[PDF] Design And Applications Of Fluorescent Protein Based Biosensors For Live Cell Imaging eBook

Author : Yidan Ding
Publisher :
Page : 174 pages
File Size : 26,82 MB
Release : 2014
Category : Biosensors
ISBN :

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Fluorescent proteins (FPs) are essential tools of biochemical research. Traditionally, FPs have been utilized as markers of gene expression, protein localization, and organelle structure. In recent years, however, FPs have gained in popularity as active biosensors of cellular activity, in which the fluorescence intensity or colour of a FP chromophore is modulated in response to a change in its environment. Current methods for converting FPs into active biosensors of live cell biochemistry remain few in number and are technically challenging. In addition, a growing demand has emerged for spectrally orthogonal biosensors for monitoring multiple biological parameters in a single live cell. A challenge in realizing this goal is that the broad spectral profiles of FPs have limited the number of biosensors that can be used together. In this thesis I describe the engineering of new FP-based biosensors and further validating them via live cell multiparameter imaging. The first class of FP-based biosensors addressed in this thesis is Förster resonance energy transfer (FRET) biosensors. I described our efforts to use optimized spectrally distinct FRET-based biosensors to image Ca2+ dynamics in two distinct subcellular compartments as well as Ca2+ and caspase-3 activity in the same subcellular compartment. Although the inherently ratiometric response of FRET biosensor permits quantitative measurements, the fact that two FPs are involved in each FRET pair present a challenge with respect to their application in multiparameter imaging. To overcome this issue, we engineered intensiometric biosensors based on the recently introduced dimerization-dependent fluorescent protein (ddFP) technology. I demonstrate that ddFP-based protease biosensors enable the reporting of protease activity either by the intensiometric loss of the initially bright fluorescence or with dramatic green-to-red and red-to-green colour switches and translocation from the cytoplasm to the nucleus. In addition to detection of caspase activities, we also achieved specific highlighting of the mitochondria-associated membrane (MAM) using ddFP technology. Attempts to detect polyADP-ribose and polyubiquitin using ddFP technology were ultimately unsuccessful. Overall, this work serves to expand the scope of current FRET and ddFP-based biosensor designs and applications.

Author : Jin Zhang
Publisher : Humana
Page : 0 pages
File Size : 48,90 MB
Release : 2016-08-23
Category : Science
ISBN : 9781493960149

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In Fluorescent Protein-Based Biosensors: Methods and Protocols, experts in the field have assembled a series of protocols describing several methods in which fluorescent protein-based reporters can be used to gain unique insights into the regulation of cellular signal transduction. Genetically encodable fluorescent biosensors have allowed researchers to observe biochemical processes within the endogenous cellular environment with unprecedented spatiotemporal resolution. As the number and diversity of available biosensors grows, it is increasingly important to equip researchers with an understanding of the key concepts underlying the design and application of genetically encodable fluorescent biosensors to live cell imaging. Written in the 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 protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Fluorescent Protein-Based Biosensors: Methods and Protocols promises to be a valuable resource for researchers interested in applying current biosensors to the study of biochemical processes in living cells as well as those interested in developing novel biosensors to visualize other cellular phenomena.

Author :
Publisher : Academic Press
Page : 449 pages
File Size : 34,91 MB
Release : 2012-12-31
Category : Science
ISBN : 012386951X

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One of the major challenges of modern biology and medicine consists in finding means to visualize biomolecules in their natural environment with the greatest level of accuracy, so as to gain insight into their properties and behaviour in a physiological and pathological setting. This has been achieved thanks to the design of novel imaging agents, in particular to fluorescent biosensors. Fluorescence Biosensors comprise a large set of tools which are useful for fundamental purposes as well as for applications in biomedicine, drug discovery and biotechnology. These tools have been designed and engineered thanks to the combined efforts of chemists and biologists over the last decade, and developed hand in hand together with imaging technologies. This volume will convey the many exciting developments the field of fluorescent biosensors and reporters has witnessed over the recent years, from concepts to applications, including chapters on the chemistry of fluorescent probes, on technologies for monitoring protein/protein interactions and technologies for imaging biosensors in cultured cells and in vivo. Other chapters are devoted to specific examples of genetically-encoded reporters, or to protein and peptide biosensors, together with examples illustrating their application to cellular and in vivo imaging, biomedical applications, drug discovery and high throughput screening. Contributions from leading authorities Informs and updates on all the latest developments in the field

Author : Ahmed Saied F. Belal
Publisher :
Page : 124 pages
File Size : 13,83 MB
Release : 2014
Category : Biosensors
ISBN :

