Download or read book Droplet based Microfluidics for the Genotype phenotype Mapping of Model Enzymes written by Dany Chauvin and published by . This book was released on 2017 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The question of how sequence encodes proteins' function is essential to understand molecular evolution but still remains elusive.Droplet-based microfluidics allows to use micro-metric droplets as reaction vessels to separately assay enzyme variants at the kHz frequency. It also provides an elegant solution to couple the genotype with the product of the catalytic activity of enzymes. Sorting droplets on demand and sequencing their content enables to map the genotype of millions of enzyme variants to their phenotype in a single experiment.First, I developed a cell-free microfluidic work-flow to carry out genotype-phenotype mapping of Streptomyces griseus aminopeptidase (SGAP). Single enzyme variant genes are encapsulated and amplified in droplets, expressed, and assayed against a fluorogenic substrate. Incompatibilities between gene amplification, expression and assay reactions, constrain to execute each one of those steps successively and to dilute the product of each reaction by droplet electro-coalescence. I show that a work-flow in which (i) genes are encapsulated and amplified into 0.2 pL droplets, (ii) expressed using cell-free expression reagents in 2 pL droplets and (iii) assayed with a fluorogenic substrate in 20 pL droplets, allows to measure SGAP variants activity with high contrast. To optimize the SGAP droplet assay, I also developed in collaboration with Dr. Johan Fenneteau (Laboratory of Organic Chemistry, ESPCI Paristech), a hydrophilic rhodamine based substrate, characterized by limited exchange of the released fluorophore between droplets.Second, I developed an in vivo microfluidic work-flow on Ratus norvegicus trypsin (rat trypsin), in which Bacillus subtilis secretion abilities are used to simplify the microfluidic work-flow. Single B. subtilis cells are encapsulated in 20 pL droplets where they secrete trypsin variants as fusion proteins with a fluorescent expression-level reporter. The variants are assayed by droplet electro-coalescence with 2 pL droplets containing a trypsin fluorogenic substrate. Trypsin variants catalytic efficiency can be directly measured in droplets, by normalizing the total trypsin activity by the expression-level reporter fluorescence. This is the first time a high-throughput protein assay work-flow gives the opportunity to directly measure the catalytic efficiency of enzyme variants at the kHz frequency. A method to carry out saturated mutagenesis on the rat trypsin gene was also developed. Together with deep sequencing, the developed experimental work-flow will allow to perform the first quantitative genotype-phenotype mapping of all single point mutants of the rat trypsin protein.
Download or read book Directed Enzyme Evolution written by Frances H. Arnold and published by Springer Science & Business Media. This book was released on 2008-02-02 with total page 381 pages. Available in PDF, EPUB and Kindle. Book excerpt: Directed evolution comprises two distinct steps that are typically applied in an iterative fashion: (1) generating molecular diversity and (2) finding among the ensemble of mutant sequences those proteins that perform the desired fu- tion according to the specified criteria. In many ways, the second step is the most challenging. No matter how cleverly designed or diverse the starting library, without an effective screening strategy the ability to isolate useful clones is severely diminished. The best screens are (1) high throughput, to increase the likelihood that useful clones will be found; (2) sufficiently sen- tive (i. e. , good signal to noise) to allow the isolation of lower activity clones early in evolution; (3) sufficiently reproducible to allow one to find small improvements; (4) robust, which means that the signal afforded by active clones is not dependent on difficult-to-control environmental variables; and, most importantly, (5) sensitive to the desired function. Regarding this last point, almost anyone who has attempted a directed evolution experiment has learned firsthand the truth of the dictum “you get what you screen for. ” The protocols in Directed Enzyme Evolution describe a series of detailed p- cedures of proven utility for directed evolution purposes. The volume begins with several selection strategies for enzyme evolution and continues with assay methods that can be used to screen enzyme libraries. Genetic selections offer the advantage that functional proteins can be isolated from very large libraries s- ply by growing a population of cells under selective conditions.
