The Factory Phenotype

      Each year, 300 to 500 million cases of malaria are diagnosed worldwide, of which 1.5 to three million, mostly in children, result in death. Drugs to treat malaria are too expensive for people in developing countries, hence the lack of proper treatment and the high mortality rate. Fortunately, a new, much less expensive anti-malarial drug will surface in the market in 2012, thanks to synthetic biologist Jay Keasling and his team at UC Berkeley. Developing this product is just one among many goals of the Synthetic Biology Engineering Research Center (SynBERC), where scientists are working to create efficient biofuels, biosensors, and cures for cancer and HIV.

      Synthetic biology in the past

      Synthetic biology has its roots in early research on DNA manipulation. In 1970, Daniel Nathans, Werner Arber, and Hamilton Smith identified restriction endonucleases, enzymes that can cut DNA strands at specific locations, which bacteria use as a defense mechanism against invading viruses. The discovery eventually led to the development of recombinant DNA techniques, which allow scientists to combine DNA sequences and introduce DNA from one organism into another. The term “synthetic biology” was first coined in 1974 by Waclaw Szybalski, now an oncology professor at University of Wisconsin-Madison Medical School, as he foresaw that the ability to build DNA sequences would eventually allow bioengineers to build “synthetic organisms,” whose traits would be chosen by humans for useful applications.

      Bringing ideas under one roof

      In the summer of 2006, armed with a five-year, 16 million dollar grant from the National Science Foundation (NSF), bioengineers at UC Berkeley and other institutions across the United States formed SynBERC, a collaborative funding initiative whose primary goal is to bring together experts in multiple disciplines to develop synthetic biology. In addition to its research efforts, the program also has a strong educational component. In Berkeley, SynBERC members reach out to local high school and undergraduate students with unique summer programs and competitions focusing on synthetic biology.

      Synthetic biology today

      The exact definition of synthetic biology varies depending on whom you ask, since each researcher’s expertise and current project shape her view on the subject. The rapid growth of the field also makes it hard to label it with any specific stamp: a definition today may not apply to synthetic biology tomorrow. The backgrounds of synthetic biologists at SynBERC today are perhaps the best representation of this diverse and dynamic field of research.

      Three main groups of scientists are joining the field: the protein engineers, who work with amino-acid building blocks; the metabolic engineers, whose focus is to regulate processes in cells to increase a cell’s production of certain substances; and the computer scientists, whose knowledge of manipulating and interpreting large datasets is critical.

      The numerous research projects of synthetic biology today have one goal in common: finding standardized processes to design biological systems with practical functions for our society’s benefits.

      Standardization is the key

      As director of SynBERC, Professor Jay Keasling in the Department of Chemical and Biomolecular Engineering at UC Berkeley views synthetic biology as “the industrialization of biotechnology.” Biotechnology, he claims, originally grew out of pharmaceuticals that were heavily restricted by patents, where each invention is independently made by individuals and is not readily available to be built upon by others. To overcome this obstacle to rapid progress, synthetic biology done at SynBERC is an open source, where scientists are free to learn from and build upon each other’s results to speed up the research process.

      Bioengineers have worked with different applications that, while sharing the same technical foundations and roadblocks, used tools that were particular to each research problem. Thus, synthetic biologists wants to find a general procedure and to identify the circuitry rules that would guarantee a working system regardless of its application. A common but oversimplified analogy is that synthetic biology puts together biological parts just as electrical engineering and computer engineering put together an electronic circuit.

      Part, device, chassis

      As director of SynBERC, Professor Jay Keasling hopes to engineer organisms to produce pathogen-fighting compounds.

      As director of SynBERC, Professor Jay Keasling hopes to engineer organisms to produce pathogen-fighting compounds.
      Credit: Kate Spohr

      Just as an electrical engineer needs resistors, capacitors, and inductors to make an electronic device, a synthetic biologist needs biological parts to build a biological device, usually in the form of bacteria or yeast that can produce certain chemicals or reactions to the environment. Biological parts include protein-encoding DNA sequences and promoters, sequences that facilitate the expression of a gene. The parts need to be assembled and powered by an appropriate chassis to produce the desired outputs. This theoretical picture, however simple, encounters three main difficulties in practice.

