic agents Bordetella pertussis localize to tumors after Christopher A. Voigt is in the Department of In the campy 1960s science fiction movie intravenous injection and can impart a ther- Pharmaceutical Chemistry and California Fantastic Voyage, a team of doctors and their apeutic effect1. To improve efficacy, strains Institute for Quantitative Biomedical Research submarine are shrunk and injected into a of S. typhimurium have been engineered (QB3), University of California, San Francisco, patient’s artery. Using a laser, they vaporize to require adenosine for growth, convert San Francisco, California 94143-2540, USA. a blood clot, after which they are expelled a prodrug into an active form and amelio- Jay D. Keasling is in the Department of Chemical through the patient’s tears. Keeping in mind rate septic shock. In addition, the Pawlek Engineering and the Berkeley Center for the goal of actually making an automated group demonstrated that the bacteria can be Synthetic Biology at the University of California, miniature therapeutic robot, there has been remotely controlled after being administered Berkeley, Berkeley, California 94720-3224, USA. considerable progress in engineering micro- to a patient by using the inducers tetracycline e-mail: [email protected] or scopic robots and computers and in harness- and mitomycin C to elicit a response in engi- [email protected] ing existing biological systems. neered bacteria thriving in a tumor.

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Programming bacteria to have therapeutic teria, yeast and the plant Artemisia annua, proliferation or differentiation7. Christopher functions will require a toolbox of cellular sen- Jay Keasling of UCB created a bacterium that Chen demonstrated that the RhoA GTPase sors that respond to microenvironments in the synthesizes large quantities of amorphadiene, can be engineered to control the differentia- body, genetic circuits to integrate this informa- a precursor of the antimalarial drug artemis- tion of stem cells into fat (constitutively off) tion and the ability to engineer the interaction inin4. The production of amorphadiene was or bone (constitutively on) owing to its role between a bacterium and mammalian cell. maximized by (i) codon optimizing the plant in regulating cell contractility. These outputs For example, bacteria localized to a tumor are enzyme amorphadiene cyclase using DNA can be engineered to respond to heterologous exposed to high cell densities and anaerobic synthesis (142-fold improvement), (ii) incor- signals. For example, Wendell Lim of UCSF conditions, which could be used as cues for the poration of a heterologous yeast mevalonate engineered the DH domain of intersectin, release of a therapeutic. Chris Voigt of UCSF pathway (30-fold) and (iii) optimization of the which controls filapodia formation, to respond demonstrated the linkage of anaerobic and intragenic messenger RNA (mRNA) structures to phosphorylation by protein kinase A (PKA). cell density sensors to the induction of inva- of the synthetic mevalonate operon (seven- A synthetic switch was constructed by fusing sin, which enables non-pathogenic bacteria to fold). These improvements have brought the DH to a PDZ domain and a PDZ substrate that adhere to and invade mammalian cells. Under cost of artemisinin production to a sufficient can be phosphorylated by PKA. This fusion these conditions, the bacteria attacked a wide level to broaden its use in the developing protein was then introduced into cells and was variety of cancerous cell lines in culture. world. A similar engineering approach could induced by the addition of the PKA-activator Viruses can also have therapeutic properties. be used to optimize other therapeutic terpenes cAMP, which led to the controllable formation By serendipity, Craig Meyers (Pennsylvania such as taxol, which is used for cancer treat- of filapodia. State University) discovered that the harmless ment, and prostratin, which is used in clinical human virus AAV2 kills a remarkably broad trials for HIV treatment. Biosensors range of cancer cell lines but does not affect pri- RNA has the capability to bind to small mol- mary cells. AAV has also been used as a vector Programming stem cells ecules and regulate gene expression, making for gene therapy. Patients with cancers affected Stem cells are able to proliferate and differ- it an ideal substrate for designing biosensors. by AAV tend not to have existing immunity to entiate into many different cell types and are Christina Smolke (California Institute of AAV, increasing the potential as a therapeu- a natural repair mechanism in the body. An Technology) demonstrated that cell-based bio- tic agent. However, the wild-type virus has a understanding of the mechanisms of this sensors can be built by linking a RNA aptamer number of drawbacks, including preexisting process will enable the use of stem cells as a that binds a small molecule