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The ability to detect disease early and deliver precision therapy would be transformative for the treatment of human illnesses. To achieve these goals, biosensors that can pinpoint when and where diseases emerge are needed. Rapid advances in synthetic biology are enabling us to exploit the information-processing abilities of living cells to diagnose disease and then treat it in a controlled fashion. For example, living sensors could be designed to precisely sense disease biomarkers, such as by-products of inflammation, and to respond by delivering targeted therapeutics in situ. Here, we provide an overview of ongoing efforts in microbial biosensor design, highlight translational opportunities, and discuss challenges for enabling sense-and-respond precision medicines. [ABSTRACT FROM AUTHOR]

• Applications of CRISPR have been extended by engineering or discovering novel Cas proteins to cleave RNA and perform DNA/RNA base substitutions. • Multiple orthogonal ligand-mediated or light-mediated regulation of CRISPR/Cas will form the basis of biocomputing for synthetic biology. • Irreversible and heritable changes in DNA sequence created by CRISPR/Cas are being used for lineage tracing and forming biological memory. • CRISPR screens are integrated with single cell RNA sequencing and lineage tracing for robust and diverse readout. Since its first demonstration for mammalian gene editing, CRISPR/Cas technology has been widely adopted in research, industry, and medicine. Beyond indel mutations induced by Cas9 activity, recent advances in CRISPR/Cas have enabled DNA or RNA base editing. In addition, multiple orthogonal methods for the spatiotemporal regulation of CRISPR/Cas activity and repurposed Cas proteins for the visualization and relocation of specific genomic loci in living cells have been described. By harnessing the versatility of CRISPR/Cas-based devices and gene circuits, synthetic biologists are developing memory devices for lineage tracing and technologies for unbiased, high-throughput interrogation of combinatorial gene perturbations. We envision that such approaches will enable researchers to gain deeper insights into the translation of genotypes to phenotypes in healthy and diseased states. [ABSTRACT FROM AUTHOR]

Natural life is encoded by evolvable, DNA-based memory. Recent advances in dynamic genome-engineering technologies, which we collectively refer to as in vivo DNA writing, have opened new avenues for investigating and engineering biology. This Review surveys these technological advances, outlines their prospects and emerging applications, and discusses the features and current limitations of these technologies for building various genetic circuits for processing and recording information in living cells. [ABSTRACT FROM AUTHOR]

As the blueprint of life, the natural exploits of DNA are admirable. However, DNA should not only be viewed within a biological context. It is an elegantly simple yet functionally complex chemical polymer with properties that make it an ideal platform for engineering new nanotechnologies. Rapidly advancing synthesis and sequencing technologies are enabling novel unnatural applications for DNA beyond the realm of genetics. Here we explore the chemical biology of DNA nanotechnology for emerging applications in communication and digital data storage. Early studies of DNA as an alternative to magnetic and optical storage mediums have not only been promising, but have demonstrated the potential of DNA to revolutionize the way we interact with digital data in the future. [ABSTRACT FROM AUTHOR]

Biological computation is a major area of focus in synthetic biology because it has the potential to enable a wide range of applications. Synthetic biologists have applied engineering concepts to biological systems in order to construct progressively more complex gene circuits capable of processing information in living cells. Here, we review the current state of computational genetic circuits and describe artificial gene circuits that perform digital and analog computation. We then discuss recent progress in designing gene networks that exhibit memory, and how memory and computation have been integrated to yield more complex systems that can both process and record information. Finally, we suggest new directions for engineering biological circuits capable of computation. [ABSTRACT FROM AUTHOR]

Many natural biological systems--such as biofilms, shells and skeletal tissues--are able to assemble multifunctional and environmentally responsive multiscale assemblies of living and non-living components. Here, by using inducible genetic circuits and cellular communication circuits to regulate Escherichia coli curli amyloid production, we show that E. coli cells can organize self-assembling amyloid fibrils across multiple length scales, producing amyloid-based materials that are either externally controllable or undergo autonomous patterning. We also interfaced curli fibrils with inorganic materials, such as gold nanoparticles (AuNPs) and quantum dots (QDs), and used these capabilities to create an environmentally responsive biofilm-based electrical switch, produce gold nanowires and nanorods, co-localize AuNPs with CdTe/CdS QDs to modulate QD fluorescence lifetimes, and nucleate the formation of fluorescent ZnS QDs. This work lays a foundation for synthesizing, patterning, and controlling functional composite materials with engineered cells. [ABSTRACT FROM AUTHOR]

Summary: Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks. [Copyright &y& Elsevier]

Over the past decade, synthetic biology has emerged as an engineering discipline for biological systems. Compared with other substrates, biology poses a unique set of engineering challenges resulting from an incomplete understanding of natural biological systems and tools for manipulating them. To address these challenges, synthetic biology is advancing from developing proof-of-concept designs to focusing on core platforms for rational and high-throughput biological engineering. These platforms span the entire biological design cycle, including DNA construction, parts libraries, computational design tools, and interfaces for manipulating and probing synthetic circuits. The development of these enabling technologies requires an engineering mindset to be applied to biology, with an emphasis on generalizable techniques in addition to application-specific designs. This review aims to discuss the progress and challenges in synthetic biology and to illustrate areas where synthetic biology may impact biomedical engineering and human health. [ABSTRACT FROM AUTHOR]

