Are the Biomedical Sciences Ready for Synthetic Biology? 2020 PDF

BioMol Concepts 2020; 11: 23–31 Mini-Review Open Access Maxwell S. DeNies, Allen P. Liu, Santiago Schnell* Are the biomedical sciences ready for synthetic biology? https://doi.org/10.1515/bmc-2020...

BioMol Concepts 2020; 11: 23–31 Mini-Review Open Access Maxwell S. DeNies, Allen P. Liu, Santiago Schnell* Are the biomedical sciences ready for synthetic biology? https://doi.org/10.1515/bmc-2020-0003 received November 19, 2019; accepted January 2, 2020. Introduction Abstract: The ability to construct a functional system Discovery and engineering are two research approaches from its individual components is foundational to commonly used to understand the natural world. To understanding how it works. Synthetic biology is a date, much of our knowledge of physiology is derived broad field that draws from principles of engineering from research using a discovery mindset, which focuses and computer science to create new biological systems on unraveling a system to understand how it works. or parts with novel function. While this has drawn well- Individual components of a system are removed or deserved acclaim within the biotechnology community, modified, and observations of how the system changes are application of synthetic biology methodologies to study recorded and synthesized to create a model of the system. biological systems has potential to fundamentally change Due to the complexity of biological systems, oftentimes it how biomedical research is conducted by providing is not possible to measure all effects simultaneously, thus researchers with improved experimental control. While this strategy can be riddled with potential side effects and the concepts behind synthetic biology are not new, we limitations (technical, metrological, and biological). Yet, present evidence supporting why the current research this approach is essential and the initial step on the path environment is conducive for integration of synthetic to mechanistic understanding as it is difficult to infer how biology approaches within biomedical research. In this a system works without first identifying the individual perspective we explore the idea of synthetic biology as components. An engineer takes the opposite approach; a discovery science research tool and provide examples using well-characterized individual components to build of both top-down and bottom-up approaches that have the system from scratch. This approach is structured on already been used to answer important physiology well-known design principles and iterative improvements questions at both the organismal and molecular level. that are implemented over time. In theory, as the design evolves, we develop a greater understanding of how the Keywords: engineering approaches; design principles; overall system works. One common theme that defines mechanisms; bottom-up discovery; top-down discovery the synthetic biology field is the application of this engineering mindset to biology. Biological systems have been redesigned by repackaging individual parts into novel combinations with unique or desirable properties, *Corresponding author: Santiago Schnell, Cellular and Molecular often with applied science implications. Scientists are Biology Graduate Program, University of Michigan Medical only now beginning to take action and use synthetic School, Ann Arbor, Michigan, USA; Department of Molecular & biology as a tool for research discovery [1–6]. Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan, USA; Department of Computational Medicine & Bioinformatics, University of Michigan Medical School, Ann Arbor, Michigan, USA, E-mail: [email protected] What is synthetic biology? Maxwell S. DeNies, Cellular and Molecular Biology Graduate Program, University of Michigan Medical School, Ann Arbor, Synthetic biology is a broad field that is difficult to Michigan, USA Allen P. Liu, Cellular and Molecular Biology Graduate Program, define. Functionally, it encompasses (1) the design and University of Michigan Medical School, Ann Arbor, Michigan, USA, construction of new biological entities such as enzymes, Department of Mechanical Engineering, University of Michigan, genetic circuits (i.e. genetic logic gates that mimic Ann Arbor, Michigan, USA, Department of Biomedical Engineering, electrical circuits) and cells, (2) the reconstruction of University of Michigan, Ann Arbor, Michigan, USA, Department of existing biological systems with the goal of understanding Biophysics, University of Michigan, Ann Arbor, Michigan, USA Open Access. © 2019 Maxwell S. DeNies et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution-NonCommercial NoDerivatives 4.0 License. 