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Synthetic biology allows us to reuse, repurpose, and reconfigure biological systems to address society's most pressing challenges. Developing biotechnologies in this way requires integrating concepts across disciplines, posing challenges to educating students with diverse expertise. We created a framework for synthetic biology training that deconstructs biotechnologies across scales—molecular, circuit/network, cell/cell-free systems, biological communities, and societal—giving students a holistic toolkit to integrate cross-disciplinary concepts towards responsible innovation of successful biotechnologies. We present this framework, lessons learned, and inclusive teaching materials to allow its adaption to train the next generation of synthetic biologists. Developing biotechnologies to address society's challenges requires integrating concepts across disciplines, posing challenges to educating students with diverse expertise. In this Perspective the authors create a framework for synthetic biology training that deconstructs biotechnologies across spatial scales. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease (IBD) is a spectrum of autoimmune diseases affecting the gastrointestinal tract characterized by a relapsing and remitting course of gut mucosal inflammation. Disease flares can be difficult to predict, and the current practice of IBD disease activity surveillance through endoscopy is invasive and requires medical expertise. Recent advancements in synthetic biology raise the possibility that symbiotic microbes can be engineered to selectively detect disease biomarkers used in current clinical practice. Here, we introduce an engineered probiotic capable of detecting the clinical gold standard IBD biomarker, calprotectin, with sensitivity and specificity in IBD patients. Specifically, we identified a bacterial promoter in the probiotic strain Escherichia coli Nissle 1917 (EcN) which exhibits a specific expression increase in the presence of calprotectin. Using murine models of colitis, we show that the reporter signal is activated in vivo during transit of the GI tract following oral delivery. Furthermore, our engineered probiotic can successfully discriminate human patients with active IBD from those in remission and without IBD using patient stool samples, where the intensity of reporter signal quantitatively tracks with clinical laboratory-measured levels of calprotectin. Our pilot study sets the stage for probiotics that can be engineered to detect fecal calprotectin for precise noninvasive disease activity monitoring in IBD patients. [ABSTRACT FROM AUTHOR]

The widespread view of bacteria as strictly pathogenic has given way to an appreciation of the prevalence of some beneficial microbes within the human body (1-3). It is perhaps inevitable that some bacteria would evolve to preferentially grow in environments that harbour disease and thus provide a natural platform for the development of engineered therapies (4-6). Such therapies could benefit from bacteria that are programmed to limit bacterial growth while continually producing and releasing cytotoxic agents in situ (7-10). Here we engineer a clinically relevant bacterium to lyse synchronously at a threshold population density and to release genetically encoded cargo. Following quorum lysis, a small number of surviving bacteria reseed the growing population, thus leading to pulsatile delivery cycles. We used microfluidic devices to characterize the engineered lysis strain and we demonstrate its potential as a drug delivery platform via co-culture with human cancer cells in vitro. As a proof of principle, we tracked the bacterial population dynamics in ectopic syngeneic colorectal tumours in mice via a luminescent reporter. The lysis strain exhibits pulsatile population dynamics in vivo, with mean bacterial luminescence that remained two orders of magnitude lower than an unmodified strain. Finally, guided by previous findings that certain bacteria can enhance the efficacy of standard therapies (11), we orally administered the lysis strain alone or in combination with a clinical chemotherapeutic to a syngeneic mouse transplantation model of hepatic colorectal metastases. We found that the combination of both circuit-engineered bacteria and chemotherapy leads to a notable reduction of tumour activity along with a marked survival benefit over either therapy alone. Our approach establishes a methodology for leveraging the tools of synthetic biology to exploit the natural propensity for certain bacteria to colonize disease sites., In order to control population levels and facilitate drug delivery using bacteria, we engineered a synchronized lysis circuit (SLC) using coupled positive and negative feedback loops that have previously been [...]

The study of bacterial ion channels has provided fundamental insights into the structural basis of neuronal signalling; however, the native role of ion channels in bacteria has remained elusive. Here we show that ion channels conduct long-range electrical signals within bacterial biofilm communities through spatially propagating waves of potassium. These waves result from a positive feedback loop, in which a metabolic trigger induces release of intracellular potassium, which in turn depolarizes neighbouring cells. Propagating through the biofilm, this wave of depolarization coordinates metabolic states among cells in the interior and periphery of the biofilm. Deletion of the potassium channel abolishes this response. As predicted by a mathematical model, we further show that spatial propagation can be hindered by specific genetic perturbations to potassium channel gating. Together, these results demonstrate a function for ion channels in bacterial biofilms, and provide a prokaryotic paradigm for active, long-range electrical signalling in cellular communities., Communication through electrical signalling is prevalent among biological systems, with one of the most familiar examples being the action potential in neurons that is mediated by ion channels (1). For [...]

