# Synthetic biology

February 22, 2012, 3:17 pm
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Photo: Jeff Tabor

## Definitions

Synthetic biology involves the deliberate, constructive modification of cells, organisms, populations – or their major subsystems – so as to achieve human objectives. It includes artificial synthesis of the natural functional components of living systems and their non-biological analogs, genetic redesign of existing organisms, and ultimately creation of utterly new organisms de novo. In principle it puts control of the entire biosphere in human hands.

 A  UT Austin team designed and built a biofilm that could perform distributed edge detection on a light-encoded image. In theory, each cell in a lawn of bacteria would update its state in response to light input and, depending on the state of neighboring cells, decide whether or not to change color. Source: The Regisgtry of Biological Parts)

This definition, couched in the broadest possible terms, encompasses the entire range of activities that involve modifying or mimicking biological systems or their constituents. It includes artificial synthesis of of natural products such as indigo and urea, modification of agricultural species to make hybrid or genetically modified (GM) crops, and even more ancient practices such as cross-breeding between horses and donkeys to produce mules and hinneys! Such a definition describes synthetic biology sensu lato.

Since the turn of the 21st century a new, narrower focus within this area has captured the imaginations of scientists from many disciplines. In essence, they apply methods used in non-biological fields like mechanical engineering, electrical engineering and computer science to configure biological systems to achieve important practical (or sometimes merely whimsical) goals. This genetic engineering on steroids" now dominates discussions of "synthetic biology," and constitutes a new sensu stricto definition of the term.

The isolation of natural products, usually in impure form, antedates scientific chemistry. Beginning in the 18th century, serious efforts to purify and characterize compounds like tartaric acid, citric acid, uric acid, glycerol, urea, cholesterol and alkaloids such as morphine, quinine, strychnine and brucine met with success and provided useful materials for pharmceutical and other applied uses. Because these all originated in living systems, the prevailing view by the 1820s was that a special vital force (élan vital) was required for their synthesis, so it was with some consternation that chemists in 1828 learned of Friedrich Wöhler’s synthesis of urea from totally inorganic starting materials. His experiment, long heralded as the point of separation between organic chemistry and biochemistry, might also be chosen as the first example of synthetic biology – albeit rather rudimentary.

Following Wöhler, organic chemists have pursued synthesis of natural products with a vengeance, conquering ever-more-complex and challenging targets as mentioned above. These projects have had several objectives. First, they provide confirmation of a correctly assigned molecular structure; second, they exercise synthetic methodologies and stimulate creation of new ones; and third, they provide access to materials that have medicinal or other commercial value. Apropos of synthetic biology, the third point is significant. While no one would undertake chemical synthesis of sucrose as a means to replace its natural source of supply, the same is not true of many natural products used as drugs, for example, taxol. Not only does a chemical synthesis often afford a less costly, more efficient route to a bioactive natural product, but it also opens the door to synthetic variants that may have safer and/or more potent pharmaceutical properties. Thus, using the broad definition of synthetic biology to include the chemical synthesis of natural products, the complete basic pattern is already clear:

(1) isolate a piece of a biological system;

(2) synthesize it artificially for some useful application; and,

(3) make unnatural variants that have different and perhaps superior properties.

Generally the term “natural product” is applied to a (comparatively) small organic molecule with at most a few hundred atoms. This sets it apart from the study of the main molecular machinery of living organisms which consists of nucleic acid, protein and polysaccharide molecules that contain thousands of atoms. These “messy” substances did not appeal to the synthetic organic chemists who preferred to work with compounds that had sufficient volatility to distill or could be readily purified via crystallization. When synthetic polymers came along, their polydispersity and non-crystalline character also discouraged interest on the part of traditional organikers such as Nobel laureate Richard Willstätter who even questioned the existence of molecules with molecular weights greater than a few thousand daltons. Thus synthetic polymer synthesis followed its own trajectory – though in the process unintentionally produced some biomimetic materials like nylon. Synthetic studies of peptides and oligonucleotides, complex natural products related to proteins and nucleic acids, remained a relative backwater until the 1950s and ’60s, and actual synthesis of the biological macromolecules was unimaginable before that time.

Progress in biochemistry and molecular biology, however, stimulated the chemistry of polypeptides and polynucleotides – first sequence analysis and then synthesis. Using methods derived from the synthetic polymer field, poly-?-amino acids were produced in quantity, but though they served as useful models for natural proteins (displaying ?-helical and ?-sheet secondary structures, for example) they were mostly either homopolymers or random copolymers lacking any of the specific characteristics of native proteins. The polynucleotide field advanced a bit faster and further, partly because of the use of enzymes as synthetic reagents, and the spectacular achievements of the group led by Har Gobind Khorana allowed the definitive analysis of the genetic code by the late 1960s. The brute-force chemical synthesis of insulin by groups in the U.S., West Germany and China, completed in 1963, demonstrated that synthesis of a protein in the laboratory was possible, but required heroic efforts and had discouragingly low yields. Introduction of solid-phase methods, first for analysis of amino acid sequences, then for their synthesis, represented a significant advance, but yields of chains longer than 50 amino acids were not encouraging. Successful synthesis of active bovine pancreatic ribonuclease A (124 residues long) proved the concept that enzyme activity could be created through synthetic chemistry, but again the issues of yield and purity discouraged further developments.