      First, biological parts, though numerous, are hardly categorized, and their functions under various laboratory conditions have not been well tested. Over the past 50 years, data accumulated from in vivo studies, as well as advances in DNA sequencing technology and computer analysis of genomes, have given considerable insight into the function of thousands of genes. However, similar to a toolbox full of nuts, bolts, screws, and nails all scrambled together rather than organized into compartments, there has been no systematic cataloging of genes and their characteristics for genetic engineering applications.

      In December 2009, synthetic biologists Jay Keasling and Adam Arkin at UC Berkeley and Drew Endy at Stanford University launched the International Open Facility Advancing Biotechnology (BIOFAB) to professionally develop and characterize new and existing biological parts. BIOFAB is the world’s first biological design-build facility, funded by NSF in partnership with Lawrence Berkeley National Laboratory (LBL), the BioBricks Foundation (BBF), and SynBERC. Once fully operational, BIOFAB is expected to produce tens of thousands of parts each year, freely available to both academic and commercial users, while also providing the design essentials for partners like SynBERC.

      Second, biological systems are highly unpredictable. The complexities of life, such as the behavior of different possible combinations of four nucleotides A, T, C, and G in a piece of DNA, are not as well understood as the physical laws governing light bulbs and batteries. “50 percent of the time, you turn on a biological circuit, and it just doesn’t work for no good reason,” says Professor Christopher Anderson in UC Berkeley’s Department of Bioengineering.

      The third difficulty lies in constructing and testing complex multi-gene circuits. Larger circuits simply demand more effort. Most of the work in syn-bio research to date has been done manually, and an automatic process to combine the genetic parts would alleviate the load tremendously. To tackle this, Anderson and his colleagues are working to engineer bacteria to assemble DNA pieces into useful circuits and select the successfully assembled products from among the unused DNA parts. The hope is that such automated systems can shorten the time needed for one assembly stage from a few days to a few hours.

      Paging doctor bug

      Keasling and colleagues are tackling the HIV problem. Human Immunodeficiency Virus (HIV) type I, the causative agent of AIDS, may be treated with various drugs to decrease its viral strength. The virus can remain latent in a patient’s body for years. Because of its inactive state, latent HIV-1 is unaffected by current drugs and can later emerge to cause a full HIV infection or AIDS. Recent studies show that two related compounds, prostratin (12-deoxyphorbol 13-acetate) and DPP (12-deoxyphorbol 12-phenylacetate), have great potential to eliminate latent HIV. However, both only exist naturally in rare plants: prostratin is found in the bark of the Samoan mamala tree Homalanthus nutans, and DPP can only be extracted from the Moroccan spurge Euphorbia resinifera growing on the slopes of the Atlas Mountains in North Africa. As it is too inefficient to obtain the compounds for commercial HIV treatment in this way, the only hope is to engineer microorganisms to produce them. Keasling’s team is in the process of locating and cloning the genes responsible for the synthesis of prostratin and DPP in the plants.

      The biggest success contributing to UC Berkeley’s placement on the worldwide syn-bio map is the yeast that can produce an anti-malaria compound, developed in Keasling’s lab in late 2005 and recently perfected. This is the first example of synthetic biology actually producing a competitive product in the market. The malaria parasite develops inside red blood cells, where it accumulates iron, which makes it vulnerable to oxygen-based free radicals released from a scarce compound known as artemisinin. This compound is 100 percent effective against even the most lethal malaria parasite, Plasmodium falciparum. But before Keasling’s process was developed, the compound could only be extracted from the plant Artemisia annua, a type of wormwood.

      Anti-malaria drugs using plant-derived artemisinin cost $2.40 per adult course (too expensive for people in developing countries), because of the low concentration of artemisinin found in wormwood. By inserting parts of the wormwood DNA into yeast DNA and reprogramming the cell’s metabolic pathway, Keasling’s team made yeast that can convert sugar into artemisinin in just a few hours. The laboratory success led to a contract between the research group and a start-up French pharmaceutical company, Sanofi-Aventis, in March 2010. The collaboration plans on releasing the synthetic artemisinin into world markets in 2012, at merely 1.5 cents per adult course, 160 times cheaper than the current price.