24 Maxwell S. DeNies et al: Are the biomedical sciences ready for synthetic biology? mechanism, and/or (3) the redesign of existing biological Examples of top-down synthetic systems. In the first point, synthetic biology is similar to synthetic organic chemistry ; its main goal is to biology harness biological processes and knowledge to create Many investigators within the physiology and biomedical new materials or capabilities not found in nature. Those sciences community have already begun to take adopting the second and third point view synthetic biology advantage of top-down synthetic biology tools to advance as the next generation of biological reconstitution as its their research. This is most evident in optogenetics. new approaches and capabilities allow researchers to ask Pioneered by Deisseroth, Boyden, and colleagues [20–22], deeper questions pertaining to molecular mechanisms, optogenetics is one example where top-down approaches the origins of life, and the design of a minimal cell have been implemented at both the organismal and capable of harnessing the basic principles of life. molecular levels to control and modify signaling activities Another view is that synthetic biology represents the in individual cells [23, 24] as well as to gain insight into promise of systems biology, providing the tools necessary specific neuronal circuitry and brain activity function in to interpret and interrogate the molecular insights rodents [23, 25] and zebrafish. Recently, Swift et al. derived from increasingly complex systems-level data. utilized channelrhodopsin to modulate locus coeruleus Regardless of definition, the tools and concepts afforded activity in rats and showed they can predictably control by synthetic biology have great potential to contribute to sleep spindle formation and consequently interfere with the biomedical sciences. REM (rapid eye movement) and NREM (non rapid eye movement) sleep. They found that these changes directly impacted rat memory formation and that by preventing Top-down synthetic biology sleep spindle formation, rats were no longer able to reliably recall learned tasks. These experiments would The field of synthetic biology can be divided into two not have been possible without the use of their synthetic broad domains based on research strategy - top-down and biology approach as the degree of accuracy and precision bottom-up [9, 10]. Top-down approaches aim to harness required to modulate a specific part of the brain is not biological phenomenon to engineer a predictable response feasible using other methods such as drug perturbation or to an input. Compared to traditional biomedical research genetic knockdown. approaches, this allows for improved experimental While the aforementioned optogenetic approaches control over how biological processes are measured have played a major role in linking neuron activity to and/or modulated. To accomplish this, researchers specific actions, prior knowledge of the underlying often redesign and modify well-characterized biological neuronal circuitry is necessary. To overcome this components for their advantage. This could be expressing limitation, multiple groups have devised strategies that optogenetically sensitive ion channel to modulate neuron utilize calcium flux (commonly observed upon neuron function, or designing a molecular sensor to monitor activation) and light activation to investigate activity- cell signaling events in real-time. Early examples of this dependent neuronal processes. An initial iteration of methodology include the development of a genetic toggle this was CaMPARI, an engineered photo-switchable switch and a genetic oscillator in bacteria [12, 13]. fluorescent protein that irreversibly switches from green More recently, researchers have focused on modifying to red light emission upon binding calcium (i.e. neuron organisms to have new capabilities by designing genetic activation) in the presence of light (Figure 1A). As circuits that integrate multiple signals via logic gates a proof-of-concept, researchers expressed CaMPARI and couple sensing with a biological readout such ubiquitously in the Drosophila brain. By applying light to as fluorescence or biological activity [14–17]. From a specific brain regions while exposing flies to a panel of biotechnology viewpoint, considerable advancements in odors, researchers were able to identify specific neurons chimeric antigen receptor T cells (CAR-T) and other smart involved with the odor sensory response by looking for immunotherapies that contain self-regulating control neurons that had switched from green to red fluorescence elements have been made possible by similar top-down (Figure 1B, C). synthetic biology approaches [18, 19]. One limitation of this approach is that it cannot be coupled to a downstream effector. Recently, researchers in the Ting group developed a similar tool that couples neuron activity to a transcriptional response. Similarly  Maxwell S. DeNies et al: Are the biomedical sciences ready for synthetic biology? 25 Figure 1: Recent top-down synthetic biology approaches to study biology. A. Schematic representation of CaMPARI technique. B. Proposed experimental design to use CaMPARI to study activity dependent neuronal circuitry in Drosophila. C. Cartoon of Drosophila brain illustrating potential result from CaMPARI study depicted in B. As is illustrated, the entire brain is fluorescently green however, upon activation of CaMPARI specific regions of the brain responsible for a response to a stimulus are switched to red fluorescence emission. D. Schematic of the FLARE technology. E. Proposed experimental strategy illustrating FLARE’s record and replay ability in neurons. Panels for this figure were adapted from Fosque et al 2015 (panels A-C) and Wang et al 2017 (panels D&E). to CaMPARI, FLARE (Fast Light- and Activity-Regulated indicative of an activated neuron (Figure 1E). This Expression) requires high calcium and light for activity. is a powerful proof-of-concept as FLARE allowed the However, unlike CaMPAIR where activation is solely researchers to not only identify activated neurons but also linked to a change in fluorescence, FLARE activation leads the ability to manipulate them afterwards. to a change in downstream transcription. Briefly, neuron