ligand with a immunity, delivery efficiency, distribution in therapy to replace cells destroyed by disease piece of RNA that inhibits or activates trans- the body, production and cell type targeting. or to grow new tissues. Stem cells can be pro- lation8. This was beautifully demonstrated Using directed evolution, David Schaffer of grammed in several ways. First, their behav- by showing a caffeine sensor that could dif- the University of California, Berkeley (UCB) ior can be tuned by controlling the properties ferentiate between espresso, coffee and decaf. found capsid mutants that overcame some of of a well-characterized external microenvi- Andy Ellington (University of Texas at Austin) these limitations. One mutant had a 100-fold ronment. Second, synthetic circuits can be showed a similar design in which an aptamer improvement in avoiding immunity, and this introduced that control the activity of central was fused to a ribozyme such that the catalytic enhanced the delivery of the erythropoietin signaling proteins. activity was regulated by the binding of a small gene to mice. David Schaffer presented the construction molecule9. Niles Pierce (California Institute of Therapeutic viruses could be engineered of a fully characterized external matrix to Technology) demonstrated that a biosensor to interfere with an infection of harmful grow neural stem cells. One of the gel poly- can be constructed by fusing a DNA aptamer

© 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology viruses. Adam Arkin (UCB) outlined a strat- mers was modified to display a small domain to a domain that triggers a hybridization chain egy to engineer a lentivirus that interacts with (RGD) from the large protein laminen and was reaction, which produces a nucleotide polymer HIV-infected cells to reduce the HIV viral shown to elicit a similar proliferative and dif- that can be detected by colorimetry, gels or setpoint in the blood and thus prevent the ferentiation response in the cells. Christopher fluorescence10. All of these sensors involved transgression of the disease to AIDS2,3. They Chen (University of Pennsylvania) elegantly simple, modular designs that mechanically link propose to gut HIV to produce a harmless demonstrated that mesenchymal stem cell the input with an output response. lentivirus, remove the capsid genes such that commitment to different lineages can be con- Synthetic sensors can also be constructed it requires a coinfection with HIV in a cell trolled by varying the mechanical environ- by fusing an extracellular input domain of a to reproduce, and add components to turn ment encountered by the cells. For example, protein to an intracellular signal transducer off HIV. They described a theoretical model fat cells were generated when the adhesion domain. This strategy was used by the synthetic showing the parameter regime under which was limited to a small island, whereas bone biology team from the University of Texas at the therapeutic virus spreads and reduces cells were formed when the cells were allowed Austin and UCSF, who designed a strain of the HIV viral setpoint to a level that does not to spread on a larger island5. The Chen group E. coli that can record a pattern of light that cause AIDS. also showed that the shapes of multicellular is shown on a bacterial lawn (‘bacterial pho- sheets can be used to direct proliferation at tography’), as described by Edward Marcotte. Microbial drug factories specific locations (for example, at corners and The light sensor was constructed by fusing Tools from synthetic biology will have an valleys) owing to localized tensile stresses gen- the extracellular domain of a cyanobacterial impact on metabolic engineering, in which the erated by the shapes6. phytochrome with the intercellular domain of goal is to create an organism that produces a A number of signaling proteins can be used an E. coli histidine kinase. A similar approach maximum of a desired chemical. One appli- as ‘switches’ in controlling stem cell prolif- was described by John Spudich (University cation is to engineer bacteria to synthesize a eration and differentiation. David Schaffer of Texas at Houston) in the construction of drug or precursor compound inexpensively. showed that the sonic hedgehog protein (Shh) a phototactic strain of E. coli11. Spudich also By combining metabolic pathways from bac- controls whether neural stem cells undergo described a procedure for engineering E. coli