It would be fair to say that by 1970, chemists’ ability to synthesize biological macromolecules was still quite limited. All this changed dramatically with the advent of recombinant DNA technology in the 1970s. This is not the place to summarize the now-familiar methodologies from this research field, but it’s important to note their results in the context of synthetic biology since they have totally transformed the possibilities of this field. Briefly, recombinant DNA techniques allow:

1. Isolation of defined natural sequences of DNA (genes or gene fragments) and amplification of these sequences by cloning or polymerase chain reaction (PCR).
2. Similar manipulation of artificial DNA fragments synthesized by solid-phase methods.
3. Insertion of isolated or designed DNA sequences into vectors, followed by transfer into host organisms to express encoded protein or RNA molecules.
4. Isolation and manipulation of the expressed molecules so as to recover them in their native forms with biological capabilities.

Though these procedures are not without difficulties (which in some cases can be extreme), the bottom line is that almost any DNA, RNA or protein molecule can be produced (together with a very large number of variants) in a reasonable time, in comparatively large amounts and at reasonable cost.

Using such recombinant DNA methods, many very useful therapeutic proteins – insulin, factor VIII, Cerazyme, etc. are now produced in quantity. Proteins for other purposes as well as RNA ribozymes can likewise be obtained by using engineered organisms as factories to produce them. Since these methodologies can be used for “protein engineering,” i.e., production of designed variants of natural proteins or even totally synthetic proteins, they represent a well-established, mature branch of synthetic biology. In the RNA field, the capacity to screen randomly generated sequences for desired properties (production of “aptamers”), amplify them, then iterate the process constitutes “directed evolution” – drawing still more on the biological model.

The fact that recombinant techniques are not limited to manipulations of single genes, combined with the obvious analogies of gene-expression and regulatory systems to computer hardware and software components, has now begun to inspire much more ambitious redesign of cells (usually bacteria) as demonstrated in two recent meetings (“Synthetic Biology 101,” at MIT in 2004, and "Synthetic Biology 201" at Berkeley in 2006). These projects aim primarily at developing an understanding of modules (“biobricks”) that can be assembled to perform particular functions (tricks!) in response to appropriate signals. Two now classic examples involved (a) the “repressilator,” that combines several operator/repressor elements in a system that generates oscillatory production of a green fluorescent protein reporter, and (b) the opposed expression of gene products from two different promoters to produce a “genetic toggle switch” that can be stably flipped from one state to another by external signals. In these cases, mathematical analysis guided the construction of the artificial genetic circuit. Extensions of this approach look very promising, and efforts are now underway to provide “tunable promoters” that can be used to adjust gene expression in subtle ways for use in such small-network engineered cells. Such sophistication may prove vital in projects that attempt to “rewire” cells to carry out entire biosynthetic processes involving multiple enzymatic steps – as best illustrated by the (almost-complete) program to engineer Escherichia coli (E. coli) to synthesize the anti-malarial drug artemisinin in Jay Keasling’s laboratory at Berkeley (funded by US$42M from the Gates Foundation!). The motivations for experiments like these are diverse. In some cases they are just playful demonstrations of the correctness of the molecular biology results and the feasibility of combining modular units into functioning systems. Many also aim at serious applications, however, and the more complex the modifications of the host cell, the more interesting and potentially useful the results. Until recently, a perceived limitation was the size of the plasmid DNA needed to incorporate all the transferred genes, to say nothing of the anticipated challenge of synthesizing an entire bacterial genome. This brings up a critical technology: accurate synthesis of very large DNA molecules. Progress in this area (based on photo-programmable microfluidic chips) has been very substantial recently, however, as demonstrated by the synthesis of a DNA fragment encoding all 21 proteins of the E. coli small ribosomal subunit. At a projected cost of 20,000 bases per dollar using this methodology, the complete E. coli genome would cost a mere$2500!