Download or read book Genotype Phenotype Coupling written by Stefan Zielonka and published by Springer Nature. This book was released on 2023-07-05 with total page 410 pages. Available in PDF, EPUB and Kindle. Book excerpt: This detailed new edition broadens the scope of the first edition by moving beyond classical display technologies. This book explores methodologies for the generation of natively paired antibody libraries, single cell technologies, alternative scaffolds, and in silico antibody sequence assessments are described. The application of these methods may allow for a generation of improved therapeutics and diagnostic reagents in a shorter time frame. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Genotype Phenotype Coupling: Methods and Protocols, Second Edition serves as an ideal guide for researchers seeking to expand their knowledge of antibody-based therapeutics.
Download or read book Enzyme Engineering and Evolution General Methods written by and published by Academic Press. This book was released on 2020-09-05 with total page 362 pages. Available in PDF, EPUB and Kindle. Book excerpt: This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. - Provides the authority and expertise of leading contributors from an international board of authors - Presents the latest release in the Methods in Enzymology series
Download or read book Molecular Epidemiology written by Paul A. Schulte and published by Academic Press. This book was released on 2012-12-02 with total page 609 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book will serve as a primer for both laboratory and field scientists who are shaping the emerging field of molecular epidemiology. Molecular epidemiology utilizes the same paradigm as traditional epidemiology but uses biological markers to identify exposure, disease or susceptibility. Schulte and Perera present the epidemiologic methods pertinent to biological markers. The book is also designed to enumerate the considerations necessary for valid field research and provide a resource on the salient and subtle features of biological indicators.
Download or read book Hydrolytic Reactions written by Marcel Florkin and published by Elsevier. This book was released on 2014-06-28 with total page 280 pages. Available in PDF, EPUB and Kindle. Book excerpt: Comprehensive Biochemisty, Volume 16: Hydrolytic Reactions; Cobamide and Biotin Coenzymes treats both the chemistry and function of the coenzymes. This book discusses the thermodynamic and kinetic aspects of enzyme catalysis, hydrolytic enzymes displaying "active center characteristics, chelation and stereochemical considerations in enzyme catalysis, and biological oxidation mechanisms. The recommendations of the Enzyme Commission of the International Union of Biochemistry and classified list of enzymes are also covered. This publication likewise elaborates the spectral characteristics of the cobamide coenzymes and mode of binding of biotin. Other topics include the classification of carboxyl esterases, structure and reactivity of phosphorylated metabolites, and mechanism of action of chymotrypsin. This volume is a good reference for biochemists and specialists concerned with enzymes.
Download or read book Functional Metagenomics Tools and Applications written by Trevor C. Charles and published by Springer. This book was released on 2017-10-09 with total page 256 pages. Available in PDF, EPUB and Kindle. Book excerpt: In this book, the latest tools available for functional metagenomics research are described.This research enables scientists to directly access the genomes from diverse microbial genomes at one time and study these “metagenomes”. Using the modern tools of genome sequencing and cloning, researchers have now been able to harness this astounding metagenomic diversity to understand and exploit the diverse functions of microorganisms. Leading scientists from around the world demonstrate how these approaches have been applied in many different settings, including aquatic and terrestrial habitats, microbiomes, and many more environments. This is a highly informative and carefully presented book, providing microbiologists with a summary of the latest functional metagenomics literature on all specific habitats.
Download or read book Industrial Biocatalysis written by Peter Grunwald and published by CRC Press. This book was released on 2014-12-11 with total page 1201 pages. Available in PDF, EPUB and Kindle. Book excerpt: Biocatalysis has become an essential tool in the chemical industry and is the core of industrial biotechnology, also known as white biotechnology, making use of biocatalysts in terms of enzymes or whole cells in chemical processes as an alternative to chemical catalysts. This shift can be seen in the many areas of daily life where biocatalysts-with
Download or read book The Pangenome written by Hervé Tettelin and published by Springer Nature. This book was released on 2020-04-30 with total page 311 pages. Available in PDF, EPUB and Kindle. Book excerpt: This open access book offers the first comprehensive account of the pan-genome concept and its manifold implications. The realization that the genetic repertoire of a biological species always encompasses more than the genome of each individual is one of the earliest examples of big data in biology that opened biology to the unbounded. The study of genetic variation observed within a species challenges existing views and has profound consequences for our understanding of the fundamental mechanisms underpinning bacterial biology and evolution. The underlying rationale extends well beyond the initial prokaryotic focus to all kingdoms of life and evolves into similar concepts for metagenomes, phenomes and epigenomes. The book’s respective chapters address a range of topics, from the serendipitous emergence of the pan-genome concept and its impacts on the fields of microbiology, vaccinology and antimicrobial resistance, to the study of microbial communities, bioinformatic applications and mathematical models that tie in with complex systems and economic theory. Given its scope, the book will appeal to a broad readership interested in population dynamics, evolutionary biology and genomics.
