Programming Life: Inquiry & Engineering Through Synthetic Biology

The report of the first chimeric DNA molecule in 1968 (1) ushered in a new age for experimental biology and biotechnology. The ability to propagate DNA obtained, in principle, from virtually any organism within the cytoplasm of Escherichia coli (2) set the stage for sequencing of genes and genomes. This advance enabled researchers not only to connect a mutant phenotype with the corresponding genotype, but also paved the way for the industrial production of medically important proteins such as insulin. The in vitro construction of recombinant DNA thus became a cornerstone method in the functional and biochemical characterization of genes and proteins.

The five decades following the birth of molecular cloning have witnessed an incredible scaling-up of molecular biology due, in large part, to the development of high-throughput technologies in nucleic acid sequencing and macromolecular analysis. But long absent from the resulting explosion of information has been the ability to rationally recreate, in the laboratory, the regulatory complexity of the very gene networks forming the basis cellular behavior. In short, we know a great deal about the “code of life” but are only now beginning to be able to program with it. This aspiration has, in part, given birth to the rapidly developing field of synthetic biology, which aims to unite the rigor of engineering with the design and construction of recombinant nucleic acids, with which to study and understand the behavior of genetic circuits as well as utilize them for technological ends.

Peter Weigle, Ph.D., New England Biolabs, Inc. and Jennifer Redig, Ph.D., BiteSize Bio

What is Synthetic Biology?

Though a comprehensive definition of synthetic biology is elusive, one may characterize it as a “build to understand” approach to biology (3). A quote by the famous theoretical physicist Richard Feynman epitomizes a theme characteristic of the field – “What I cannot create, I do not understand.” How does this sentiment relate to recombinant DNA? Imagine beginning with a repertoire of well-characterized DNA “parts” encoding biological functions such as receptors, promoters, activators, repressors, terminators and reporter genes (or other outputs) – and attempting to rearrange them into configurations designed to direct a biological system (typically, a cellular “chassis”) to accomplish a desired task. Think a pollution detecting E. coli cell that expresses green fluorescent protein (GFP) in the presence of arsenic and then self-destructs after a given period of time, or an engineered implantable human cell line that undergoes a preset number of cell divisions and then secretes insulin at precisely regulated levels in response to extracellular glucose concentrations.

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Synthetic biology draws some of its inspiration from the engineering disciplines of control theory and digital circuit-design. In the illustrated example (A), an assemblage of biological components ideally functions to convert two chemical inputs (IPTG and arabinose) into an output: the expression of the fluorescent reporter protein GFP. Two promoters (PBAD and Plac are each constitutively repressed until induced by their cognate chemical signals (arabinose and IPTG, respectively). Each operon expresses half of a two-part transcriptional activator (the SicA and InvF gene products) which together activate the transcription of the GFP under the control of PsicA. Expression of the reporter only occurs in the presence of both inputs. The DNA circuit can be represented abstractly as a logic gate implementing the Boolean “AND” operation and is shown with the associated truth table (B). Higher order circuits (C) can be created by combinations of modular genetic gates; in this example, three AND gates convert four inputs into a single output. Figure content adapted from Brophy and Voigt (2014).

Such re-purposed cells would be described, in synthetic biology parlance, as “genetic devices.” These devices are designed for multi-step behaviors, and relative to earlier examples of genetic engineering, their design is necessarily complex. How cells can be programmed for such functions is neither intuitive nor obvious. Here, synthetic biology has made a radical departure from previous forms of genetic engineering by borrowing engineering concepts from control theory and digital computing as a framework upon which to design genetic circuits for programming cellular behaviors. A genetic implementation of one such “simple” computational operation, the Boolean AND gate, is shown in Figure 1. Higher order combinations of multiple kinds of genetically encoded Boolean operations, and other types of synthetic gene circuits, have been constructed to perform a variety of simple computational tasks, including edge-detection, cell to cell communication, and counting of signal inputs (4).

Going from a circuit schematic to a working genetic device is guided by an engineering paradigm: the design-build-test cycle (Figure 2, p. 4). A key tool in this process is computer-aided mathematical modeling. Unlike their electrical counterparts, genetic circuits operate under conditions that dominate the cellular environment. A model attempting to describe and predict the behavior of a genetic device must accurately incorporate a range of parameters such as diffusion, binding equilibria, networks of protein/DNA interactions, and dynamic reactant concentrations; a variety of deterministic and stochastic approaches have been employed to accomplish this goal (5). As such, the model embodies a sophisticated hypothesis about how the device might work. The genetic device is prototyped (e.g., synthetic DNA is assembled and transformed into the cell) and its behavior evaluated in terms of the model. What is learned during each stage is used to improve the performance of the device in subsequent rounds of the cycle – through changes to the device itself, as well as through refinements to the model.