      Also on the medical frontier is Anderson’s new technique to combat cancer cells. The idea is to inject E. coli bacteria that can find and destroy cancerous cells into the bloodstream. The bacteria were first engineered to recognize tumors, which are characterized by high cell density and anaerobic growth. E. coli naturally possesses several genes, such as formate dehydrogenase (fdhF), that are strongly expressed when the bacteria are in a low-oxygen environment. Anderson’s team then inserted a genetic circuit (lux) from the bacterium Vibrio fischeri to allow E. coli to distinguish between regions with low cell densities and those with high cell densities. Parallel to engineering these sensing circuits, the team inserted the inv gene from the bacteria Y. pseudotuberculosis into E. coli to allow E. coli to produce invasin, a protein that binds the bacteria to mammalian cells. Finally, the sensing circuits (fdhF and lux) were linked with the inv gene. The result is that only the low-oxygen, high-density environment of the tumor can trigger the bacteria to produce invasin, which in turn causes the cancerous cells to engulf the bacteria. The next step is to engineer E. coli to release a toxin once inside the cancerous cells to kill them. By putting DNA pieces from different bacteria into E. coli, Anderson’s team hopes to create intelligent toxin-carriers that actively look for and destroy tumors without affecting healthy cells. Currently, the separate parts have been built and tested successfully, and with further examination, it may be possible to apply the same technique to different applications with shared properties. For instance, researchers can use the parts developed by Anderson to deliver medicine to HIV-infected white blood cells.

      Energy applications

      The rare plant  Euphorbia resinifera produces DPP, a compound SynBERC  hopes to be able to synthesize for HIV treatment. Credit: Chris Smith

      The rare plant Euphorbia resinifera produces DPP, a compound SynBERC hopes to be able to synthesize for HIV treatment.
      Credit: Chris Smith

      Following medical applications, the second most socially driven research area in synthetic biology is biofuels. The Berkeley SynBERC team carries out studies on this frontier at the Joint BioEnergy Institute (JBEI), a LBL-led scientific partnership between UC Berkeley, UC Davis, the Carnegie Institution for Science, Lawrence Livermore National Laboratory, and Sandia National Laboratory. Scientists at JBEI engineer microbes similar to yeast that can transform sugars from engineered plants into hydrocarbon-based fuels, such as butanol. The current highest production level for yeast is 2.5 mg of butanol per liter of growing substrate. Although this is still much lower than the 500 mg/l yield from engineered bacteria, researchers believe that yeast has high industrial value because of its robust growth and resistance to contamination.

      Furthermore, Keasling announced in May 2010 that his team had successfully engineered E. coli to produce a biodiesel from hemicellulose, a sugar polymer present in large quantities in plant biomass. The engineered bacteria digest hemicellulose in such a way that the sugar is transformed into diesel, which is secreted by the cells and floats to the top of the culture where it can be collected. With very little purification, this diesel is ready for use.

      Reaching out for more talents

      As any ambitious goal requires a tremendous amount of work, SynBERC relies on collective effort to thrive. In terms of expertise, the field welcomes researchers from not only the biological sciences and engineering disciplines, but also humanities and anthropology. At SynBERC’s Human Practices Lab (HPL), anthropology professor Paul Rabinow and his colleagues ponder the relations among biotechnological advances, politics, and security. As Gaymong Bennett, assistant director of HPL, put it, “the job of the ethicists and lawyers among others is to set up regulations on how your stuff can get used outside of the lab.” Such study is necessary whenever there are concerns about the potential impacts of a newly developed technology.

      In terms of finance and educational outreach, five institutions – UC Berkeley, UC San Francisco, MIT, Harvard University, and Prairie View A&M University in Texas – and about twenty independent companies collaborate on funding and directing the research at SynBERC. As Anderson views it, the industry exposure also shows synthetic biology students the broad range of job opportunities available in the field, an important motivation for the pursuers of syn-bio study.