In addition to optogenetic approaches, researchers activation results in a calcium influx, which leads to the recently developed a cellular memory system (MEMOIR) coupling of a TEV protease to the membrane tethered that records cellular history using a fluorescent readout LOV domain-protected transcription factor (Figure 1D).. One of the major limitations of lineage tracing At this time, steric hindrance prevents protease cleavage experiments is that spatial information is lost. To overcome and functional transcription factor release (Figure 1D). this issue, the researchers introduced a series of identical However, upon light activation, this is relieved and DNA sequences (i.e. coined scratchpads) that each had protease cleavage results in the release of a functional an associated unique barcode identification sequence. transcription factor that is able to activate transcription of Next they used the stochastic gene editing capabilities a desired target (Figure 1D). Compared to CaMPARI, of CRISPR-Cas9 to randomly and irreversibly mutate the FLARE allows researchers access to additional tools originally identical scratch pads to generate of record beyond fluorescent proteins to study downstream neuron of cellular history. As a readout, single molecule RNA activities. An interesting application of this is illustrated fluorescence in situ hybridization probes (smFISH) were in Figure 1E. In this example, researchers coupled FLARE used to simultaneously detect scratchpads and unique activity to the transcription of an Opsin (light sensitive barcodes in single cells allowing them to determine protein) and after 18hrs Opsin was activated using red light the lineage of individual cells within a mouse embryo to trigger an increase in calcium indicator fluorescence – stem cell population simultaneously via direct imaging 26 Maxwell S. DeNies et al: Are the biomedical sciences ready for synthetic biology?. Future applications of this approach could include approaches within the organoid space are nicely review modifying this system to record other cellular events and by Morsut and Davies. could be accomplished by coupling CRISPR-Cas9 activity At the molecular level, recent research has focused to biological sensor to measure a specific biological on developing liposomes (asymmetric or symmetric process. phospholipid double emulsion vesicles) with defined At the single cell physiology level, several groups input-output relationships [44, 45]. This is the initial have engineered sensors and other synthetic constructs in phase in the development of synthetic organelles or mammalian cells to detect changes in cell signaling [30– cells and represents a major first step in overcoming 33], as well as interrogate the role of protein localization the limitation of traditional bulk in vitro reconstitution in regulating these processes [34, 35]. One clever use of experiments. In contrast to traditional approaches, by synthetic biology to study cell signaling dynamics, pattern conducting experiments within liposomes, researchers formation, and differentiation was the development are able to better recapitulate the spatiotemporal of the synthetic Notch (SynNotch) circuit. Using regulation of a cell or organelle, which is necessary for the Notch receptor as a base (due to the simplicity and understanding more complicated biological mechanisms direct transcription response to ligand activation), the that have spatiotemporal elements. In addition to Lim lab replaced the extracellular ligand-binding motif democratizing liposome generation, microfluidic devices and intracellular transcription-regulating motif with have also been used to generate nested liposomes that user-defined protein domains that recognize “synthetic more closely resemble cellular structures. For more ligands”. The result was a chimeric receptor capable of information, a nice review of the properties of liposomes sensing an exogenous extracellular signal and translating and polymersomes as well as generation methods was it into a desirable transcriptional output. Due to the recently published by Ridear et al.. direct transcription response of these chimeric receptors, Improved reliability and utility of cell-free expression co-expressing different circuits is readily possible. systems (i.e. coupled transcription-translation reactions Applications of this approach have facilitated research (TXTL)) , are a second major advancement within into cell signaling circuitry, pattern formation and cell bottom-up synthetic biology that has great potential to differentiation in both cell culture and organoid models impact biomedical research. While cell-free lysates have [36–41]. been used for more than 50 years , recent advancements have made these approaches increasingly feasible. Cell-free expression systems are composed of a crude cell Bottom-up synthetic biology lysate without the nucleus or plasma membrane fractions. Lysates are then supplemented with genetic circuits, RNA polymerase as well as metabolic building blocks In contrast to top-down approaches, bottom-up synthetic (NTPs/Amino Acids) that allow for the simultaneous biology aims to assemble biological systems from scratch. transcription and translation of a target gene (Figure Unlike natural systems where it can be difficult to 2A). The inclusion of lipid membranes and organelles, predict experimental crosstalk and outcomes, bottom-up such as ER microsomes, in mammalian cell-free lysates approaches provide