Download or read book Microfluidics and Nanofluidics written by Mohsen Sheikholeslami Kandelousi and published by BoD – Books on Demand. This book was released on 2018-08-22 with total page 320 pages. Available in PDF, EPUB and Kindle. Book excerpt: In the present book, various applications of microfluidics and nanofluidics are introduced. Microfluidics and nanofluidics span a broad array of disciplines including mechanical, materials, and electrical engineering, surface science, chemistry, physics and biology. Also, this book deals with transport and interactions of colloidal particles and biomolecules in microchannels, which have great importance to many microfluidic applications, such as drug delivery in life science, microchannel heat exchangers in electronic cooling, and food processing industry. Furthermore, this book focuses on a detailed description of the thermal transport behavior, challenges and implications that involve the development and use of HTFs under the influence of atomistic-scale structures and industrial applications.
Download or read book Advanced Healthcare Materials written by Ashutosh Tiwari and published by John Wiley & Sons. This book was released on 2014-05-09 with total page 421 pages. Available in PDF, EPUB and Kindle. Book excerpt: Offers a comprehensive and interdisciplinary view of cutting-edge research on advanced materials for healthcare technology and applications Advanced healthcare materials are attracting strong interest in fundamental as well as applied medical science and technology. This book summarizes the current state of knowledge in the field of advanced materials for functional therapeutics, point-of-care diagnostics, translational materials, and up-and-coming bioengineering devices. Advanced Healthcare Materials highlights the key features that enable the design of stimuli-responsive smart nanoparticles, novel biomaterials, and nano/micro devices for either diagnosis or therapy, or both, called theranostics. It also presents the latest advancements in healthcare materials and medical technology. The senior researchers from global knowledge centers have written topics including: State-of-the-art of biomaterials for human health Micro- and nanoparticles and their application in biosensors The role of immunoassays Stimuli-responsive smart nanoparticles Diagnosis and treatment of cancer Advanced materials for biomedical application and drug delivery Nanoparticles for diagnosis and/or treatment of Alzheimers disease Hierarchical modelling of elastic behavior of human dental tissue Biodegradable porous hydrogels Hydrogels in tissue engineering, drug delivery, and wound care Modified natural zeolites Supramolecular hydrogels based on cyclodextrin poly(pseudo)rotaxane Polyhydroxyalkanoate-based biomaterials Biomimetic molecularly imprinted polymers
Download or read book The Perfect Slime written by Hans-Curt Flemming and published by IWA Publishing. This book was released on 2016-09-15 with total page 336 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Perfect Slime presents the latest state of knowledge and all aspects of the Extracellular Polymeric Substances, (EPS) matrix – from the ecological and health to the antifouling perspectives. The book brings together all the current material in order to expand our understanding of the functions, properties and characteristics of the matrix as well as the possibilities to strengthen or weaken it. The EPS matrix represents the immediate environment in which biofilm organisms live. From their point of view, this matrix has paramount advantages. It allows them to stay together for extended periods and form synergistic microconsortia, it retains extracellular enzymes and turns the matrix into an external digestion system and it is a universal recycling yard, it protects them against desiccation, it allows for intense communication and represents a huge genetic archive. They can remodel their matrix, break free and eventually, they can use it as a nutrient source. The EPS matrix can be considered as one of the emergent properties of biofilms and are a major reason for the success of this form of life. Nevertheless, they have been termed the “black matter of biofilms” for good reasons. First of all: the isolation methods define the results. In most cases, only water soluble EPS components are investigated; insoluble ones such as cellulose or amyloids are much less included. In particular in environmental biofilms with many species, it is difficult to impossible isolate, separate the various EPS molecules they are encased in and to define which species produced which EPS. The regulation and the factors which trigger or inhibit EPS production are still very poorly understood. Furthermore: bacteria are not the only microorganisms to produce EPS. Archaea, Fungi and algae can also form EPS. This book investigates the questions, What is their composition, function, dynamics and regulation? What do they all have in common?
