Synthetic Biology Devices for in Vitro and in Vivo Diagnostics Shimyn Slomovica,1, Keith Pardeeb,1, and James J

SPECIAL FEATURE: PERSPECTIVE PERSPECTIVE SPECIAL FEATURE: Synthetic biology devices for in vitro and in vivo diagnostics Shimyn Slomovica,1, Keith Pardeeb,1, and James J. Collinsa,b,c,d,2 aInstitute for Medical Engineering & Science, Department of Biological Engineering, and Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA 02139; bWyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115; cHarvard-MIT Program in Health Sciences and Technology, Cambridge, MA 02139; and dBroad Institute of MIT and Harvard, Cambridge, MA 02142 Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved June 16, 2015 (received for review May 6, 2015) There is a growing need to enhance our capabilities in medical and environmental diagnostics. Synthetic biologists have begun to focus their biomolecular engineering approaches toward this goal, offering promising results that could lead to the development of new classes of inexpensive, rapidly deployable diagnostics. Many conventional diagnostics rely on antibody-based platforms that, although exquisitely sensitive, are slow and costly to generate and cannot readily confront rapidly emerging pathogens or be applied to orphan diseases. Synthetic biology, with its rational and short design-to-production cycles, has the potential to overcome many of these limitations. Synthetic biology devices, such as engineered gene circuits, bring new capabilities to molecular diagnostics, expanding the molecular detection palette, creating dynamic sensors, and untethering reactions from laboratory equipment. The field is also beginning to move toward in vivo diagnostics, which could provide near real-time surveillance of multiple pathological conditions. Here, we describe current efforts in synthetic biology, focusing on the translation of promising technologies into pragmatic diagnostic tools and platforms. synthetic biology | diagnostics | biosensing | synthetic gene networks | nanobiotechnology Synthetic biology employs a forward-engi- on this diversity of sensors and regulatory to the electrode, producing a steady current neering approach to create new molecular elements, incorporating them into gene net- flow that correlated to photosynthetic activity function. In the field’s earliest stages, engi- works and applying their own design crite- (5). This activity and the ensuing current were neering principles guided the design and con- ria of selection for use within novel synthetic disrupted by herbicides and, therefore, could struction of synthetic gene regulatory circuits, architectures. Here, we describe how this report their presence in tested water. In another such as toggle switches and ring oscillators rationale is being applied to confront the example, Zymomonas mobilis was immobilized (1, 2), which quickly led to the develop- growing need for novel diagnostic tools on a pH electrode, and its native glucose– ment of logic, sensor, counter, and timer and capabilities. fructose metabolic activity was used to measure elements (3). These capabilities continue sugar levels via hydrogen ion production (6). to grow in complexity and now include Whole-Cell Biosensing These native cell-based technologies car- biomolecular circuits that can interrogate Hybrid Devices. Synthetic biology was rec- ried important advantages over abiotic sen- both intra- and intercellular spaces and, ognized early on as an opportunity to engi- sors based on purified antibodies or nucleic in response, direct downstream activity neer organisms that could serve as whole-cell acid hybridization, including lower cost, im- of other engineered components, as well biosensors in what may be viewed as an ex- proved stability, and the capacity to generate as endogenous cellular elements. Although tensionoftheuseofanimalsentinelsfor higher biocatalytic activity (7, 8). However, there is not a clear consensus regarding a environmental sensing throughout history. whereas these early biosensing devices were definition of the boundaries of synthetic Examples of the latter include caged canaries remarkable, they suffered from poor selec- biology, it is widely accepted that squarely warning of toxic gases in coal mines, the tivity and interference from culturing condi- within them is the aspiration to use syn- centuries-old practice of using hogs to locate tions (9). As the genetic modification of thetic circuitry and other engineered com- rare truffles, or training dogs to detect illicit microorganisms became more practical, syn- ponents to create novel functions inside materials, explosives, or even cancer (4). In thetic whole-cell biosensors were engineered cells. In the context of diagnostics, syn- the years preceding the emergence of syn- such that the microorganism