Looking ahead, some of the possibilities are:

• Further development of cells as macromolecule factories. These would allow large-scale production of de novo-designed proteins as prime sources of diagnostic and therapeutic pharmaceuticals and industrial enzymes. Ribozyme production will doubtless also follow suit as will expression of engineered eukaryotic proteins in a variety of systems that allow needed post-translational modifications.
• Successful metabolic engineering of prokaryotic cells to synthesize a wide range of useful low-molecular-weight products (à la artemisinin). Such systems may also enable conversion of biomass to substitutes for petroleum – an effort already underway at the Venter Institute.
• Fancier “circuits” that can act as computational devices. The downside of such “gooey computing” is significant, though as tests of cellular regulatory and network theories, these efforts may bear some fruit. Studies on the synchronization of “sloppy clocks” in cell populations may greatly contribute to the understanding of circadian rhythms – a clinically significant field.
• Modified cells “tasked” with therapeutic and other applied practical roles. Phytoremediation already has made substantial progress and could be supplemented or replaced by “smart” strains of bacteria that could respond to the types and amounts of pollutants. Merger of modified-cell biology with various themes in nanotechnology may also occur.
• Synthetic prokaryotes. As notably proposed by J. Craig Venter as long ago as 1999, there are no apparent obstacles in the way of creating totally artificial prokaryotic organisms (perhaps to be classified in a new genus Venteria?). Building on the experiences of chimeric strains with genes from multiple biological species as well as the effort to define a “minimal genome,” this project has already spawned at least one new company (Synthetic Genomics) under Venter’s leadership.
• Synthetic eukaryotes (probably a form of yeast). Manipulation of nitrogen fixation, photosynthesis, hydrocarbon production and other such processes may be initial targets in the design of artificial nucleated, organelle-containing cells. Uncertainties in the step to multicellularity limit predictions about eventual artificial plants and animals to the realm of science fiction for the time being.
• Can synthetic biology cross with stem-cell research? Cloning? Again, though such suggestions remain only in the visionary stages, it isn’t unreasonable to suggest that the ability to manipulate stem cells and embryos could be combined with the ability to create novel genes and produce striking products (perhaps photosynthetic farm animals that require much less feed...).

## Ethical Issues

As with any new scientific/technological advance, synthetic biology raises a number of serious questions in the minds of the public — and the scientists themselves — concerning its potential for harm as well as benefits. These questions tend to fall into three major categories.

"Playing God." For many people, a technology with the potential to create entirely novel living organisms — to say nothing of making major modifications in existing species, including Homo sapiens — threatens to transgress sacred limits. Like Prometheus, synthetic biologists bid fair ultimately to put god-like power in human hands. Not surprisingly, the hero of Mary Shelley's famous 1818 Gothic horror novel Frankenstein (subtitled The Modern Prometheus) gets mentioned repeatedly in discussions of synthetic biology. Where does legitimate ambition end and hubris begin? Clearly, practical, working limits must be set that have support from the majority of the general public. The synthetic biology research community has recognized this fact and already has a tradition of openness about its plans and projects. Since the more melodramatic possibilities lie fairly far in the future, a solid, confidence-building track record can be established before the most controversial issues arise. Of course, the public must make the effort to understand the basic technical details in order to contribute constructively to the process of creating sound regulations to control the genie.

Safety. Vital concerns for the welfare of researchers, the public, and the environment are easier to address than the hubris issue. Thanks to more than 30 years of experience working with recombinant DNA (and an even longer period of microbiological research on dangerous pathogens) a wealth of safeguards already exists to cope with many anticipated laboratory hazards. These include prevention of unintended releases of dangerous organisms to the environment. Physical isolation procedures (BL2, BL3 and BL4 laboratory standards) are well-established, understood and effective. Beyond that comes biological isolation — steps taken to insure that the products of genetic manipulation are restricted to the cells and habitats for which they were designed. Many possibilities exist to accomplish such biological containment and these have great promise especially in the area of preventing harmful disturbances to the environment. Synthetic biologists have learned from the problems (both real and perceived) encountered in the introduction of genetically modified crops, that such ecologically complex processes require detailed and sophisticated planning. New regulations and standards must be developed with care and strictly enforced. How to manage this on a global scale poses a challenging task.

Malevolence. A broad consensus holds that it is much easier to construct new or modified pathogens than to build most other destructive devices — nuclear weapons, for example. Hence, in a time of great concern about terrorism, a field like synthetic biology that promises to increase our ability to modify or create all types of organism, raises major concerns about preventing abuse by terrorists. The anthrax attack(s) of 2001 in the U.S. set a disturbing precedent. This topic has been extensively discussed in the synthetic biology community, particularly at the conferences held at MIT in 2004 (Synthetic Biology 1.0) and at Berkeley in 2006 (Synthetic Biology 2.0). Two main themes emerged from these discussions: (1) the culture of the synthetic biology community has been deliberately very transparent and open — a situation that is expected to continue and that should work strongly against malicious attempts to pervert the science; and (2) the particular need in synthetic biology for rapid, large-scale DNA synthesis affords an easily monitored "check point" where efforts to make pathogens could be detected. This second factor depends upon cooperation from the (relatively few) companies that provide commercial synthesis of DNA. Representatives of most of these companies attended Synthetic Biology 2.0 and described the steps they have taken (some of which are government-mandated) to screen orders to make sure that they do not support unethical projects. Further efforts to strengthen and systematize such screening are currently underway.