Bacteriophage therapy for bacterial infections is a concept with an extensive but controversial history. There has been a recent resurgence of interest into bacteriophages owing to the increasing incidence of antibiotic resistance and virulent bacterial pathogens. Despite these efforts, bacteriophage therapy remains an underutilized option in Western medicine due to challenges such as regulation, limited host range, bacterial resistance to phages, manufacturing, side effects of bacterial lysis, and delivery. Recent advances in biotechnology, bacterial diagnostics, macromolecule delivery, and synthetic biology may help to overcome these technical hurdles. These research efforts must be coupled with practical and rigorous approaches at academic, commercial, and regulatory levels in order to successfully advance bacteriophage therapy into clinical settings. [ABSTRACT FROM AUTHOR]

Synthetic biology is focused on the rational construction of biological systems based on engineering principles. During the field's first decade of development, significant progress has been made in designing biological parts and assembling them into genetic circuits to achieve basic functionalities. These circuits have been used to construct proof-of-principle systems with promising results in industrial and medical applications. However, advances in synthetic biology have been limited by a lack of interoperable parts, techniques for dynamically probing biological systems and frameworks for the reliable construction and operation of complex, higher-order networks. As these challenges are addressed, synthetic biologists will be able to construct useful next-generation synthetic gene networks with real-world applications in medicine, biotechnology, bioremediation and bioenergy. [ABSTRACT FROM AUTHOR]

Synthetic gene networks can be constructed to emulate digital circuits and devices, giving one the ability to program and design cells with some of the principles of modern computing, such as counting. A cellular counter would enable complex synthetic programming and a variety of biotechnology applications. Here, we report two complementary synthetic genetic counters in Escherichia coli that can count up to three induction events: the first, a riboregulated transcriptional cascade, and the second, a recombinase-based cascade of memory units. These modular devices permit counting of varied user-defined inputs over a range of frequencies and can be expanded to count higher numbers. [ABSTRACT FROM AUTHOR]

Antimicrobial drug development is increasingly lagging behind the evolution of antibiotic resistance, and as a result, there is a pressing need for new antibacterial therapies that can be readily designed and implemented. In this work, we engineered bacteriophage to overexpress proteins and attack gene networks that are not directly targeted by antibiotics. We show that suppressing the SOS network in Escherichia coli with engineered bacteriophage enhances killing by quinolones by several orders of magnitude in vitro and significantly increases survival of infected mice in vivo. In addition, we demonstrate that engineered bacteriophage can enhance the killing of antibiotic-resistant bacteria, persister cells, and biofilm cells, reduce the number of antibiotic-resistant bacteria that arise from an antibiotic-treated population, and act as a strong adjuvant for other bactericidal antibiotics (e.g., aminoglycosides and β-lactams). Furthermore, we show that engineering bacteriophage to target non-SOS gene networks and to overexpress multiple factors also can produce effective antibiotic adjuvants. This work establishes a synthetic biology platform for the rapid translation and integration of identified targets into effective antibiotic adjuvants. [ABSTRACT FROM AUTHOR]

Synthetic biology involves the engineering of biological organisms by using modular and generalizable designs with the ultimate goal of developing useful solutions to real-world problems. One such problem involves bacterial biofilms, which are crucial in the pathogenesis of many clinically important infections and are difficult to eradicate because they exhibit resistance to antimicrobial treatments and removal by host immune systems. To address this issue, we engineered bacteriophage to express a biofilm-degrading enzyme during infection to simultaneously attack the bacterial cells in the biofilm and the biofilm matrix, which is composed of extracellular polymeric substances. We show that the efficacy of biofilm removal by this two-pronged enzymatic bacteriophage strategy is significantly greater than that of nonenzymatic bacteriophage treatment. Our engineered enzymatic phage substantially reduced bacterial biofilm cell counts by ≈4.5 orders of magnitude (≈99.997% removal), which was about two orders of magnitude better than that of nonenzymatic phage. This work demonstrates the feasibility and benefits of using engineered enzymatic bacteriophage to reduce bacterial biofilms and the applicability of synthetic biology to an important medical and industrial problem. [ABSTRACT FROM AUTHOR]

Abstract: In mammalian cochleas, outer hair cells (OHCs) produce mechanical amplification over the entire audio-frequency range (up to 100kHz). Under the ‘somatic electromotility’ theory, mechano-electrical transduction modulates the OHC transmembrane potential, driving an OHC mechanical response which generates cycle-by-cycle mechanical amplification. Yet, though the OHC motor responds up to at least 70kHz, the OHC membrane RC time constant (in vitro upper limit ∼1000Hz) reduces the potential driving the motor at high frequencies. Thus, the mechanism for high-frequency amplification with slow OHCs has been a two-decade-long mystery. Previous models fit to experimental data incorporated slow OHCs but did not explain how the OHC time constant limitation is overcome. Our key contribution is showing that negative feedback due to organ-of-Corti functional anatomy with adequate OHC gain significantly extends closed-loop system bandwidth and increases resonant gain. The OHC gain-bandwidth product, not just bandwidth, determines if high-frequency amplification is possible. Due to the cochlea’s collective traveling-wave architecture, a single OHC’s gain need not be great. OHC piezoelectricity increases the effectiveness of negative-feedback but is not essential for amplification. Thus, emergent closed-loop network dynamics differ significantly from open-loop component dynamics, a generally important principle in complex biological systems. [Copyright &y& Elsevier]