      Get them while they’re young…

      At the high school level, SynBERC and JBEI, among other laboratories in the Bay Area, open doors both figuratively and literally to host six students and two high school teachers each year in an eight week paid summer internship known as the Introductory College Level Experience in Microbiology (iCLEM). The program, founded in 2008 by two of JBEI’s postdoctoral researchers, Clem Fortman and James Carothers, gives the interns an opportunity to work with high-tech lab equipment and take part in the biofuel research at JBEI.

      The goal of iCLEM is not to have results churned out by the end of each summer, but to arm the students and high school teachers with lab experience and research skills, while showing them the opportunities available in this new scientific field. Oakland Unity High School science department head Rowan Driscoll, iCLEM 2009 participant, praised the opportunity: “As a biology and chemistry teacher, I want to make education as real and as practical as possible. I want to open doors, open eyes, and help my students find avenues to use science to make themselves useful to society.”

      For both teachers and students, the research does not stop when the internship ends. Berkeley High School science teacher Kate Trimlett, also a participant in iCLEM 2009, decided to start teaching about biofuels in her classroom, organizing her students to collect and analyze bacteria from a compost heap to test their ability to break down cellulose. Such classroom outreach embodies the goal of iCLEM: to raise the level of interest in science of Bay Area high school students, and, hopefully, to inspire many of them to become future synthetic biologists. As Kate Spohr, SynBERC’s education and outreach coordinator, said, “We want to get them while they’re young.”

      …And they will revolutionize the way science is done

      Beyond its presence in high school classrooms, SynBERC offers unparalleled opportunities for undergraduates to make a meaningful contribution to syn-bio research. Each year, five students are chosen to form the Berkeley International Genetically Engineered Machines (iGEM) competition team. The team is given a set of biological parts, with which they will build biological systems and operate them in living cells. This is not just a menial practice project, but, “part of cutting edge syn-bio research,” says postdoctoral researcher Mariana Leguia. “The results they get will be used to advance the field.” The iGEM team works full time in the summer, then present their results to other iGEM teams in an international competition held at MIT every November. Since its creation in 2004, the event has become an effective method to introduce undergraduates to synthetic biology research, and several winners have gone on to prestigious research institutions for their graduate study.

      Undergraduates on the Berkeley International Genetically Engineered Machines (iGEM) teamhave the opportunity to participate in some of the most cutting-edge research in the field.

      Undergraduates on the Berkeley International Genetically Engineered Machines (iGEM) team have the opportunity to participate in some of the most cutting-edge research in the field.
      Credit: Peg Skorpinski

      Berkeley iGEM teams have won gold medals in the past five competitions with their innovations such as Clonebots (2008), a collection of devices and DNA strains to aid part synthesis and analysis, and Bactoblood (2007), a cost-effective red blood cell substitute constructed from engineered E. coli.

      This year, the Berkeley iGEM team sets out to genetically engineer choanoflagellates (see “Redrawing the branches,” current issue), single-celled organisms whose DNA is intractable by current methods in genetic engineering. “Since choanoflagellates eat bacteria, we plan on designing some bacteria that can do the job for us,” says bioengineering major Christoph Neyer. The bacteria contain a set of proteins which are designed to insert a gene into the genome of the choanoflagellate once the bacteria get inside the choanoflagellate’s food vacuole.

      If carried out successfully, the experiment will lay the foundation for a new technique in genetic manipulation of choanoflagellates. “These species are interesting because they are the closest living relative to the microbial ancestor that became the first multicellular animal,” says iGEM team member Conor McClune. To be able to genetically manipulate these little creatures would allow scientists to learn more about the transition from single-celled organisms to multicellular ones, an important step in evolution history.

      More importantly, projects like this are the best representation of SynBERC and synthetic biology research in general, in which two main themes dominate. First, doing science is a collaborative effort in which participants are responsible for inventing individual parts, which are then standardized. Second, these parts must be open source and available to anyone who is interested, accelerating future developments. Once these two requirements are met, the possible applications are limitless, or as Amy Kristofferson, a fourth-year molecular and cell biology student, puts it, “[synthetic biology] is creating all sorts of applications for genetic engineering that are in turn revolutionizing the way in which science is done.”

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