Two groups have made organized efforts to address the ethical concerns of scientific biology. At the National Institutes of Health (NIH) the National Science Advisory Board for Biosecurity (NSABB), comprised of outside experts as well as ex officio members representing 15 federal government departments, has the charge of making recommendations "on ways to minimize the possibility that knowledge and technologies emanating from vitally important biological research will be misused to threaten public health or national security." Information concerning the activities of this board, including a July 2006 "Draft Guidance Document" is available on the internet (see Further Reading). The second group, funded by a grant from the Alfred P. Sloan Foundation, consists of investigators from the Massachusetts Institute of Technology (Drew Endy), the J. Craig Venter Institute in Rockville, MD (Robert M. Friedman), and the Center for Strategic and International Studies in Washington, D.C. (Gerald L. Epstein). This group focuses strictly on "the societal impacts of synthetic genomics" and has worked for more than a year collecting information from scientists, policymakers and the general public. Some interim information about this project (plus valuable links to other websites) can be found on the internet (see Further Reading). A final report on the project is due out shortly.

• Ball, P. (2004) “Synthetic Biology: starting from scratch,” Nature 431(#7009) 624-626 (October 7, 2004).
• Benner, S. A. and Sismour, A. M. (2005) “Synthetic Biology,” Nature Reviews Genetics, 6(7), 533-543 (July 2005).
(Good review from the chemist’s perspective – written for readers with a background in biochemistry or molecular biology. Stresses the contrasts between analytical and synthetic approaches to biology.)
• Church, G. (2005) "Let us go forth and safely multiply," Nature 438 (#7067) 423 (November 24, 2005).
(A clear, detailed statement of the need for the synthetic biology research community to take safety issues seriously and to practice "discipline and openness." A more-or-less consensus view.)
• Endy, D. (2005) “Foundations for engineering biology,” Nature 438 (#7067) 449-453 (November 24, 2005).
(The case for synthetic biology (sensu stricto) described by one of the leaders in the field. For readers with general scientific literacy (but not necessarily specialists).)
• Ferber, D. (2004) “Microbes Made to Order,” Science 303(2), 158-161 (January 9, 2004).
(“News Focus” on synthetic biology for the general reader.)
• Forster, A. C. and Church, G. M. (2006) “Towards Synthesis of a Minimal Cell,” Molecular Systems Biology doi:10.1038/msb4100090 (August 22, 2006).
(A first pass at designing “a chemical system capable of replication and evolution, fed only by small-molecule nutrients.” Of general interest, but requires a rudimentary knowledge of molecular biology to understand fully.)
• Gibbs, W. W. (2004) "Synthetic Life," Scientific American 290(5), 74-81 (May 2004).
(Clear, authoritative introduction to synthetic biology, written in layperson’s language.)
• Heinemann, M. and Panke, S. (2006) “Synthetic biology -- putting engineering into biology,” Bioinformatics. 2006 Sep 5; [Epub ahead of print].
(Well-written and referenced current review that emphasizes engineering principles and the computational tools required to implement them in biological systems.)
(Links to information concerning the activities of this board, including a July 2006 "Draft Guidance Document".)
• Ro, D-K., Paradise, E. M., Ouellet, M., Fisher, K. J., Newman, K. L., Ndungu, J. M., Ho, K. A., Eachus, R. A., Ham, T. S., Kirby, J., Chang, M. C. Y., Withers, S. T., Shiba, Y., Sarpong, R., and Keasling, J. D. (2006) "Production of the antimalarial drug precursor artemisinic acid in engineered yeast," Nature 440(#7086), 940-943 (April 13, 2006).
(Highly technical report of the successful completion of the initial phase of metabolic engineering of Sacharomyces cerevisiae so as to produce the key precursor of artemisinin, the crucial ingredient in triple-drug antimalarial therapy. Further optimization of the biosynthesis, together with industrial scale-up, should make this a poster-child project that demonstrates the potential societal benefits of synthetic biology.)
• Silver, L. M. (2006) Challenging Nature: The Clash of Science and Spirituality a the New Frontiers of Life, 464 pp. (Ecco/Harper Collins, New York, 2006).
(An extensive analysis of the forces in modern society that oppose the scientific modification of living organisms for human purposes on the grounds that Nature should remain inviolate. Silver, a Princeton biologist and author of Remaking Eden (1997), lays out the case for thoughtful and constructive use of modern biotechnology to meet human needs. In the process he presents detailed arguments to refute positions of the religious right and the eco-environmentalist left.)