Synthetic biology has the potential to transform cell‐ and gene‐based therapies for a variety of diseases. Sophisticated tools are now available for both eukaryotic and prokaryotic cells to engineer cells to selectively achieve therapeutic effects in response to one or more disease‐related signals, thus sparing healthy tissue from potentially cytotoxic effects. This report summarizes the Keystone eSymposium "Synthetic Biology: At the Crossroads of Genetic Engineering and Human Therapeutics," which took place on May 3 and 4, 2021. Given that several therapies engineered using synthetic biology have entered clinical trials, there was a clear need for a synthetic biology symposium that emphasizes the therapeutic applications of synthetic biology as opposed to the technical aspects. Presenters discussed the use of synthetic biology to improve T cell, gene, and viral therapies, to engineer probiotics, and to expand upon existing modalities and functions of cell‐based therapies. [ABSTRACT FROM AUTHOR]

Successful implementation of adoptive cell therapy (ACT) of cancer requires comprehensively addressing biological and practical challenges. This approach has been largely overlooked, resulting in a gap between the potential of ACT and its actual effectiveness. We summarize the most promising technical strategies in creating an "ideal" ACT product, focusing on chimeric antigen receptor (CAR)-engineered cells. Since many requirements for effective ACT are common to most cancers, what we outline here might have a broader impact. [ABSTRACT FROM AUTHOR]

The gut microbiome has a significant impact on health and disease and can actively contribute to obesity, diabetes, inflammatory bowel disease, cardiovascular disease, and neurological disorders. We do not yet have the necessary tools to fine-tune the microbial communities that constitute the microbiome, though such tools could unlock extensive benefits to human health. Here, we provide an overview of the current state of technological tools that may be used for microbiome engineering. These tools can enable investigators to define the parameters of a healthy microbiome and to determine how gut bacteria may contribute to the etiology of a variety of diseases. These tools may also allow us to explore the exciting prospect of developing targeted therapies and personalized treatments for microbiome-linked diseases. The microbiome plays a fundamental role in our health and disease. Engineering the microbiome might enable studying the contribution of individual microbes and generating potential therapies against metabolic (e.g., phenylketonuria and chronic kidney disease), inflammatory, and immunological diseases, among others. Current methods for probing the microbiome include fecal microbiota transplantation and the use of antibiotics, probiotics, and prebiotics; these can produce broad alterations in the microbiome and its reciprocal interactions with the immune system. Novel tools are needed to precisely reprogram microbial communities in order to improve health and disease outcomes (among other applications). [ABSTRACT FROM AUTHOR]

The increased incidence of bacterial resistance to available antibiotics represents a major global health problem and highlights the need for novel anti-infective therapies. Antimicrobial peptides (AMPs) represent promising alternatives to conventional antibiotics. AMPs are versatile, have almost unlimited sequence space, and can be tuned for broad-spectrum or specific activity against microorganisms. However, several obstacles remain to be overcome in order to develop AMPs for medical use, such as toxicity, stability, and bacterial resistance. We lack standard experimental procedures for quantifying AMP activity and do not yet have a clear picture of the mechanisms of action of AMPs. The rational design of AMPs can help solve these issues and enable their use as new antimicrobials. Here we provide an overview of the main physicochemical features that can be engineered to achieve enhanced bioactivity and describe current strategies being used to design AMPs. Unlabelled Image • Urgency for new antimicrobial agents to treat antibiotic-resistant microorganism-caused infections • Efficient design tools are needed for rational design of promising antibiotic candidates. • Physicochemical features are the basis for the most relevant activity descriptors of antimicrobial peptides. • Structure–activity relationships studies enable a wide range of commercial applications for antimicrobial peptides. [ABSTRACT FROM AUTHOR]

Synthetic biology aims to introduce new sense-and-respond capabilities into living cells, which would enable novel therapeutic strategies. The development of regulatory elements, molecular computing devices, and effector screening technologies has enabled researchers to design synthetic gene circuits in many organisms, including mammalian cells. Engineered gene networks, such as closed-loop circuits or Boolean logic gate circuits, can be used to program cells to perform specific functions with spatiotemporal control and restoration of homeostasis in response to the extracellular environment and intracellular signaling. In addition, genetically modified microbes can be designed as local delivery of therapeutic molecules. In this review, we will discuss recent advances in therapeutic applications of synthetic gene circuits, as well as challenges and future opportunities for biomedicine. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) have evolved through billions of years as part of our innate immune system. These agents are produced by various cells throughout the human body and play important roles in our ability to respond to infections. In this review, we outline evidence linking AMP expression with a range of inflammatory and autoimmune human diseases. Finally, we highlight the promise of endogenous AMP induction for the treatment of disease (i.e., host-directed therapy) and briefly mention the different peptide drugs that are currently undergoing clinical trials. [ABSTRACT FROM AUTHOR]

An excerpt is presented of an article published online at dx.doi.org/10.1126/science.1256272 regarding the use of genomic DNA as an artificial data storage medium via Synthetic Cellular Recorders Integrating Biological Events (SCRIBE) architecture using single-stranded DNA (ssDNA).

