Programming Cellular Function
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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. 304 VOLUME 1 NUMBER 6 NOVEMBER 2005 NATURE CHEMICAL BIOLOGY MEETING REPORT 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.