superior experimental control by have made the synthesis and functional reconstitution clearly defining experimental inputs and assumptions. of difficult-to-purify cell membrane proteins increasingly Organoids are one area of bottom-up synthetic biology possible (Figure 2A, B). Additionally, while in vitro in which groups of cells rather than proteins are utilized reconstitution has been used for years , the ability to as building blocks to reconstitute a synthetic tissue or encapsulate cell-free lysate with specific genetic circuits, organ. Introducing control elements via synthetic within liposomes or other artificial structures, have gene circuits within organoids allows researchers the made this technology increasingly useful as a model to ability to more closely mimic developmental processes study minimal cells or membrane bound organelle-like such as differentiation and pattern formation [3, 42, 43]. structures with spatiotemporal resolution. By comparing synthetic to natural processes, researchers are able to better understand the evolutionary origins and conserved mechanisms underlying these processes with greater control than methods that rely on systematically breaking the system to understand how components interact. Recent advancements using synthetic biology  Maxwell S. DeNies et al: Are the biomedical sciences ready for synthetic biology? 27 Figure 2: Bottom-up synthetic biology approaches to study biology. A. Cell-free expression system schematic. Cell lysate is collected from a desired cell type – bacterial or mammalian – and supplemented amino acids, energy mix (NTPs), cofactors, salts, RNA polymerase, and your desired cell-free expression system compatible plasmid. Incubation a specified temperature results in the production of the desired protein (encoded by the plasmid). Experiments can be conducted in bulk or encapsulated within double emulsion vesicles (i.e. liposomes) B. Experimental strategy for the investigation of the SUN1 and SUN2 topology on artificial nuclear membranes. Initially silica beads are incubated with SUVs to generate model artificial nuclear membranes (ANMs). Afterwards, ANMs are incubated with cell-free lysate containing plasmids encoding the SUN1 or SUN2 genes, which upon protein synthesis, leads to the functional reconstitution of the SUN proteins on ANMs. Panel B was adapted from Majumder et al 2018. Examples of bottom-up synthetic an external stimulus is translated to intra-vesicular biology information. For many applications, this is critical as the ability to transfer information between different compartments is foundational to many biological systems. Bottom-up synthetic biology approaches have been used Bottom-up synthetic biology has also been used to to create artificial biological functions in liposomes that recapitulate natural processes to better understand the mirror natural biological processes. By encapsulating cell- molecular mechanism of a particular process. Researchers free lysate within liposomes, researchers have been able to recently used a mammalian cell-free expression system generate artificial input and output relationships within a to study the nuclear membrane architecture of the LINC synthetic vesicle. Early advances in this space have used complex (Linker of Nucleoskeleton and Cytoskeleton) on mechanical sensing as a proof-of-principle application. artificial nuclear membranes (ANMs) not readily feasible In one example, cell-free lysate with a nested genetic using traditional cell biology techniques. WT and circuit was encapsulated within phospholipid liposomes. mutant SUN1 and SUN2 proteins (components of LINC The genetic circuit encoded a mechanosensitive channel complex) were individually and co-expressed using membrane protein (MscL) and G-GECO, a genetically mammalian cell-free lysate and allowed to integrate into encoded calcium sensor. When liposomes were exposed ANMs (Figure 2B). Utilizing a protease protection assay to hypo-osmotic shock, an influx of calcium through and comparing WT and mutant receptors, the authors activated MscL channels was reversibly detected by were able to reveal topology differences between SUN1 G-GECO fluorescence. Due to the membrane lipid and SUN2. To test the functionality of this artificial charge, diffusion of calcium into liposomes was solely model of the LINC complex, the authors reconstituted dependent on MscL expression and osmotic shock. KASH-binding SUN proteins for the first time. This study This example highlights two important proof-of-principle showcases the potential of using synthetic platforms to concepts that exemplify of the utility of bottom-up better understand molecular mechanisms and highlights synthetic biology. Firstly, it showcases that difficult-to- how recent advancements in synthetic biology are purify membrane proteins can be functionally produced responsible for growth in this area. using the cell-free expression system. Secondly, while this is a simple mechanism, it is an example of where 28 Maxwell S. DeNies et al: Are the biomedical sciences ready for synthetic biology? Cell-free expression systems have also been used either include or have been influenced by RNA sequencing for rapid prototyping. Recent work from the Noireaux data sets and the power of this technology is beginning to lab showcased the utility this technology to rapidly test be realized within the biomedical sciences. CRISPR-Cas efficacy in vitro before verifying results in There are many parallels between the