The gut microbiome is essential for processing complex food compounds and synthesizing nutrients that the host cannot digest or produce, respectively. New model systems are needed to study how the metabolic capacity provided by the gut microbiome impacts the nutritional status of the host, and to explore possibilities for altering host metabolic capacity via the microbiome. Here, we colonized the nematode Caenorhabditis elegans gut with cellulolytic bacteria that enabled C. elegans to utilize cellulose, an otherwise indigestible substrate, as a carbon source. Cellulolytic bacteria as a community component in the worm gut can also support additional bacterial species with specialized roles, which we demonstrate by using Lactobacillus plantarum to protect C. elegans against Salmonella enterica infection. This work shows that engineered microbiome communities can be used to endow host organisms with novel functions, such as the ability to utilize alternate nutrient sources or to better fight pathogenic bacteria. Synopsis: A microbe‐host interaction model is developed by colonizing C. elegans with functional bacteria that allow digesting long‐chain cellulose. Direct benefits include increased host larval yield and protection of other gut species against pathogens.Heterologous bacteria (e.g. Pseudomonas cellulosa) in the gut can help C. elegans to digest cellulose, an otherwise indigestible carbon substrate.Cellulolytic bacteria can also support other bacterial species with specialized roles: Lactobacillus protected the worms against Salmonella infection, and interspecies synergy between P. cellulosa and L. plantarum conferred benefit to the host.Engineered microbiome communities may provide host organisms with novel functions, including the ability to use complex nutrient sources and to fight pathogens.C. elegans colonized with bacteria provides a model system for studying microbiome‐host interactions. [ABSTRACT FROM AUTHOR]

High-order interactions among components of interconnected genetic networks regulate complex functions in biological systems, but deciphering these interactions is challenging. New strategies are emerging to decode these combinatorial genetic interactions across a wide range of organisms. Here, we review advances in multiplexed and combinatorial genetic perturbation technologies and high-throughput profiling platforms that are enabling the systematic dissection of complex genetic networks. These rapidly evolving technologies are being harnessed to probe combinatorial gene functions in functional genomics studies and have the potential to advance our understanding of how genetic networks regulate sophisticated biological phenotypes, to generate novel therapeutic strategies, and to enable the engineering of complex artificial gene networks. [ABSTRACT FROM AUTHOR]

The microbial community that lives on and in the human body exerts a major impact on human health, from metabolism to immunity. In order to leverage the close associations between microbes and their host, development of therapeutics targeting the microbiota has surged in recent years. Here, we discuss current additive and subtractive strategies to manipulate the microbiota, focusing on bacteria engineered to produce therapeutic payloads, consortia of natural organisms and selective antimicrobials. Further, we present challenges faced by the community in the development of microbiome therapeutics, including designing microbial therapies that are adapted for specific geographies in the body, stable colonization with microbial therapies, discovery of clinically relevant biosensors, robustness of engineered synthetic gene circuits and addressing safety and biocontainment concerns. Moving forward, collaboration between basic and applied researchers and clinicians to address these challenges will poise the field to herald an age of next-generation, cellular therapies that draw on novel findings in basic research to inform directed augmentation of the human microbiota. [ABSTRACT FROM AUTHOR]

The ability to record molecular events in vivo would enable monitoring of signaling dynamics within cellular niches and critical factors that orchestrate cellular behavior. We present a self-contained analog memory device for longitudinal recording of molecular stimuli into DNA mutations in human cells. This device consists of a self-targeting guide RNA (stgRNA) that repeatedly directs Streptococcus pyogenes Cas9 nuclease activity toward the DNA that encodes the stgRNA, enabling localized, continuous DNA mutagenesis as a function of stgRNA expression. We demonstrate programmable and multiplexed memory storage in human cells triggered by exogenous inducers or inflammation, both in vitro and in vivo. This tool, Mammalian Synthetic Cellular Recorder Integrating Biological Events (mSCRIBE), provides a distinct strategy for investigating cell biology in vivo and enables continuous evolution of targeted DNA sequences. [ABSTRACT FROM AUTHOR]

The microbiome contributes to the development and maturation of the immune system. In response to commensal bacteria, intestinal CD4+ T lymphocytes differentiate into functional subtypes with regulatory or effector functions. The development of small intestine intraepithelial lymphocytes that coexpress CD4 and CD8aa homodimers (CD4IELs) depends on the microbiota. However, the identity of the microbial antigens recognized by CD4+ T cells that can differentiate into CD4IELs remains unknown. We identified b-hexosaminidase, a conserved enzyme across commensals of the Bacteroidetes phylum, as a driver of CD4IEL differentiation. In a mouse model of colitis, b-hexosaminidase-specific lymphocytes protected against intestinal inflammation. Thus, T cells of a single specificity can recognize a variety of abundant commensals and elicit a regulatory immune response at the intestinal mucosa. [ABSTRACT FROM AUTHOR]

The role of the microbiome in health and disease is attracting the attention of researchers seeking to engineer microorganisms for diagnostic and therapeutic applications. Recent progress in synthetic biology may enable the dissection of host–microbiota interactions. Sophisticated genetic circuits that can sense, compute, memorize, and respond to signals have been developed for the stable commensal bacterium Bacteroides thetaiotaomicron , dominant in the human gut. In this review, we highlight recent advances in expanding the genetic toolkit for B. thetaiotaomicron and foresee several applications of this species for microbiome engineering. We provide our perspective on the challenges and future opportunities for the engineering of human gut-associated bacteria as living therapeutic agents. [Display omitted] [ABSTRACT FROM AUTHOR]