Cells that reside within a community can cooperate and also compete with each other for resources. It remains unclear how these opposing interactions are resolved at the population level. Here we investigate such an internal conflict within a microbial (Bacillus subtilis) biofilm community: cells in the biofilm periphery not only protect interior cells from external attack but also starve them through nutrient consumption. We discover that this conflict between protection and starvation is resolved through emergence of long-range metabolic co-dependence between peripheral and interior cells. As a result, biofilm growth halts periodically, increasing nutrient availability for the sheltered interior cells. We show that this collective oscillation in biofilm growth benefits the community in the event of a chemical attack. These findings indicate that oscillations support population-level conflict resolution by coordinating competing metabolic demands in space and time, suggesting new strategies to control biofilm growth., Cooperation and competition are complex social interactions that can have critical roles in biological communities. Cooperative behaviour often increases the overall fitness of the population through processes such as division [...]

Protease competition is used to produce rapid and tunable coupling of genetic circuits, enabling a coupled clock network that can encode independent environmental cues into a single time series output, a form of frequency multiplexing in a genetic circuit context. Engineered biocircuits that can stand the noise A major challenge in synthetic biology is that of integrating individual regulatory modules into larger circuits in a noisy and highly interconnected cellular environment. Jeff Hasty and colleagues tackle this problem by recruiting a host-cell protein-degradation system to synchronize genetic circuits and obtain delay times much shorter than those achieved by transcription-based coupling. They then apply this post-translational engineering scheme to synchronize cellular clocks and enable bacterial colonies to respond to independent environmental cues in 'frequency multiplexing'. One promise of synthetic biology is the creation of genetic circuitry that enables the execution of logical programming in living cells. Such 'wet programming' is positioned to transform a wide and diverse swathe of biotechnology ranging from therapeutics and diagnostics to water treatment strategies. Although progress in the development of a library of genetic modules continues apace.sup.1,2,3,4, a major challenge for their integration into larger circuits is the generation of sufficiently fast and precise communication between modules.sup.5,6. An attractive approach is to integrate engineered circuits with host processes that facilitate robust cellular signalling.sup.7. In this context, recent studies have demonstrated that bacterial protein degradation can trigger a precise response to stress by overloading a limited supply of intracellular proteases.sup.8,9,10. Here we use protease competition to engineer rapid and tunable coupling of genetic circuits across multiple spatial and temporal scales. We characterize coupling delay times that are more than an order of magnitude faster than standard transcription-factor-based coupling methods (less than 1 min compared with ~20-40 min) and demonstrate tunability through manipulation of the linker between the protein and its degradation tag. We use this mechanism as a platform to couple genetic clocks at the intracellular and colony level, then synchronize the multi-colony dynamics to reduce variability in both clocks. We show how the coupled clock network can be used to encode independent environmental inputs into a single time series output, thus enabling frequency multiplexing (information transmitted on a common channel by distinct frequencies) in a genetic circuit context. Our results establish a general framework for the rapid and tunable coupling of genetic circuits through the use of native 'queueing' processes such as competitive protein degradation., Author(s): Arthur Prindle [sup.1] , Jangir Selimkhanov [sup.1] , Howard Li [sup.1] , Ivan Razinkov [sup.1] , Lev S. Tsimring [sup.2] , Jeff Hasty [sup.1] [sup.2] [sup.3] Author Affiliations: (1) [...]

Although there has been considerable progress in the development of engineering principles for synthetic biology, a substantial challenge is the construction of robust circuits in a noisy cellular environment. Such an environment leads to considerable intercellular variability in circuit behaviour, which can hinder functionality at the colony level. Here we engineer the synchronization of thousands of oscillating colony 'biopixels' over centimetre-length scales through the use of synergistic intercellular coupling involving quorum sensing within a colony and gas-phase redox signalling between colonies. We use this platform to construct a liquid crystal display (LCD)-like macroscopic clock that can be used to sense arsenic via modulation of the oscillatory period. Given the repertoire of sensing capabilities of bacteria such as Escherichia coli, the ability to coordinate their behaviour over large length scales sets the stage for the construction of low cost genetic biosensors that are capable of detecting heavy metals and pathogens in the field., Synthetic biology can be broadly broken down into the 'top-down' synthesis of genomes (1) and the 'bottom-up' engineering of relatively small genetic circuits (2-10). In the field of genetic circuits, [...]