While synthetic biology shares many of the tools and reagents with hypothesis-driven experimental biology and molecular biology, it follows a fundamentally different approach. Many of the techniques in a molecular biologist’s repository (e.g., oligo synthesis, genome editing) may not exist in their current form were it not for synthetic biology. Conversely, synthetic biologists can build upon discoveries made by molecular biologists. In essence, synthetic biologists assemble genetic components in order to execute an “artificial” function, and in the process of getting it to work, the engineered genetic construct becomes itself an object of study and yields basic principles for application to subsequent designs.

Chemical engineering in vivo

A practical definition of synthetic biology must also include the latest developments in industrial fermentation and metabolic engineering. Even a cursory survey of papers and journals covering synthetic biology shows a significant number of reports describing synthetic biology to synthesize fuels, chemicals and materials. Historically, this technology began as an outgrowth of beer and wine making, after it was discovered that fermentation could also be used to produce economically valuable solvents and organic acids (6). With the advent of greatly expanded sequence databases, inexpensive DNA synthesis, and genome engineering methods, it has become increasingly practical to do chemical synthesis in vivo. Designer metabolic pathways utilizing genes encoding enzymes derived from any of the domains of life can inserted into microbes such as Saccharomyces cerevisiae or E. coli, endowing them with the ability to convert cheap chemical inputs, such as starch- or cellulose-derived sugars, into more commercially valuable chemicals.

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Figure 2. The synthetic biology workflow is iterative.
The products of synthetic biology applied to industrial fermentation are already in the marketplace and new products are in the works. Large agro-chemical companies such as Cargill® have established plants for the conversion of starch to platform chemicals such as 3-hydroxypropanoate, which can serve as an intermediate for many other commodity chemicals. The engineering of E. coli and Saccharomyces to produce the anti-malarial precursor artemisinic acid by Amyris® is a landmark achievement in synthetic biology and metabolic engineering. Elements of their engineered biosynthetic pathway have subsequently been repurposed to produce fuels and high value chemicals (7), while Ginkgo Bioworks™ (8) and companies such as Joule® (9) are engineering microbes to mitigate greenhouse gases such as methane and CO2 and produce valuable products, including biofuels. Industrial biosynthesis is not limited to pharmaceuticals and commodity-scale chemicals. Companies such as Evolva® are working on ways to engineer yeast to produce vanillin, stevia and even the flavor components of saffron (10), while Pronutria® is working to efficiently convert CO2 to feed and medicinal nutrients (11).

The First Synthetic Gene Circuits

In 2000, the first synthetic circuits were made when Gardner, Cantor and Collins created a genetic toggle switch (12), and Elowitz and Leibler engineered a repressilator, a synthetic genetic regulatory network designed from scratch to produce stable oscillations of gene expression (13). Both of these circuits were model-based, but both also needed experimental fine-tuning to achieve agreement between model and experimental output.

These experiments were quickly followed by “The First International Meeting on Synthetic Biology” or SB1.0, which was held in 2004 at MIT (14). Attended by biologists, chemists, physicists, engineers and computer scientists, the goal of this conference was to bring together those scientists interested in creating and characterizing synthetic biological systems. This meeting, and smaller ones like it, laid down the foundation of a new, emerging discipline.

A Community to Build From

As the synthetic biology discipline grew, it quickly became clear that there needed to be a more efficient way to assemble genetic parts and circuits (4). Without established methods for assembly and testing, researchers were forced to ad hoc experimental designs, wasting time and money by designing, testing and redesigning constructs. To combat these issues, a public repository, the Registry of Standard Biological Parts (RSBP), was founded at MIT by Tom Knight and Drew Endy. The goal of this repository is to catalog and develop genetic parts into ‘BioBricks®’ that could be used for the assembly of larger circuits. The Bio- Brick standard was developed to ensure that parts could be easily shared and used among synthetic biologists by requiring submitted parts to conform to a simplified cloning scheme utilizing four restriction enzymes. However, it became quickly clear that the task of populating the Registry with biological parts, and the work of characterizing them to establish their utility, would dwarf the resources of the relatively small numbers of labs devoted to synthetic biology. Out of this daunting mission, and the need to sustainably train a new generation of synthetic biologists, the International Genetically Engineered Machine (iGEM) competition was born (15).