Synthetic DNA has great propensity for efficiently and stably storing non-biological information. With DNA writing and reading technologies rapidly advancing, new applications for synthetic DNA are emerging in data storage and communication. Traditionally, DNA communication has focused on the encoding and transfer of complete sets of information. Here, we explore the use of DNA for the communication of short messages that are fragmented across multiple distinct DNA molecules. We identified three pivotal points in a communication—data encoding, data transfer & data extraction—and developed novel tools to enable communication via molecules of DNA. To address data encoding, we designed DNA-based individualized keyboards (iKeys) to convert plaintext into DNA, while reducing the occurrence of DNA homopolymers to improve synthesis and sequencing processes. To address data transfer, we implemented a secret-sharing system—Multiplexed Sequence Encoding (MuSE)—that conceals messages between multiple distinct DNA molecules, requiring a combination key to reveal messages. To address data extraction, we achieved the first instance of chromatogram patterning through multiplexed sequencing, thereby enabling a new method for data extraction. We envision these approaches will enable more widespread communication of information via DNA. [ABSTRACT FROM AUTHOR]

Genetic circuits, composed of complex networks of interacting molecular machines, enable living systems to sense their dynamic environments, perform computation on the inputs, and formulate appropriate outputs. By rewiring and expanding these circuits with novel parts and modules, synthetic biologists have adapted living systems into vibrant substrates for engineering. Diverse paradigms have emerged for designing, modeling, constructing, and characterizing such artificial genetic systems. In this paper, we first provide an overview of recent advances in the development of genetic parts and highlight key engineering approaches. We then review the assembly of these parts into synthetic circuits from the perspectives of digital and analog logic, systems biology, and metabolic engineering, three areas of particular theoretical and practical interest. Finally, we discuss notable challenges that the field of synthetic biology still faces in achieving reliable and predictable forward-engineering of artificial biological circuits. [ABSTRACT FROM AUTHOR]

Current antibiotics tend to be broad spectrum, leading to indiscriminate killing of commensal bacteria and accelerated evolution of drug resistance. Here, we use CRISPR-Cas technology to create antimicrobials whose spectrum of activity is chosen by design. RNA-guided nucleases (RGNs) targeting specific DNA sequences are delivered efficiently to microbial populations using bacteriophage or bacteria carrying plasmids transmissible by conjugation. The DNA targets of RGNs can be undesirable genes or polymorphisms, including antibiotic resistance and virulence determinants in carbapenem-resistant Enterobacteriaceae and enterohemorrhagic Escherichia coli. Delivery of RGNs significantly improves survival in a Galleria mellonella infection model. We also show that RGNs enable modulation of complex bacterial populations by selective knockdown of targeted strains based on genetic signatures. RGNs constitute a class of highly discriminatory, customizable antimicrobials that enact selective pressure at the DNA level to reduce the prevalence of undesired genes, minimize off-target effects and enable programmable remodeling of microbiota. [ABSTRACT FROM AUTHOR]

New therapeutic strategies are needed to treat infections caused by drug-resistant bacteria, which constitute a major growing threat to human health. Here, we use a high-throughput technology to identify combinatorial genetic perturbations that can enhance the killing of drug-resistant bacteria with antibiotic treatment. This strategy. Combinatorial Genetics En Masse (CombiGEM), enables the rapid generation of high-order barcoded combinations of genetic elements for high-throughput multiplexed characterization based on next-generation sequencing. We created ~34,000 pairwise combinations of Escherichia coli transcription factor (TF) overexpression constructs. Using lllumina sequencing, we identified diverse perturbations in antibiotic-resistance phenotypes against carbapenem-resistant Enterobacteriaceae. Specifically, we found multiple TF combinations that potentiated antibiotic killing by up to 106-fold and delivered these combinations via phagemids to increase the killing of highly drug-resistant E. coli harboring New Delhi metallo-beta-lactamase-1. Moreover, we constructed libraries of three-wise combinations of transcription factors with >4 million unique members and demonstrated that these could be tracked via next-generation sequencing. We envision that CombiGEM could be extended to other model organisms, disease models, and phenotypes, where it could accelerate massively parallel combinatorial genetics studies for a broad range of biomedical and biotechnology applications, including the treatment of antibiotic-resistant infections. [ABSTRACT FROM AUTHOR]

Since their discovery, bacteriophages have contributed enormously to our understanding of molecular biology as model systems. Furthermore, bacteriophages have provided many tools that have advanced the fields of genetic engineering and synthetic biology. Here, we discuss bacteriophage-based technologies and their application to the study of infectious diseases. New strategies for engineering genomes have the potential to accelerate the design of novel phages as therapies, diagnostics, and tools. Though almost a century has elapsed since their discovery, bacteriophages continue to have a major impact on modern biological sciences, especially with the growth of multidrug-resistant bacteria and interest in the microbiome. [ABSTRACT FROM AUTHOR]

Logic and memory are essential functions of circuits that generate complex, state-dependent responses. Here we describe a strategy for efficiently assembling synthetic genetic circuits that use recombinases to implement Boolean logic functions with stable DNA-encoded memory of events. Application of this strategy allowed us to create all 16 two-input Boolean logic functions in living Escherichia coli cells without requiring cascades comprising multiple logic gates. We demonstrate long-term maintenance of memory for at least 90 cell generations and the ability to interrogate the states of these synthetic devices with fluorescent reporters and PCR. Using this approach we created two-bit digital-to-analog converters, which should be useful in biotechnology applications for encoding multiple stable gene expression outputs using transient inputs of inducers. We envision that this integrated logic and memory system will enable the implementation of complex cellular state machines, behaviors and pathways for therapeutic, diagnostic and basic science applications. [ABSTRACT FROM AUTHOR]