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to use light for energy by incorporating the Rational biomolecular design Selection strategies also have their pit- light-driven proton pump proteorhodopsin in There have been tremendous advances in the falls because cells are very good at finding their membranes. ability to design the sequence of a biological unintended pathways to survive. This prin- polymer to fold into a target structure. Tanja ciple emerged in a project described by Andy Genetic circuits Kortemme (UCSF) presented a computational Ellington in which a selection scheme that Programming cells will require the integration algorithm that generates pairs of proteins that intended to create a ribozyme-based genetic of signals from multiple sensors. Receiving bind to each other15. This is done in silico by circuit ended up creating novel secondary information from multiple sources provides destabilizing the interface and then reen- structures that deactivated the circuit, which greater specificity when linking gene expres- gineering the partner to optimize the bind- had unintended toxicity. It was found that the sion to an environmental niche. An AND gate, ing affinity. This algorithm was successfully hairpins deactivated ribosomes—a principle in which the output is activated only when all applied to the redesign of a PDZ binding inter- that he is now using to regulate the number of inputs are on, is a particularly useful circuit. action, which could be used to create circuits ribosomes per cell as a mechanism to control Presenters described several new designs for and rewire signaling pathways. cellular noise. AND gates using DNA, RNA and protein com- In creating synthetic cells, it will be impor- ponents that functioned in vitro, within bacteria tant to understand how the composition of Ethics, security and society and in eukaryotic cells. Using principles from lipids in the membranes affects the physics At the symposium, four talks directly addressed DNA computing, Ehud Shapiro (Weizmann of micelle formation. This was explored by ethical, safety and security issues. Laurie Zoloth Institute of Science) demonstrated an AND gate Mark Sansom (Oxford University), who used (Northwestern University), a bioethicist, spoke that identifies four ‘symptom’ inputs (abnormal molecular dynamics simulations to study the about the ethical concerns raised by the work, up- or downregulation of specific mRNA levels) self-assembly of lipids and membrane proteins how they arise from larger social responses to and releases a nucleotide drug in response12. into micelles and bilayers16. science and technology, how such concerns To program bacteria, an AND gate that Niles Pierce described algorithms that can give raise to public discourse and how that dis- responds to the activation of two input pro- be used to design nucleotide sequences to course can lead to varying models of regula- moters would be useful in linking the outputs program synthetic mechanical functions into tion. Paul Rabinow (UCB), an anthropologist, from multiple biosensors and various path- RNA and DNA. As an example, he showed the discussed safety and security concerns in light ways. Chris Voigt showed such a circuit, in design of a system of DNA fragments that are of the history of prior regulation of science. which an input drives an activator modified to able to self-assemble into a motor analogous Two scientists, Andy Ellington (University of contain an amber stop codon, and the second to kinesin and take the first steps along a piece Texas, Austin) and Roger Brent (Molecular promoter drives the transcription of the trans- of DNA17. This work opens up the possibility Sciences Institute), advocated the importance fer RNA (tRNA) amber suppressor. In eukary- that mechanical functions can be programmed of paying attention to student training and to otic cells, logic gates can be built by rearranging into cells using nucleotide sequences. articulating possible future applications that protein-protein interaction domains. Wendell promise great benefit to society. The following Lim built a variety of logic blocks using Directed evolution as a tool is a summary of the themes from these presen- modular domains (such as PDZ and SH3). A Intelligent designers sometimes resort to using tations and the discussions afterward. relatively small library of designs resulted in a in vitro evolution as a rapid method to engi- All new technologies can be used to cause broad range of logic gate behaviors, including neer a biological system. At the symposium, harm. Moreover, errors, mistakes, and, indeed, AND, OR and antagonistic functions13. This directed evolution emerged as a common dual use (meaning the use for both social work was extended to create a three-input method that was used successfully to improve welfare and for weaponry) of all technol-

