Gábor Balázsi, Ph.D. from the Louis and Beatrice Laufer Center for Physical and Quantitative Biology and the Department of Biomedical Engineering at Stony Brook University in the U.S., shares his perspective on the field of synthetic biology in terms of the past, present and future
“What I cannot create, I do not understand” – wrote Richard Feynman on his blackboard decades ago. We all know from experience that struggling to create something such as a working radio, a meal or even the cheque book balance can reveal strengths and weaknesses in our understanding. Almost all the machines and tools we use, from corkscrews to cell phones to rockets highlight our understanding of non-living, physical and chemical systems. On the other hand, despite millennia of observing the living world, human-built living devices do not exist – or at least not until recently. That is about to change now, as a new generation of scientists started engineering biology, establishing the new field of synthetic biology. Synthetic biologists aim to create artificial biological systems for specific purposes by modifying and assembling the molecular components of living cells in new ways.
It all started with two scientific papers in 2000 Nature 403(6767):335-42 (2000) reporting on switches and oscillators constructed from genes. These new “gene circuits” did not just emerge from trial and error: their capabilities were mathematically predictable. In other words, the gene circuits had designable behaviours, just like engineers design new machines on paper or within a computer beforehand. Many other gene circuits followed, including pulse detectors, light sensors, counters, memory devices, dimmers and logic gates Nature Reviews Microbiology 12, 381–390 (2014). As the complexity and number of such biological devices increased, discussions and hopes about their potential uses intensified, including medical and industrial applications. The hope for practical applications stimulated the emergence of a second branch of synthetic biology, focused on metabolic engineering and bioproduct fabrication, starting with the successful synthesis of an otherwise expensive anti-malarial drug precursor, by moving appropriate plant genes into bacteria and then yeast in 2003 and 2006 Nature 440(7086):940-3 (2006). A third branch emerged as well, aimed to synthesise and edit entire genomes. Whole-genome engineering started with viruses, then continued with bacteria and yeast Science 351 (6280):aad6253 (2016); Science 355(6329): 1040-4 (2017), reflecting the increasing ease of DNA synthesis. From these achievements, it is conceivable that fully synthetic human genomes will eventually follow, although it is unclear when.
The three branches of synthetic biology have somewhat different goals, methods and promises. Closest to mindful engineering is perhaps novel gene circuit development, which relies most on mathematics for design and performance prediction. The other two branches (metabolic and whole-genome engineering) are closer to automation and tend to rely on biotechnology more than mathematics. In Feynman’s terms, gene circuit development exemplifies human creation of novel bio-devices, whereas the other two branches are closer to recreating, copying nature’s own, existing designs. Practical benefits from gene circuit development may be less straightforward and farther-away than the benefits from the other two branches, which are already enabling the cost-effective synthesis of pharmaceuticals, biofuels and cosmetics and are attracting the attention of industry and start-ups. Nonetheless, we will focus here on synthetic gene circuits, to increase public understanding of how they work and appreciation of why they may be important.
Gene circuit development relies on the ability to cut, copy and paste DNA, the stringy hereditary material of every living cell containing a code written in four letters: A, C, T, G. Each gene is a piece of DNA, carrying the code the cell uses to produce one of its thousands of proteins. The types and amounts of proteins present in a cell determine what the cell does – whether it sits in specific tissues or moves away, whether it resists external stresses, or whether it produces certain biochemical compounds. Adding, deleting or editing a gene adds, removes or alters the corresponding protein, respectively.
However, modifying certain genes can have indirect effects: besides the protein they encode, the levels of some other proteins will change. Typically, these indirectly-acting genes encode transcription factor proteins that bind to promoter regions near other genes to either promote (activate) or slow (repress) their protein synthesis. Pairing such genes and the corresponding promoters in new, artificial configurations enables connecting them in predesigned ways, creating gene circuits. For example, pairing a gene with a promoter it represses gives rise to a simple negative feedback gene circuit that can be used to precisely control cellular protein levels Proc. Natl. Acad. Sci. USA. 106(13):5123-8 (2009). Pairing a gene with a promoter repressed by another gene and vice versa results in a genetic toggle switch Nature 403 (6767):335-42 (2000). To become functional, the DNA sequence of gene circuits must be introduced into living cells that can make the proteins corresponding to each gene.
Biotechnology is increasingly capable of manipulating DNA strings. Currently, the most exciting advances in biotechnology involve precisely cutting, editing, writing and reassembling DNA sequences. Longer and longer synthetic DNA pieces can be ordered for falling prices, meaning that gene circuit engineering is simpler and more accessible by the day. However, the ability to reliably predict the capabilities and robustness of newly assembled synthetic gene circuits has not kept pace. Unlike in electronics where circuit components are typically standardised, with known tolerances and error rates, little of that holds for genetic components. Part of the problem is biological knowledge gaps – we still do not know exactly the function of each stretch of DNA sequence. Moreover, even known DNA functions can change depending on the host cell’s genome or the environment.
Efforts have been underway to simplify gene circuit sequences, minimise host genomes and standardise genetic parts to eliminate some of that uncertainty, making gene circuit behaviour predictable by engineering software. However the exact details we aim to predict matter – it is unlikely that we can predict the levels of every protein in every single cell, despite their importance. Second, even if gene circuit function is predictable in some precisely-defined cell type and environment, the predictions may fall apart as soon as one of these factors (such as cell type) changes. Third, the behaviour of the initially intact gene circuit may change as mutations randomly arise, as they inevitably do in nature. Overall, major challenges await to be addressed before synthetic biology can deliver on some of its promises.
Once some of these difficulties are resolved, future opportunities for synthetic gene circuits are abundant, almost infinite – considering the immense number of cell types and possible applications. Gene circuits can sense and report on almost anything that happens inside living cells or can act as control knobs that allow humans to interact with cells and drive cell behaviours. Knowing what cells do and manipulating their behaviour could revolutionise cell, tissue and organ transplantation and engineering, stem cell- and immunotherapies, as well as gene therapies. It could also aid environmental remediation, biomaterial synthesis and biofuel production and it can contribute to cosmic travel and defence applications.
Supporting basic synthetic gene circuit research is crucial to enable the future advances we desire. Currently, gene circuit development is the branch of synthetic biology that could lead to the broadest range of applications from medicine to ecology to agriculture. Yet, the interest of the private sector in gene circuits is modest because the deliverables are less immediate than for metabolic engineering and bioproduct synthesis. While some nationwide funding initiatives for basic synthetic biology research exist, such as the UK’s Biotechnology and Biological Sciences Research Council (BBSRC) prioritising the field, the U.S. Office of Naval Research or the NSF-funded multi-university consortium Synberc, they are insufficient and do not distinguish between the three branches of synthetic biology. Since the area is still young, it is difficult to recruit reviewers for synthetic biology proposals. Synthetic biology proposals submitted to national funding agencies are typically reviewed by panels of traditional biologists, along with traditional biology grants, leading to low success rates. This situation is unlikely to change unless definite, unorthodox steps are taken to support this highly promising field, especially the design and basic, quantitative characterisation of synthetic gene circuits, which has attracted less industrial interest and support than the other branches of synthetic biology.
Overall, synthetic gene circuits could be exactly the tools we will need to address many societal challenges we will be increasingly facing – including ageing, cancer, drug-resistant microbes, environmental pollution – but whether we will have these tools when such challenges truly arise depends on what we do right now.
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