Natural microbial sensing circuits can be rewired into new gene networks to build living sensors that detect and respond to disease‐associated biomolecules. However, synthetic living sensors, once ingested, are cleared from the gastrointestinal (GI) tract within 48 h; retaining devices in the intestinal lumen is prone to intestinal blockage or device migration. To localize synthetic microbes and safely extend their residence in the GI tract for health monitoring and sustained drug release, an ingestible magnetic hydrogel carrier is developed to transport diagnostic microbes to specific intestinal sites. The magnetic living hydrogel is localized and retained by attaching a magnet to the abdominal skin, resisting the peristaltic waves in the intestine. The device retention is validated in a human intestinal phantom and an in vivo rodent model, showing that the ingestible hydrogel maintains the integrated living bacteria for up to seven days, which allows the detection of heme for GI bleeding in the harsh environment of the gut. The retention of microelectronics is also demonstrated by incorporating a temperature sensor into the magnetic hydrogel carrier. [ABSTRACT FROM AUTHOR]

Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods. We use the targeted reversetranscription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. RLR simultaneously introduces many genomic variants, producing pooled and barcoded variant libraries addressable by targeted deep sequencing. We use RLR for pooled phenotyping of synthesized antibiotic resistance alleles, demonstrating quantitative measurement of relative growth rates. We also perform RLR using the sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for causal variants, demonstrating that RLR is uniquely suited to utilize large pools of natural variation. Using ssDNA produced in vivo for pooled experiments presents avenues for exploring variation across the genome. [ABSTRACT FROM AUTHOR]

[Display omitted] • Synthetic biology enables efficient phage genome engineering. • Phages with tunable host range and antimicrobial payload delivery enhance efficacy. • Phage design principles may be guided by computational approaches in the future. The antimicrobial and therapeutic efficacy of bacteriophages is currently limited, mostly due to rapid emergence of phage-resistance and the inability of most phage isolates to bind and infect a broad range of clinical strains. Here, we discuss how phage therapy can be improved through recent advances in genetic engineering. First, we outline how receptor-binding proteins and their relevant structural domains are engineered to redirect phage specificity and to avoid resistance. Next, we summarize how phages are reprogrammed as prokaryotic gene therapy vectors that deliver antimicrobial 'payload' proteins, such as sequence-specific nucleases, to target defined cells within complex microbiomes. Finally, we delineate big data- and novel artificial intelligence-driven approaches that may guide the design of improved synthetic phage in the future. [ABSTRACT FROM AUTHOR]

Background: Iron is essential for bacterial survival. Bacterial siderophores are small molecules with unmatched capacity to scavenge iron from proteins and the extracellular milieu, where it mostly occurs as insoluble Fe3+. Siderophores chelate Fe3+ for uptake into the cell, where it is reduced to soluble Fe2+. Siderophores are key molecules in low soluble iron conditions. The ability of bacteria to synthesize proprietary siderophores may have increased bacterial evolutionary fitness; one way that bacteria diversify siderophore structure is by incorporating different polyamine backbones while maintaining the catechol moieties. Results: We report that Serratia plymuthica V4 produces a variety of siderophores, which we term the siderome, and which are assembled by the concerted action of enzymes encoded in two independent gene clusters. Besides assembling serratiochelin A and B with diaminopropane, S. plymuthica utilizes putrescine and the same set of enzymes to assemble photobactin, a siderophore found in the bacterium Photorhabdus luminescens. The enzymes encoded by one of the gene clusters can independently assemble enterobactin. A third, independent operon is responsible for biosynthesis of the hydroxamate siderophore aerobactin, initially described in Enterobacter aerogenes. Mutant strains not synthesizing polyamine-siderophores significantly increased enterobactin production levels, though lack of enterobactin did not impact the production of serratiochelins. Knocking out SchF0, an enzyme involved in the assembly of enterobactin alone, significantly reduced bacterial fitness. Conclusions: This study shows the natural occurrence of serratiochelins, photobactin, enterobactin, and aerobactin in a single bacterial species and illuminates the interplay between siderophore biosynthetic pathways and polyamine production, indicating routes of molecular diversification. Given its natural yields of diaminopropane (97.75 μmol/g DW) and putrescine (30.83 μmol/g DW), S. plymuthica can be exploited for the industrial production of these compounds. [ABSTRACT FROM AUTHOR]

In the era where antibiotic resistance is considered one of the major worldwide concerns, bacteriophages have emerged as a promising therapeutic approach to deal with this problem. Genetically engineered bacteriophages can enable enhanced anti-bacterial functionalities, but require cloning additional genes into the phage genomes, which might be challenging due to the DNA encapsulation capacity of a phage. To tackle this issue, we designed and assembled for the first time synthetic phages with smaller genomes by knocking out up to 48% of the genes encoding hypothetical proteins from the genome of the newly isolated Pseudomonas aeruginosa phage vB_PaeP_PE3. The antibacterial efficacy of the wild-type and the synthetic phages was assessed in vitro as well as in vivo using a Galleria mellonella infection model. Overall, both in vitro and in vivo studies revealed that the knock-outs made in phage genome do not impair the antibacterial properties of the synthetic phages, indicating that this could be a good strategy to clear space from phage genomes in order to enable the introduction of other genes of interest that can potentiate the future treatment of P. aeruginosa infections. [ABSTRACT FROM AUTHOR]