© 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology AND gate. In addition, it was shown that the metabolic pathways, mRNA switches, viruses ogy, including synthetic biology, is inevitable. transfer function of a protein switch could be and genetic circuits. Screening libraries of However, the potential for both malevolent tuned from a graded to a switch-like response genetic circuits requires a different strategy and beneficent use cannot unilaterally pre- by increasing the number of domains associ- from evolving a single protein or enzyme. clude basic research. Using this as an argu- ated with one input. Christina Smolke com- Circuits are characterized by their transfer ment to stop research progress would have bined a theophylline-binding RNA sensor with function, which captures their output over a halted all technology since the development an RNA hairpin structure that acts as a tem- range of input conditions. At a minimum, this of metallurgy. The problem of dual use, or of perature sensor to produce a circuit in yeast requires screening in two conditions: in the unintended consequences, is not intrinsic to that responds to a small-molecule ligand only presence and absence of input signal. synthetic biology, but rather due to a widening above certain temperatures. Several new positive-negative selection of the number of people that can work with Obtaining programmable multicellular strategies were proposed that are able both DNA. Given that technology such as recombi- organization will require the use of genetic to search through large libraries and to iden- nant DNA has been widely used for 25 years, circuits that control cell-cell communication. tify functional circuits. Yohei Yokobayashi the questions are, how can we minimize the Lingchong You (Duke University) described a (University of California, Davis) demon- risks? How do we best prepare for contingen- genetic circuit that regulates bacterial popula- strated a dual selection where the ON and cies? Have new technologies emerged that tion density using a quorum sensor linked to OFF states of a switch activated the expres- require revisiting previous strategies? the expression of a toxic protein14. Moving up sion of different antibiotic resistance genes. As applied to recombinant DNA, these con- a level in complexity, he demonstrated the use This strategy was applied to the evolution of a cerns were addressed by the 1975 Asilomar of two quorum sensors to create a predator- genetic inverter. Chris Voigt presented a posi- meeting. Asilomar succeeded for two reasons. prey system. These circuits could be used to tive-negative selection strategy that used the First, it reduced the danger to a question of program temporal-spatial patterns into cells, Yersinia pseudotuberculosis protein invasin as safety, which converted the issue from a politi- which has applications in designing biofilms an output and mammalian cells as sequester- cal one to a technical and hence manageable and growing synthetic tissue. ing devices. one. Scientists at the meeting then devised

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a scheme involving graduated levels of con- political, social and theoretical stances across cal components. In addition, of course, cells tainment and laid out a series of experiments the American spectrum. Political skepticism have natural photosynthetic processes that can that could give answers that would support arises from the political left, which raises con- be harnessed in these applications. the relaxation of the level of containment. cerns about any genetic alteration, and the Fine chemical synthesis and therapeutics. However, given the current focus on security, political right, which raises questions about Keasling’s work illustrates the possible utility the attempt to reduce questions of risk to the scientific project itself. The argument can of developing cells that can make precursors questions of safety is not sufficient. Second, be summarized as follows: first, there is a view to one sort of chemical backbone and poten- they articulated the societal benefits that can that nature is a morally good, hierarchical and tial great utility of such cells in synthesizing come from recombinant DNA. They identi- stable system that is instantiated in large part drugs that are directed against malaria, a fied three: gene therapy, crops that will fix in DNA. Natural order can be violated when, disease of the developing world. Others may their own nitrogen and production of recom- for example, the species barrier is broken down well extend it to other backbones and thus to binant protein drugs, including insulin and in the creation of chimeric or recombinant other diseases. It may also be possible to use interferon. Even though only one of these DNA. Similarly, building synthetic biological biological components to create smart drug applications has so far been successful, its real systems suggests a mastery over finitude, and delivery devices. benefits have helped to allay fear. Following some raise the fear that such a mastery is an Research. A potential 5- to 10-year project the example of Asilomar, there needs to be a attempt to avoid core aspects of the human might be to make logic and output (DNA writ- coherent (and articulate) scientific community condition—suffering and death—which, they ing) circuitry to develop a combination cell in synthetic biology that works to mitigate the argue, is a definition of our species. Many fear cycle