Training the next generation of bioengineers

Since its inception in 2004, iGEM has evolved into a highly successful vehicle for training and showcasing a new generation of biological engineers using the synthetic biology framework. In 2014, iGEM hosted its 10th annual Jamboree, with over 4,000 participants from across the globe presenting projects that detailed their efforts to model, build and test genetic devices. Students competed in a variety of tracks such as Food, Medicine, Manufacturing and Information Processing. Each team was also asked to demonstrate that they have considered the impact and implications of the technologies they are developing through dialog with relevant stakeholders. Teams were supported by various organizations, including NEB. To date, more than 28,000 student competitors have participated in this engineering competition.

A rapidly maturing field

Synthetic Biology as a discipline continues to grow rapidly. Recent synthetic biology developments include:

Circuits Get Complex – In the early 2000s, DNA circuits continued to advance. More elements were added (16), and sensing became more diversified (17,18). Additionally, RNA, not just DNA, was used in circuit generation (19–21).

‘Synthetic’ Used to Investigate ‘Native’ – Beginning in 2009, designed circuits were used to understand native systems through compare/contrast schemes (22) of engineered versus native systems. Synthetic Biology was not just limited to engineering new biology; it was also used to investigate and understand native biology.

Commercially Valuable Products are Made – In the 2000s, amino acid biosynthesis was used to produce commercially valuable products such as isobutanol (23,24), biodiesel (25) and gasoline (26). These experiments were a logical extension of fermentation biotechnology and highlighted synthetic biology’s commercial and environmental promise.

Assembly of a Whole Bacterial Genome in Yeast – In 2008, researchers were able to take advantage of yeast’s remarkable ability to recombine overlapping DNA fragments to assemble an entire genome in a single step. This method allowed for speedier assembly of DNA molecules than previous methods (27,28). New Genome Editing Tools Emerge – Beginning in 2010, zinc finger nucleases gave way to more precise genome editing tools, from TALENS (29) to, in 2013, CRISPR/Cas9 (30) systems. This empowered synthetic and molecular biologists to create and explore as never before. In addition, a catalytically inactive form of Cas9, known as dCas9, has further enhanced the usefulness of the CRISPR/Cas9 system by enabling both activation and repression of transcription in yeast and mammalian cells, allowing modulation of endogenous gene expression (31).

First “Artificial Cell” Engineered – In 2010, Craig Venter and colleagues demonstrated just how far the discipline of synthetic biology had come when they published a paper disclosing the recreation of a Mycoplasma mycoides cell controlled by a chemically-synthesized genome (32).

Therapies Engineered – In 2010, Fussenegger and colleagues engineered a synthetic circuit that, when inserted into the genome of a mouse mutant bred to develop hyperuricemia, was able to maintain uric acid homeostasis, essentially correcting an inborn metabolic defect (33). This demonstrated the therapeutic promise of synthetic biology.

Ongoing Challenges

Synthetic Biology is a young field, but it has achieved much in a short time period. However, like all disciplines, it continues to face challenges.

Measurement, Robustness and Predictability – Aspects of synthetic biology still remain an art. Genetic circuits often require much “tweaking” in order to get them to function in the context for which they were designed. Further principles governing the function of genetic circuits will have to be elucidated to improve the interoperability of genetic parts in multiple contexts.

Cells are Not Exactly Digital – Though incredibly powerful as a guiding framework for designing, building, and testing genetic circuits, the digital circuit metaphor has limits. Biological systems differ from electronic ones in fundamental ways, and modeling genetic regulation remains under determined. Synthetic biology researchers continue to incorporate new ideas and theories to describe, model and predict genetic circuit behaviors. A new and promising area utilizes analogies to analog circuitry (34).

Ethics and Safety
– Synthetic Biology, and indeed all genetic engineering, has provoked concern over potential misuse, intentional or accidental. There is active discussion regarding potential impacts (35). Built-in forms of biological containment are also an active area of investigation, including the refinement of genetic “kill switches”, which ideally would ensure that genetic devices could not survive outside of the laboratory or factory. Government policy has and will continue to weigh in: information on the ethics of synthetic biology can be found in the 2010 Presidential Bioethics Commission report on synthetic biology (36). As with other technologies, a scientifically literate public is a requirement for nuanced and effective dialog.

Future Directions

The past sixty years have seen incredible scientific and technological advances based on the ability to compose in DNA. DNA-driven technologies will continue to absorb developments and ways of thinking from diverse fields. Advances in materials sciences, nanotechnology, microfluidics, automated liquid handling, indeed all the applied sciences, will drive new applications using cellular systems and even biological technologies beyond the cell (37,38). The proliferation and use of these technologies will continue to impact our lives. With prudence and foresight, they may prove indispensible to our survival.

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