Novel antibiotics are urgently needed to combat multidrug-resistant pathogens. Venoms represent previously untapped sources of novel drugs. Here we repurposed mastoparan-L, the toxic active principle derived from the venom of the wasp Vespula lewisii, into synthetic antimicrobials. We engineered within its N terminus a motif conserved among natural peptides with potent immunomodulatory and antimicrobial activities. The resulting peptide, mast-MO, adopted an α-helical structure as determined by NMR, exhibited increased antibacterial properties comparable to standard-of-care antibiotics both in vitro and in vivo, and potentiated the activity of different classes of antibiotics. Mechanism-of-action studies revealed that mast-MO targets bacteria by rapidly permeabilizing their outer membrane. In animal models, the peptide displayed direct antimicrobial activity, led to enhanced ability to attract leukocytes to the infection site, and was able to control inflammation. Permutation studies depleted the remaining toxicity of mast-MO toward human cells, yielding derivatives with antiinfective activity in animals. We demonstrate a rational design strategy for repurposing venoms into promising antimicrobials. [ABSTRACT FROM AUTHOR]

The powerful biological adhesion systems of barnacles, mussels, and other marine animals have led to bio-inspired adhesives with impressive performance characteristics. However, lacking the capacities that only cells possess (e.g., self-regeneration, environmental responsiveness, etc.), these artificial materials cannot exploit the full potential of the natural adhesive systems that inspired them. Here, we developed "functional cellular glues" made of Bacillus subtilis biofilms—tightly associated bacteria encased in an extracellular matrix—that contain both an engineered amyloid protein functionalized with a mussel foot protein and an engineered hydrophobin-like protein. We demonstrate proof-of-concept for both tunable adhesion performance via inducible enzymatic modification and for improved adhesion through metal ion-assisted curing. By conceptualizing biofilms themselves—rather than individual material components—as adhesives, we have developed an evolvable engineering platform that opens the door to make smart living glues with dynamic, self-healing, and other previously unattainable material properties. [ABSTRACT FROM AUTHOR]

Infections caused by Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa foremost among them, constitute a major worldwide health problem. Bioinformatics methodologies are being used to rationally design new antimicrobial peptides, a potential alternative for treating these infections. One of the algorithms used to develop antimicrobial peptides is the Joker, which was used to design the peptide PaDBS1R6. This study evaluates the antibacterial activities of PaDBS1R6 in vitro and in vivo , characterizes the peptide interaction to target membranes, and investigates the PaDBS1R6 structure in contact with mimetic vesicles. Moreover, we demonstrate that PaDBS1R6 exhibits selective antimicrobial activity against Gram-negative bacteria. In the presence of negatively charged and zwitterionic lipids the structural arrangement of PaDBS1R6 transits from random coil to α-helix, as characterized by circular dichroism. The tertiary structure of PaDBS1R6 was determined by NMR in zwitterionic dodecylphosphocholine (DPC) micelles. In conclusion, PaDBS1R6 is a candidate for the treatment of nosocomial infections caused by Gram-negative bacteria, as template for producing other antimicrobial agents. Unlabelled Image • PaDBS1R6 presented in-helix conformation when in contact with anionic lipid membranes • Antibacterial selectivity of PaDBS1R6 against Gram-negative bacteria • PaDBS1R6 was active in vivo in murine intraperitoneal and skin infection models. [ABSTRACT FROM AUTHOR]

The article provides an overview of seventeen articles on topics related to biotransformation and bioactivation reactions published in 2018 which includes the article on drug optimization "Structure-function-guided Exploration of the Antimicrobial Peptide Polybia-CP Identifies Activity Determinants and Generates Synthetic Therapeutic Candidate," by Marcelo D.T. Torres, Cibele N. Pedron, Yasutomi Higashikuni, Robin M. Kramer, Marlon H. Cardoso, Karen G.N. Oshiro, Octavio L. Franco, Pedro I. Silva Junior, Fernanda D. Silva, Vani X. Oliveira Junior, Timothy K. Lu,and Cesar de la Fuente-Nunez, which appeared in the 2017 1:221 issue of "Community Biology;" an article on metabolites and drug metabolizing enzymes "Hydrolytic Metabolism of Cyanopyrrolidine DPP-4 Inhibitors Mediated by Dipeptidyl Peptidases," by Kong F, Pang X, Zhao J, Deng P, Zheng M,Zhong D, Chen X in "Drug Metabolism and Disposition;" and "N-Methylation of BI 187004 by thiol S-methyltransferase," by Hlaing H. Maw, Xingzhong Zeng, Scot Campbell, Mitchell E. Taub and Aaron M. Teitelbaum," from the volume 48 , 2018 issue of "Drug Metabolism Disposition."