counter and lineage trace that can be used risks while convincingly communicating real- a ‘slippery slope’ in which such technology to identify the developmental lineage of every istically possible benefits. could redefine humanity in disturbing, unjust cell in the body of a mammalian organism. Training programs will train a cadre of young or mechanistic ways. Others note that dual use These inspirational goals could serve to synthetic biologists that have the tools necessary and mistakes are inevitable and warn against shape the field and to define credible, social to handle emerging threats, humility in the face catastrophic secondary effects with enormous benefits for the work. Even modest success of the risks, an understanding of the problem consequences. Lastly, others warn about the in one of these applications would have a and a commitment to education, prudence and conflict of interests inherent in science that is tremendous impact on society. self-regulation. This bottom-up structure is ulti- linked to patent, profits and markets, fearing 1. Pawelek, J., Low, K.B. & Bermudes, D. Lancet Oncol. mately better than trying to limit the technology that the marketplace will distort the science. 4, 548–556, 2003 (2003). itself, leaving us with a small group of trained Roger Brent made the point that convincing 2. Weinberger, L., Schaffer, D.V. & Arkin, A.P. J. Virol. 77, researchers and no regulatory structure. Several the public of the benefits of synthetic biology 10028–10036 (2003). 3. Weinberger, L.S., Burnett, J.C., Toettcher, J.E., Arkin, educational programs are being built to address will require the articulation of a core set of A.P. & Schaffer, D.V. Cell 122, 169–182 (2005). this challenge. Drew Endy (Massachusetts applications, similar to those that arose out of 4. Martin, V.J.J., Pitera, D.J., Withers, S.T., Newman, J.D. & Keasling, J.D. Nat. Biotechnol. 21, 796–802 Institute of Technology) presented an under- Asilomar, on which the nascent community (2003). graduate class that brings hands-on experience might actually deliver. It would be helpful 5. McBeath, R., Pirone, D., Nelson, C.M., Bhadriraju, K. in designing, building and testing cells that can in this regard to diminish the hype that has & Chen, C.S. Dev. Cell 6, 483–495 (2004). 6. Nelson, C.M. et al. Proc. Natl. Acad. Sci. USA 102, perform synthetic tasks. There is also an annual entered into these discussions, some of which 11594–11599 (2005). nationwide genetic engineering competition has arisen in order to attract funding. False 7. Lai, K., Kaspar, B.K., Gage, F.H. & Schaffer, D.V. Nat. (iGEM) in which teams of students compete promises will erode public confidence and the Neurosci. 6, 21–27 (2003). 8. Bayer, T.S. & Smolke, C.D. Nat. Biotechnol. 23, 337– to engineer a synthetic organism. This year, credibility of researchers among other scien- 343 (2005).

© 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology fourteen schools are participating. As an edu- tists. Keeping this in mind, a series of chal- 9. Levy, M. & Ellington, A.D. Chem. Biol. 9, 417–426 (2002). cational tool, these programs have focused on lenges were proposed. 10. Dirks, R.M. & Pierce, N.A. Proc. Natl. Acad. Sci. USA toy problems in cellular control. To create a base Bulk chemical synthesis. The future chemical 101, 15275–15278 (2004). to deal with emerging problems, we will need to industry will use glucose as a feedstock, not oil. 11. Jung, K.H., Spudich, E.N., Trivedi, V.D. & Spudich, J.L. J. Bacteriol. 183, 6365–6371 (2001). expand the curriculum to include applications Accelerating the decrease in the price of glu- 12. Benenson, Y., Gil, B., Ben-Dor, U., Adar, R. & Shapiro, such as vaccine development, energy produc- cose will hasten the day when it becomes less E. Nature 429, 423–429 (2004). tion and materials. expensive than hydrocarbons, and it would 13. Dueber, J.E., Yeh, B.J., Chak, K. & Lim, W.A. Science 301, 1904–1908 (2003). All research will also need a careful public be useful for synthetic biologists to hasten the 14. You, L., Cox, R.S., Weiss, R. & Arnold, F.H. Nature 428, system for oversight. It is important to under- intersection of those two trend lines: the ‘green 868–871 (2004). 15. Kortemme, T. et al. Nat. Struct. Mol. Biol. 11, 371–379 stand the reasonable public understanding chemical industry’ takeoff point. (2004). of the risk inherent in this research. Laurie Energy. Cells and organisms can be used to 16. Bond, P.J., Cuthbertson, J.M., Deol, S.S., & Sansom, Zoloth presented a list of the reasons behind produce fuels, and electron-handling machin- M.S. J. Am. Chem. Soc. 126, 15948–15949 (2004). the public’s concern about synthetic biology. ery from photosynthetic reaction centers 17. Shin, J.S. & Pierce, N.A. J. Am. Chem. Soc. 126, The arguments for caution emerge from many might find use in fuel cells built from biologi- 10834–10835 (2004).

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