research a more complex experimental system. To do so, environment that enabled the rise of high-throughput CRISPR-Cas machinery was expressed using the cell-free sequencing technologies and the current environment expression system with linear DNA targets and guide in the synthetic biology. Facilitated by the development RNAs. Using a fluorescent readout for CRISPR efficacy of software tools that aid in genetic circuits and sensors they were able to quickly screen guide RNAs for efficacy. design [55, 56], new molecular cloning strategies to This approach was further used to characterize the effects generate difficult-to-construct plasmids, and increased of CRISPR inhibitory proteins as well as to quickly identify utility of high-throughput screening technologies, have CRISPR PAM sequences without the need for transfection all enable researchers ability to test new ideas with optimization - a common nuisance when characterizing unprecedented speed [56, 58]. Decreasing costs of these new Cas proteins. Furthermore, by removing services via commercial suppliers have also played a an additional layer of variability when conducting role in this trend. Additionally, for bottom-up synthetic experiments in cells or an organism, this approach is able biology, reproducible and accessible cell-free expression to distinguish between CRISPR-Cas or application-specific system lysates are now widely accessible through both issues such as transfection efficacy or unintended side commercial suppliers (NEB PURExpress and Thermo effects. Fisher Expressway) as well as via standardized protocols Future applications of bottom-up synthetic biology that make producing a variety of cell lysates in-house may permit investigation of proteins that are lethal or feasible for every lab [48, 59–65]. These developments have to limit crosstalk between signaling cascades that are rapidly increased the user-friendliness of using synthetic difficult to untangle. Additionally, immunodepletion of biology methodologies for academic researchers. Given specific proteins or isolation of specific organelles from these recent advancements, it is surprising that synthetic cell-free lysates (pioneered with Xenopus egg extracts biology approaches are not more commonly used research [52, 53]) may provide researchers with the ability to more tools within the biomedical research community. easily study how a protein or process interacts within the larger system without long-term side effects such as transcriptional reprogramming possible with knockdown or genetic knockout experiments. Challenges facing synthetic biology approaches within physiology Are we ready to adopt synthetic There are several challenges that we still face when applying synthetic biology methodologies to new biology in basic science? Lessons systems. As is true with any engineering process or assay, from the rise of high-throughput standardization and component modularity remain areas of improvement. Central to this is the ability to measure sequencing technologies things accurately and reliably. Recognizing the importance of measurement accuracy and reproducibility to synthetic As noted by others [1–5, 54], the biotechnology applications biology, a new joint initiative between NIST, Stanford, and of synthetic biology only capture a subset of its scientific industry (JIMB - https://jimb.stanford.edu/) has recently utility and that the transition from being a niche field been established with the goal of improving metrology to widely used scientific approach is necessary for the within the biological sciences and initially focusing in realization of its full potential. A similar evolution occurred synthetic biology and genomics. with rise of high-throughput sequencing (genomics, RNA For top-down approaches, off-target effects as well as sequencing etc.). Although the human genome project was specificity of reagents or genetic circuits are particularly completed in the early 2000s, for many, high-throughput important to consider. The leakiness of expression and sequencing technologies remained out of reach because of switch-like behavior of sensors can have consequential limited access to technology, required expertise, and cost. implications on the accuracy of measurements. As these methods have become increasingly accessible Researchers developing these technologies have spent over the intervening years, many research hypotheses significant time investigating these areas to limit the effects  Maxwell S. DeNies et al: Are the biomedical sciences ready for synthetic biology? 29 of these challenges by including additional “insulators” to Fellowship Program and University of Michigan Rackham genetic circuits (commonly accounted for in sensor design Pre-Doctoral Fellowship. APL acknowledges support software tools) as well as modeling the compatibility of from National Science Foundation (1612917, 1844132, and multi-layer circuit designs [55, 58]. Recognizing these 1817909). Additionally, we would like to thank Mariana limitations, researchers have utilized irreversible methods Schnell for help with making the figure illustrations. events such as cleavage or DNA mutagenesis as readouts We would also like to thank Sagardip Majumder, to decrease measurement stochasticity. Matthias Truttmann, Carol Elias, and Suzanne Moenter Areas of improvement in bottom-up synthetic biology for constructive comments during the writing of the can be further sub-divided. 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