Download or read book Synthetic Metabolic Pathways written by Michael Krogh Jensen and published by Humana Press. This book was released on 2017-11-24 with total page 354 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume outlines key steps associated with the design, building, and testing of synthetic metabolic pathways for optimal cell factory performance and robustness, and illustrates how data-driven learning from these steps can be used for rational cost-effective engineering of cell factories with improved performance. Chapters are divided into four sections focusing on the four steps of the iterative design-build-test-learn cycle related to modern cell factory engineering. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Synthetic Metabolic Pathways: Methods and Protocols aims to ensure successful results in the further study of this vital field.
Download or read book The Adaptive Seascape written by David J. Merrell and published by U of Minnesota Press. This book was released on 1994 with total page 284 pages. Available in PDF, EPUB and Kindle. Book excerpt: Based on observational and experimental data, in natural populations of plants and animals studied in the field and in the laboratory, this perspective unravels the hidden and often poorly founded assumptions underlying some of the more troublesome controversies in evolutionary biology today
Download or read book Microfluidics and Lab on a Chip written by Andreas Manz and published by Royal Society of Chemistry. This book was released on 2020-09-24 with total page 307 pages. Available in PDF, EPUB and Kindle. Book excerpt: Responding to the need for an affordable, easy-to-read textbook that introduces microfluidics to undergraduate and postgraduate students, this concise book will provide a broad overview of the important theoretical and practical aspects of microfluidics and lab-on-a-chip, as well as its applications.
Download or read book Environmental Chemicals the Human Microbiome and Health Risk written by National Academies of Sciences, Engineering, and Medicine and published by National Academies Press. This book was released on 2018-03-01 with total page 123 pages. Available in PDF, EPUB and Kindle. Book excerpt: A great number of diverse microorganisms inhabit the human body and are collectively referred to as the human microbiome. Until recently, the role of the human microbiome in maintaining human health was not fully appreciated. Today, however, research is beginning to elucidate associations between perturbations in the human microbiome and human disease and the factors that might be responsible for the perturbations. Studies have indicated that the human microbiome could be affected by environmental chemicals or could modulate exposure to environmental chemicals. Environmental Chemicals, the Human Microbiome, and Health Risk presents a research strategy to improve our understanding of the interactions between environmental chemicals and the human microbiome and the implications of those interactions for human health risk. This report identifies barriers to such research and opportunities for collaboration, highlights key aspects of the human microbiome and its relation to health, describes potential interactions between environmental chemicals and the human microbiome, reviews the risk-assessment framework and reasons for incorporating chemicalâ€"microbiome interactions.