Cyclic di-guanosine monophosphate (c-di-GMP) is a bacterial second messenger that governs the lifestyle switch between planktonic and biofilm states. While substantial investigation has focused on the proteins that produce and degrade c-di-GMP, less attention has been paid to the potential for metabolic control of c-di-GMP signaling. Here, we show that micromolar levels of specific environmental purines unexpectedly decrease c-di-GMP and biofilm formation in Pseudomonas aeruginosa. Using a fluorescent genetic reporter, we show that adenosine and inosine decrease c-di-GMP even when competing purines are present. We confirm genetically that purine salvage is required for c-di-GMP decrease. Furthermore, we find that (p)ppGpp prevents xanthosine and guanosine from producing an opposing c-di-GMP increase, reinforcing a salvage hierarchy that favors c-di-GMP decrease even at the expense of growth. We propose that purines can act as a cue for bacteria to shift their lifestyle away from the recalcitrant biofilm state via upstream metabolic control of c-di-GMP signaling. [Display omitted] • Adenosine and inosine decrease c-di-GMP and biofilm formation • When (p)ppGpp is not present, xanthosine and guanosine increase c-di-GMP • Adenosine and inosine block the effects of xanthosine and guanosine • Hierarchy in purine salvage thus favors c-di-GMP decrease Kennelly et al. demonstrate that adenosine and inosine decrease c-di-GMP and biofilm formation of Pseudomonas aeruginosa. Without (p)ppGpp, xanthosine and guanosine increase c-di-GMP, but adenosine and inosine block this increase. This work reveals that environmental purines can act as a cue to shift bacteria away from the recalcitrant biofilm state. [ABSTRACT FROM AUTHOR]

The field of synthetic biology seeks to program living cells to perform novel functions with applications ranging from environmental biosensing to smart cell‐based therapeutics. Bacteria are an especially attractive chassis organism due to their rapid growth, ease of genetic manipulation, and ability to persist across many environmental niches. Despite significant progress in bacterial synthetic biology, programming bacteria to perform novel functions outside the well‐controlled laboratory context remains challenging. In contrast to planktonic laboratory growth, bacteria in nature predominately reside in the context of densely packed communities known as biofilms. While biofilms have historically been considered environmental and biomedical hazards, their physiology and emergent behaviors could be leveraged for synthetic biology to engineer more capable and robust bacteria. Specifically, bacteria within biofilms participate in complex emergent behaviors such as collective organization, cell‐to‐cell signaling, and division of labor. Understanding and utilizing these properties can enable the effective deployment of engineered bacteria into natural target environments. Toward this goal, this review summarizes the current state of synthetic biology in biofilms by highlighting new molecular tools and remaining biological challenges. Looking to future opportunities, advancing synthetic biology in biofilms will enable the next generation of smart cell‐based technologies for use in medicine, biomanufacturing, and environmental remediation. [ABSTRACT FROM AUTHOR]

The article focuses findings of a study on programmable probiotics for detection of cancer in urine. It states that the probiotic Escherichia coli was used to develop an orally administered diagnostic that can noninvasively indicate the presence of liver metastasis, and mentions that the diagnostic generated a high-contrast urine signal, and expression of a lacZ reporter. It infers that probiotics can be programmed to safely and selectively deliver synthetic gene circuits to diseased tissues.

The role of electricity in biological systems was first appreciated through electrical stimulation experiments performed by Luigi Galvani in the 18 th century. These pioneering experiments demonstrated that the behavior of living tissues is governed by the flow of electrochemical species—an insight that gave rise to the modern field of electrophysiology. Since then, electrophysiology has largely remained a bastion of neuroscience. However, exciting recent developments have demonstrated that even simple bacteria residing in communities use electrochemical communication to coordinate population-level behaviors. These recent works are defining the emerging field of bacterial biofilm electrophysiology. To view this SnapShot, open or download the PDF. [ABSTRACT FROM AUTHOR]

It is now well established in humans that there is a bidirectional pathway of communication between the central and enteric nervous systems in which members of the microbiome participate. This microbiota–gut–brain axis (MGBA) is crucial for normal development and physiology, and its dysregulation has been implicated in a range of neurological and intestinal disorders. Investigations into the mechanistic underpinnings of the MGBA have identified serotonin as a molecule of particular interest. In this review, we highlight recent advances toward understanding the role of endogenous serotonin in microbial communities, how microbial communities bidirectionally interact with host serotonin, and potential future engineering opportunities to leverage these novel mechanisms for biomedical applications. [Display omitted] • Serotonin can interact with bacterial proteins with a range of effects. • Serotonin is used in signaling between members of the microbiome and host. • Microbiome dysbiosis is associated with many diseases that feature altered serotonin. • Advances in probiotic engineering could be used to regulate host serotonin levels. [ABSTRACT FROM AUTHOR]

Summary Bacteria residing within biofilm communities can coordinate their behavior through cell-to-cell signaling. However, it remains unclear if these signals can also influence the behavior of distant cells that are not part of the community. Using a microfluidic approach, we find that potassium ion channel-mediated electrical signaling generated by a Bacillus subtilis biofilm can attract distant cells. Integration of experiments and mathematical modeling indicates that extracellular potassium emitted from the biofilm alters the membrane potential of distant cells, thereby directing their motility. This electrically mediated attraction appears to be a generic mechanism that enables cross-species interactions, as Pseudomonas aeruginosa cells also become attracted to the electrical signal released by the B. subtilis biofilm. Cells within a biofilm community can thus not only coordinate their own behavior but also influence the behavior of diverse bacteria at a distance through long-range electrical signaling. PaperClip [ABSTRACT FROM AUTHOR]