encompassed thetic biology design efforts are typically thetic biology, animal sentinels were scaled both purpose-built analyte sensors and signal focused on building sensors that are cou- down to the essential unit of life: the living output components. Thus, rather than being pled to a measurable output. These circuits cell. Early cell-based biosensors were a phys- are the outcome of the engineered assembly of ical merger between single-cell organisms and Author contributions: S.S., K.P., and J.J.C. wrote the paper. hardware components that could transduce natural molecular components, which have The authors declare no conflict of interest. been rewarded with survival for their func- biochemical signals into a measurable elec- This article is a PNAS Direct Submission. tional performance through eons of natural tronic output. Mounted onto electrodes, the This article is part of the special series of PNAS 100th Anniversary selection. Biology is enriched with an incred- cells functioned as the sensor component of articles to commemorate exceptional research published in PNAS ible molecular diversity of sensors and regula- the larger detector. In one example, photosyn- over the last century. tors that help maintain organism homeostasis, thetic cyanobacteria were used as biocatalysts 1S.S. and K.P. contributed equally to this work. find resources, and avoid deleterious stresses. that reduced an electrochemical mediator, 2To whom correspondence should be addressed. Email: jimjc@ 3-/4- Synthetic biologists are beginning to draw [Fe(CN)6] , which then donated an electron mit.edu. www.pnas.org/cgi/doi/10.1073/pnas.1508521112 PNAS | November 24, 2015 | vol. 112 | no. 47 | 14429–14435 Downloaded by guest on September 27, 2021 constrained by naturally evolved systems, the riboregulators and Spinach RNA for small sequence in the 5′ region of the toehold. organism itself became the target of engi- molecules and target RNA sequences (21–25). Using these toehold sensors, bacteria were neering efforts (10). Designer circuits tuned RNA sensing for diagnostics recently took a programmed to detect both short synthetic for specific molecular recognition, rather than leap forward with the development of a new RNAs and full-length endogenous mRNAs a general effect on host physiology, provided class of RNA sensors called toehold switches within the cellular environment. a significant step forward for biomolecular (26). Until this point, riboregulators relied on Such RNA-sensing technology has great selectivity. In many early embodiments, the sequestration of the ribosomal binding diagnostic potential; however, while confined the signaling element relied on genetically site (RBS), via a cis-repression sequence, to within the structure of the bacterial cell, its introducing luciferase gene cassettes for bio- control the translation of the downstream accessibility to target RNA in clinical samples luminescent reporting. For instance, by fus- mRNA (Fig. 1A). In the presence of a higher is limited and, therein, its range of applica- ing the Vibrio fisheri luxCDABE cassette to a affinity RNA molecule (the transactivator), tion, as well. This example and whole-cell naphthalene degradation operon in Pseudo- the RBS is exposed and translation ensues. biosensors in general are also inherently monas fluorescens, these living biosensors Although useful, riboregulators are limited bound to biosafety concerns with regard to could sense naphthalene and salicylate bio- for diagnostic applications because the the release of genetically modified microor- availability and produce a bioluminescent transactivator must share sequence con- ganisms into certain environments. There- response (11). Over time, bacterial strains servation with the RBS, which is not a fore, one of the challenges and opportunities were genetically modified to detect arsenic in distinguishing feature of real-world mRNA that synthetic biology faces in the development natural water resources (12), as well as fam- sequences. Green et al. (26) resolved this of deployable diagnostics is to circumvent ilies of other analytes, including hydrocarbon constraint by adding a “toehold” for trigger such limitations when possible. Conse- pollutants, sugars, heavy metals, and antibi- RNA binding and positioning the RBS in quently, focus has recently been trained on otics (13). a loop region of an RNA hairpin, which practical approaches for hosting these pow- The integration of living cells with ma- prevents ribosomal binding and reporter chines was a trend that persisted through erful tools outside of the cell. As we discuss translation while eliminating the need for below, there are new technologies that have the early days of synthetic biology, with the sequence conservation

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