Download or read book Introduction to Single Cell Omics written by Xinghua Pan and published by Frontiers Media SA. This book was released on 2019-09-19 with total page 129 pages. Available in PDF, EPUB and Kindle. Book excerpt: Single-cell omics is a progressing frontier that stems from the sequencing of the human genome and the development of omics technologies, particularly genomics, transcriptomics, epigenomics and proteomics, but the sensitivity is now improved to single-cell level. The new generation of methodologies, especially the next generation sequencing (NGS) technology, plays a leading role in genomics related fields; however, the conventional techniques of omics require number of cells to be large, usually on the order of millions of cells, which is hardly accessible in some cases. More importantly, harnessing the power of omics technologies and applying those at the single-cell level are crucial since every cell is specific and unique, and almost every cell population in every systems, derived in either vivo or in vitro, is heterogeneous. Deciphering the heterogeneity of the cell population hence becomes critical for recognizing the mechanism and significance of the system. However, without an extensive examination of individual cells, a massive analysis of cell population would only give an average output of the cells, but neglect the differences among cells. Single-cell omics seeks to study a number of individual cells in parallel for their different dimensions of molecular profile on genome-wide scale, providing unprecedented resolution for the interpretation of both the structure and function of an organ, tissue or other system, as well as the interaction (and communication) and dynamics of single cells or subpopulations of cells and their lineages. Importantly single-cell omics enables the identification of a minor subpopulation of cells that may play a critical role in biological process over a dominant subpolulation such as a cancer and a developing organ. It provides an ultra-sensitive tool for us to clarify specific molecular mechanisms and pathways and reveal the nature of cell heterogeneity. Besides, it also empowers the clinical investigation of patients when facing a very low quantity of cell available for analysis, such as noninvasive cancer screening with circulating tumor cells (CTC), noninvasive prenatal diagnostics (NIPD) and preimplantation genetic test (PGT) for in vitro fertilization. Single-cell omics greatly promotes the understanding of life at a more fundamental level, bring vast applications in medicine. Accordingly, single-cell omics is also called as single-cell analysis or single-cell biology. Within only a couple of years, single-cell omics, especially transcriptomic sequencing (scRNA-seq), whole genome and exome sequencing (scWGS, scWES), has become robust and broadly accessible. Besides the existing technologies, recently, multiplexing barcode design and combinatorial indexing technology, in combination with microfluidic platform exampled by Drop-seq, or even being independent of microfluidic platform but using a regular PCR-plate, enable us a greater capacity of single cell analysis, switching from one single cell to thousands of single cells in a single test. The unique molecular identifiers (UMIs) allow the amplification bias among the original molecules to be corrected faithfully, resulting in a reliable quantitative measurement of omics in single cells. Of late, a variety of single-cell epigenomics analyses are becoming sophisticated, particularly single cell chromatin accessibility (scATAC-seq) and CpG methylation profiling (scBS-seq, scRRBS-seq). High resolution single molecular Fluorescence in situ hybridization (smFISH) and its revolutionary versions (ex. seqFISH, MERFISH, and so on), in addition to the spatial transcriptome sequencing, make the native relationship of the individual cells of a tissue to be in 3D or 4D format visually and quantitatively clarified. On the other hand, CRISPR/cas9 editing-based In vivo lineage tracing methods enable dynamic profile of a whole developmental process to be accurately displayed. Multi-omics analysis facilitates the study of multi-dimensional regulation and relationship of different elements of the central dogma in a single cell, as well as permitting a clear dissection of the complicated omics heterogeneity of a system. Last but not the least, the technology, biological noise, sequence dropout, and batch effect bring a huge challenge to the bioinformatics of single cell omics. While significant progress in the data analysis has been made since then, revolutionary theory and algorithm logics for single cell omics are expected. Indeed, single-cell analysis exert considerable impacts on the fields of biological studies, particularly cancers, neuron and neural system, stem cells, embryo development and immune system; other than that, it also tremendously motivates pharmaceutic RD, clinical diagnosis and monitoring, as well as precision medicine. This book hereby summarizes the recent developments and general considerations of single-cell analysis, with a detailed presentation on selected technologies and applications. Starting with the experimental design on single-cell omics, the book then emphasizes the consideration on heterogeneity of cancer and other systems. It also gives an introduction of the basic methods and key facts for bioinformatics analysis. Secondary, this book provides a summary of two types of popular technologies, the fundamental tools on single-cell isolation, and the developments of single cell multi-omics, followed by descriptions of FISH technologies, though other popular technologies are not covered here due to the fact that they are intensively described here and there recently. Finally, the book illustrates an elastomer-based integrated fluidic circuit that allows a connection between single cell functional studies combining stimulation, response, imaging and measurement, and corresponding single cell sequencing. This is a model system for single cell functional genomics. In addition, it reports a pipeline for single-cell proteomics with an analysis of the early development of Xenopus embryo, a single-cell qRT-PCR application that defined the subpopulations related to cell cycling, and a new method for synergistic assembly of single cell genome with sequencing of amplification product by phi29 DNA polymerase. Due to the tremendous progresses of single-cell omics in recent years, the topics covered here are incomplete, but each individual topic is excellently addressed, significantly interesting and beneficial to scientists working in or affiliated with this field.
