RESUMEN
The gut microbiome and its interactions with the host have been shown to affect several aspects of human health and disease. Investigations to elucidate these mechanisms typically involve sequence analysis of fecal samples. To support these studies, we present methods to design RNA toehold switch sensors to detect microbial and host transcripts. The sensors are embedded in paper-based, cell-free reactions that enable affordable and rapid analysis of microbiome samples.
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Microbioma Gastrointestinal , Bacterias/genética , Biomarcadores , Heces , Microbioma Gastrointestinal/genética , Humanos , ARN Ribosómico 16S/genéticaRESUMEN
Online video resources have increasingly become a common way to effectively share scientific research ideas and engage viewers at many levels of interest or expertise. While synthetic biology is a comparatively young field, it has accumulated online videos across a spectrum of content and technical depth. Such video content can be used to introduce viewers to synthetic biology, supplement college course content, teach new lab skills and entertain. Here, we compile online videos concerning synthetic biology into public YouTube playlists tailored for six different, though potentially overlapping, audiences: those wanting an introduction to synthetic biology, those wanting to get quick overviews of specific topics within synthetic biology, those wanting teaching or public lectures, those wanting more technical research lectures, those wanting to learn lab protocols and those interested in the International Genetically Engineered Machine competition.
RESUMEN
There is a need for large-scale, longitudinal studies to determine the mechanisms by which the gut microbiome and its interactions with the host affect human health and disease. Current methods for profiling the microbiome typically utilize next-generation sequencing applications that are expensive, slow, and complex. Here, we present a synthetic biology platform for affordable, on-demand, and simple analysis of microbiome samples using RNA toehold switch sensors in paper-based, cell-free reactions. We demonstrate species-specific detection of mRNAs from 10 different bacteria that affect human health and four clinically relevant host biomarkers. We develop a method to quantify mRNA using our toehold sensors and validate our platform on clinical stool samples by comparison to RT-qPCR. We further highlight the potential clinical utility of the platform by showing that it can be used to rapidly and inexpensively detect toxin mRNA in the diagnosis of Clostridium difficile infections.
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Biomarcadores/análisis , Microbioma Gastrointestinal , Papel , Biología Sintética/economía , Biología Sintética/métodos , Clostridioides difficile/genética , Clostridioides difficile/aislamiento & purificación , Biología Computacional , Heces/microbiología , Humanos , Inflamación/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Ribosómico 16S/genética , Especificidad de la EspecieRESUMEN
Hands-on demonstrations greatly enhance the teaching of science, technology, engineering, and mathematics (STEM) concepts and foster engagement and exploration in the sciences. While numerous chemistry and physics classroom demonstrations exist, few biology demonstrations are practical and accessible due to the challenges and concerns of growing living cells in classrooms. We introduce BioBits™ Explorer, a synthetic biology educational kit based on shelf-stable, freeze-dried, cell-free (FD-CF) reactions, which are activated by simply adding water. The FD-CF reactions engage the senses of sight, smell, and touch with outputs that produce fluorescence, fragrances, and hydrogels, respectively. We introduce components that can teach tunable protein expression, enzymatic reactions, biomaterial formation, and biosensors using RNA switches, some of which represent original FD-CF outputs that expand the toolbox of cell-free synthetic biology. The BioBits™ Explorer kit enables hands-on demonstrations of cutting-edge science that are inexpensive and easy to use, circumventing many current barriers for implementing exploratory biology experiments in classrooms.
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Técnicas Biosensibles/métodos , Fenómenos Fisiológicos Celulares , Enzimas/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Musa/química , Odorantes/análisis , Biología Sintética/educación , Hidrogel de Polietilenoglicol-Dimetacrilato/química , EnseñanzaRESUMEN
Synthetic gene expression is highly sensitive to intragenic compositional context (promoter structure, spacing regions between promoter and coding sequences, and ribosome binding sites). However, much less is known about the effects of intergenic compositional context (spatial arrangement and orientation of entire genes on DNA) on expression levels in synthetic gene networks. We compare expression of induced genes arranged in convergent, divergent, or tandem orientations. Induction of convergent genes yielded up to 400% higher expression, greater ultrasensitivity, and dynamic range than divergent- or tandem-oriented genes. Orientation affects gene expression whether one or both genes are induced. We postulate that transcriptional interference in divergent and tandem genes, mediated by supercoiling, can explain differences in expression and validate this hypothesis through modeling and in vitro supercoiling relaxation experiments. Treatment with gyrase abrogated intergenic context effects, bringing expression levels within 30% of each other. We rebuilt the toggle switch with convergent genes, taking advantage of supercoiling effects to improve threshold detection and switch stability.
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ADN Bacteriano/genética , ADN Superhelicoidal/genética , Redes Reguladoras de Genes , Genes Bacterianos , Genes Sintéticos , Fenómenos Biofísicos , Regulación Bacteriana de la Expresión Génica , Iniciación de la Cadena Peptídica Traduccional , Regiones Promotoras Genéticas/genética , Transcripción GenéticaRESUMEN
Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a "collateral effect" of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.
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Bacterias/aislamiento & purificación , Proteínas Bacterianas/química , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN Bacteriano/análisis , Virus del Dengue/aislamiento & purificación , Sistemas de Atención de Punto , ARN Viral/análisis , Ribonucleasas/química , Virus Zika/aislamiento & purificación , Bacterias/patogenicidad , ADN Tumoral Circulante/análisis , ADN Tumoral Circulante/genética , Dengue/diagnóstico , Virus del Dengue/genética , Humanos , Mutación , Neoplasias/genética , División del ARN , ARN Viral/genética , Virus Zika/genética , Infección por el Virus Zika/diagnósticoRESUMEN
The past several years have witnessed an increased presence of control theoretic concepts in synthetic biology. This review presents an organized summary of how these control design concepts have been applied to tackle a variety of problems faced when building synthetic biomolecular circuits in living cells. In particular, we describe success stories that demonstrate how simple or more elaborate control design methods can be used to make the behaviour of synthetic genetic circuits within a single cell or across a cell population more reliable, predictable and robust to perturbations. The description especially highlights technical challenges that uniquely arise from the need to implement control designs within a new hardware setting, along with implemented or proposed solutions. Some engineering solutions employing complex feedback control schemes are also described, which, however, still require a deeper theoretical analysis of stability, performance and robustness properties. Overall, this paper should help synthetic biologists become familiar with feedback control concepts as they can be used in their application area. At the same time, it should provide some domain knowledge to control theorists who wish to enter the rising and exciting field of synthetic biology.
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Modelos Teóricos , Biología Sintética/métodos , Humanos , Biología Sintética/tendenciasRESUMEN
Developing organisms can regulate and scale patterns to achieve consistent proportions despite immense changes in size. In this issue, Cao et al. build a model system, using engineered E. coli, to understand how regulatory dynamics can produce pattern scaling without the need for a morphogen gradient.