More than 3000 antimicrobial peptides (AMPs) have been discovered, seven of which have been approved by the U.S. Food and Drug Administration (FDA). Now commercialized, these seven peptides have ...mostly been utilized for topical medications, though some have been injected into the body to treat severe bacterial infections. To understand the translational potential for AMPs, we analyzed FDA-approved drugs in the FDA drug database. We examined their physicochemical properties, secondary structures, and mechanisms of action, and compared them with the peptides in the AMP database. All FDA-approved AMPs were discovered in Gram-positive soil bacteria, and 98% of known AMPs also come from natural sources (skin secretions of frogs and toxins from different species). However, AMPs can have undesirable properties as drugs, including instability and toxicity. Thus, the design and construction of effective AMPs require an understanding of the mechanisms of known peptides and their effects on the human body. This review provides an overview to guide the development of AMPs that can potentially be used as antimicrobial drugs.
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.
Cellular memory is crucial to many natural biological processes and sophisticated synthetic biology applications. Existing cellular memories rely on epigenetic switches or recombinases, which are ...limited in scalability and recording capacity. In this work, we use the DNA of living cell populations as genomic "tape recorders" for the analog and distributed recording of long-term event histories. We describe a platform for generating single-stranded DNA (ssDNA) in vivo in response to arbitrary transcriptional signals. When coexpressed with a recombinase, these intracellularly expressed ssDNAs target specific genomic DNA addresses, resulting in precise mutations that accumulate in cell populations as a function of the magnitude and duration of the inputs. This platform could enable long-term cellular recorders for environmental and biomedical applications, biological state machines, and enhanced genome engineering strategies. Record your memories with a DNA recorderDNA-based memory devices are not optimal for recording analog information, such as the magnitude of inputs over time. Farzadfard and Lu converted genomic DNA into a "tape recorder" within living bacterial populations (see the Perspective by Auslaender and Fussenegger). Specific DNAs were used to introduce precise mutations into genomic DNA. The stored information could be read out via reporter genes, functional assays, and DNA sequencing. This approach allowed the memorization of multiple inputs at the population level. The record could also be erased when required.Science, this issue 10.1126/science.1256272; see also p. 813
Engineered Living Hydrogels Liu, Xinyue; Inda, Maria Eugenia; Lai, Yong ...
Advanced materials,
July 1, 2022, Letnik:
34, Številka:
26
Journal Article
Recenzirano
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Living biological systems, ranging from single cells to whole organisms, can sense, process information, and actuate in response to changing environmental conditions. Inspired by living biological ...systems, engineered living cells and nonliving matrices are brought together, which gives rise to the technology of engineered living materials. By designing the functionalities of living cells and the structures of nonliving matrices, engineered living materials can be created to detect variability in the surrounding environment and to adjust their functions accordingly, thereby enabling applications in health monitoring, disease treatment, and environmental remediation. Hydrogels, a class of soft, wet, and biocompatible materials, have been widely used as matrices for engineered living cells, leading to the nascent field of engineered living hydrogels. Here, the interactions between hydrogel matrices and engineered living cells are described, focusing on how hydrogels influence cell behaviors and how cells affect hydrogel properties. The interactions between engineered living hydrogels and their environments, and how these interactions enable versatile applications, are also discussed. Finally, current challenges facing the field of engineered living hydrogels for their applications in clinical and environmental settings are highlighted.
The convergence of engineering, biology, and materials science is providing unprecedented opportunities to integrate living microbes into hydrogel matrices. This integration constructs engineered living hydrogels with the capability of performing tasks associated with living microbes such as self‐replication, self‐adaption, and environmental responsiveness.
RNA-based regulation and CRISPR/Cas transcription factors (CRISPR-TFs) have the potential to be integrated for the tunable modulation of gene networks. A major limitation of this methodology is that ...guide RNAs (gRNAs) for CRISPR-TFs can only be expressed from RNA polymerase III promoters in human cells, limiting their use for conditional gene regulation. We present new strategies that enable expression of functional gRNAs from RNA polymerase II promoters and multiplexed production of proteins and gRNAs from a single transcript in human cells. We use multiple RNA regulatory strategies, including RNA-triple-helix structures, introns, microRNAs, and ribozymes, with Cas9-based CRISPR-TFs and Cas6/Csy4-based RNA processing. Using these tools, we efficiently modulate endogenous promoters and implement tunable synthetic circuits, including multistage cascades and RNA-dependent networks that can be rewired with Csy4 to achieve complex behaviors. This toolkit can be used for programming scalable gene circuits and perturbing endogenous networks for biology, therapeutic, and synthetic biology applications.
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•Multiplexed CRISPR/Cas guide RNAs are expressed from single compact transcripts•Guide RNAs are generated from RNA polymerase II promoters with three strategies•Multistage cascades and gene circuits are created with CRISPR-TFs and microRNAs•Csy4-based RNA processing enables synchronized circuit rewiring at the RNA level
Nissim et al. describe three complementary strategies for the generation of efficient generation of functional gRNAs from RNA polymerase II promoters for CRISPR/Cas-based transcriptional regulation. Integrated with Cas6/Csy4-dependent RNA processing and mammalian RNA regulatory strategies, this toolkit can implement multiplexed gene regulation, multilayered transcriptional cascades, and RNA circuit rewiring.
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.
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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.
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.
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.
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► Zinc fingers can be used to wire orthogonal connections in yeast synthetic circuits ► Zinc finger TF design permits adjustable component properties for modulating outputs ► Protein-protein interactions can be used to engineer cooperativity in zinc finger TFs ► TF component properties can be combinatorially adjusted to reshape signal integration
A library of synthetic eukaryotic transcription factors constructed from modifiable component properties of artificial zinc fingers is capable of modulating outputs and reshaping signal integration.
A central goal of synthetic biology is to achieve multi-signal integration and processing in living cells for diagnostic, therapeutic and biotechnology applications. Digital logic has been used to ...build small-scale circuits, but other frameworks may be needed for efficient computation in the resource-limited environments of cells. Here we demonstrate that synthetic analog gene circuits can be engineered to execute sophisticated computational functions in living cells using just three transcription factors. Such synthetic analog gene circuits exploit feedback to implement logarithmically linear sensing, addition, ratiometric and power-law computations. The circuits exhibit Weber's law behaviour as in natural biological systems, operate over a wide dynamic range of up to four orders of magnitude and can be designed to have tunable transfer functions. Our circuits can be composed to implement higher-order functions that are well described by both intricate biochemical models and simple mathematical functions. By exploiting analog building-block functions that are already naturally present in cells, this approach efficiently implements arithmetic operations and complex functions in the logarithmic domain. Such circuits may lead to new applications for synthetic biology and biotechnology that require complex computations with limited parts, need wide-dynamic-range biosensing or would benefit from the fine control of gene expression.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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