Abstract Venezuelan equine encephalitis virus (VEEV), a new world alphavirus belonging to the Togaviridae family, causes periodic disease outbreaks in humans and equines with high associated mortality and morbidity. VEEV is highly infectious via the aerosol route and so has been developed as a biological weapon (Hawley and Eitzen, 2001). Despite its current classification as a category B select agent, there are no FDA approved vaccines or therapeutics to counter VEEV infections. Here we utilize a naturally occurring host defense peptide, LL-37, as a therapeutic strategy to inhibit VEEV multiplication in infected cells. LL-37 has previously demonstrated activity against several viruses by directly interacting with viral particles and indirectly by establishing an antiviral state in the host cell. We show that LL-37 exhibited potent antiviral activity against VEEV by inhibiting viral replication. Genomic RNA copies of the TC-83 strain of VEEV and viral titers were significantly reduced compared to non-treated controls. LL-37 also inhibited the virulent Trinidad Donkey (TrD) strain of VEEV. Entry assays revealed a robust reduction of viral RNA copies at the early stages of TC-83 infection. Pre-incubation of cells with LL-37 and TC-83 resulted in a strong inhibitory response, indicating that LL-37 impacts early stages of the infectious process. Confocal and electron microscopy images confirmed the aggregation of viral particles, which potentially accounts for entry prevention and hence reduced viral infection. LL-37 treatment also modulated type I interferon (IFN) expression in infected cells. LL-37 treatment dramatically increased IFNβ1 expression in treated cells in a time-dependent manner. Our results establish LL-37 as a relevant and novel potential therapeutic strategy for the treatment of VEEV infections. Highlights • Host defense peptide LL-37 is presented as an approach to treating Venezuelan equine encephalitis virus (VEEV) infection. • LL-37 exhibits robust antiviral activity with minimal toxicity. • LL-37 is found to inhibit virus entry. • LL-37 modulates type I interferon (IFN) expression in infected host cells, thereby eliciting an antiviral state. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) constitute promising alternatives to classical antibiotics for the treatment of drug-resistant infections, which are a rapidly emerging global health challenge. However, our understanding of the structure-function relationships of AMPs is limited, and we are just beginning to rationally engineer peptides in order to develop them as therapeutics. Here, we leverage a physicochemical-guided peptide design strategy to identify specific functional hotspots in the wasp-derived AMP polybia-CP and turn this toxic peptide into a viable antimicrobial. Helical fraction, hydrophobicity, and hydrophobic moment are identified as key structural and physicochemical determinants of antimicrobial activity, utilized in combination with rational engineering to generate synthetic AMPs with therapeutic activity in a mouse model. We demonstrate that, by tuning these physicochemical parameters, it is possible to design nontoxic synthetic peptides with enhanced sub-micromolar antimicrobial potency in vitro and anti-infective activity in vivo. We present a physicochemical-guided rational design strategy to generate peptide antibiotics. Marcelo D. T. Torres et al. turn toxic wasp-derived antimicrobial peptide polybia-CP into a viable antimicrobial with therapeutic activity in a mouse model. This study demonstrates that a physicochemical property-guided rational design strategy can be used to generate peptide antibiotics. [ABSTRACT FROM AUTHOR]

Abstract Many living organisms make use of diverse amyloid proteins as functional building blocks to fulfill a variety of physiological applications. This fact, along with the intrinsic self-assembly and outstanding material properties of amyloids, has prompted a significant amount of research in the synthetic design of functional amyloids to form diverse nanoarchitectures, molecular materials, and hybrid or composite materials. In particular, a new research paradigm has recently been advanced that uses synthetic biology to harness functional amyloids with cells as living materials or functional devices. Here we outline important progress in the synthetic design of functional amyloids, in the context of both non-living and living systems. We propose several important tools and underline emerging techniques and principles that might be useful in advancing the future synthetic design of functional amyloids. Graphical abstract Unlabelled Image Highlights • The identification of functional amyloids in organisms, along with intrinsic self-assembly and outstanding material properties of amyloids, prompted new research in the synthetic design of functional amyloids. • Functional amyloids are harvested to create multidimensional architectures or multifunctional molecular materials. • Functional living systems engineered based on amyloids represent a promising field in the design of functional synthetic amyloids. • The cutting-edge synthetic biology and increasing discoveries of functional amyloids will advance future synthetic design of functional amyloids. [ABSTRACT FROM AUTHOR]

At the edge of a confluent cell layer, cell-free empty space is a cue that can drive directed collective cellular migration. Similarly, contact guidance is also a robust mechanical cue that can drive cell migration. However, it is unclear which of the two effects is stronger, and how each mechanism affects collective migration. To address this question, here we explore the trajectories of cells migrating collectively on a substrate containing micropatterned grooves (10-20 μm in periodicity, 2 μm in height) compared with unpatterned control substrates. Compared with unpatterned controls, the micropatterned substrates attenuated path variance by close to 70% and augmented migration coordination by more than 30%. Together, these results show that contact guidance can play an appreciable role in collective cellular migration. Also, our result can provide insights into tissue repair and regeneration with the remodeling of the connective tissue matrix. [ABSTRACT FROM AUTHOR]

Biomolecular monitoring in the gastrointestinal tract could offer rapid, precise disease detection and management but is impeded by access to the remote and complex environment. Here, we present an ingestible micro-bio-electronic device (IMBED) for in situ biomolecular detection based on environmentally resilient biosensor bacteria and miniaturized luminescence readout electronics that wirelessly communicate with an external device. As a proof of concept, we engineer heme-sensitive probiotic biosensors and demonstrate accurate diagnosis of gastrointestinal bleeding in swine. Additionally, we integrate alternative biosensors to demonstrate modularity and extensibility of the detection platform. IMBEDs enable new opportunities for gastrointestinal biomarker discovery and could transform the management and diagnosis of gastrointestinal disease. [ABSTRACT FROM AUTHOR]