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The discovery of green fluorescent protein (GFP) from the Aequorea victoria Jellyfish followed by the extensive efforts of protein engineers to produce other fluorescent proteins (FPs) spanning the visible color spectrum made fluorescent proteins indispensable biochemical tools in the scientific community. Experimental biologists have utilized FPs as genetically encoded markers for the imaging of subcellular structures and protein dynamics in live cells. Another important application of FPs is their design for use in biosensors for either enzymes or small biological molecules of interest. The work described in this thesis is an attempt to portray different experimental designs of FP based biosensors with the final objective of either modifying previously reported or introducing novel biosensors. We addressed the FP biosensors based on the principle of intramolecular Förster resonance energy transfer (FRET) in two projects. In the first project we demonstrated a modification of a methodology of development and optimization of FRET-based biosensor for a post translational modification. The end result of this project has led to improving a previously reported protein kinase B (PkB) biosensor and the discovery of a new cyclin B1- cyclin dependent kinase 1 (Cyclin B1-CDK1) biosensor of a higher dynamic range than previously published one. In the second project our efforts were directed to develop a matrix metallo proteinase 2 (MMP2) FRET based biosensor, with the ultimate goal of using this biosensor in live cell imaging of cardiomyocytes to explore postulated MMP2 intracellular role in ischemia-reperfusion injury. We designed protein constructs based on both previously reported MMP2 substrate sequences and potential cardiac protein target sequences. After testing and characterizing the designed constructs, the expression of the best candidate in neonatal cardiomyocytes cell lines is undertaken by our collaborator Professor Richard Schulz, Department of Pharmacology, University of Alberta. In the third and final project we utilized single FP-based biosensor concept in trying to develop Hydrogen peroxide biosensors of different hues. Our efforts were fruitful in discovering two green biosensors, one is with direct and the other is with inverse response to hydrogen peroxide.

Author : Kevin F. Sullivan
Publisher : Elsevier
Page : 613 pages
File Size : 31,13 MB
Release : 2007-12-14
Category : Science
ISBN : 0080557244

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This new edition of Fluorescent Proteins presents current applications of autofluorescent proteins in cell and molecular biology authored by researchers from many of the key laboratories in the field. Starting from a current review of the broad palette of fluorescent proteins available, several chapters focus on key autofluorescent protein variants, including spectral variants, photodynamic variants as well as chimeric FP approaches. Molecular applications are addressed in chapters that detail work with single molecules, approaches to generating protein fusions and biosensors as well as analysis of protein-protein interactions in vivo by FRET, fluorescence polarization and fluorescence cross correlation techniques. A number of approaches to in vivo dynamics are presented, including FRAP, photoactivation, and 4-dimensional microscopy. Behavior of spindle components, membrane proteins, mRNA trafficking as well as analysis of cell types in tissues and in development are detailed and provide models for a wide variety of experimental approaches. In addition, several chapters deal directly with the computational issues involved in processing multidimensional image data and using fluorescent imaging to probe cellular behavior with quantitative modeling. This volume brings together the latest perspective and techniques on fluorescent proteins and will be an invaluable reference in a wide range of laboratories.

Author : Huimin Zhao
Publisher : John Wiley & Sons
Page : 41 pages
File Size : 40,97 MB
Release : 2021-08-23
Category : Science
ISBN : 3527344705

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A one-stop reference that reviews protein design strategies to applications in industrial and medical biotechnology Protein Engineering: Tools and Applications is a comprehensive resource that offers a systematic and comprehensive review of the most recent advances in the field, and contains detailed information on the methodologies and strategies behind these approaches. The authors—noted experts on the topic—explore the distinctive advantages and disadvantages of the presented methodologies and strategies in a targeted and focused manner that allows for the adaptation and implementation of the strategies for new applications. The book contains information on the directed evolution, rational design, and semi-rational design of proteins and offers a review of the most recent applications in industrial and medical biotechnology. This important book: Covers technologies and methodologies used in protein engineering Includes the strategies behind the approaches, designed to help with the adaptation and implementation of these strategies for new applications Offers a comprehensive and thorough treatment of protein engineering from primary strategies to applications in industrial and medical biotechnology Presents cutting edge advances in the continuously evolving field of protein engineering Written for students and professionals of bioengineering, biotechnology, biochemistry, Protein Engineering: Tools and Applications offers an essential resource to the design strategies in protein engineering and reviews recent applications.

Author : Martin Chalfie
Publisher : Wiley-Liss
Page : 424 pages
File Size : 45,42 MB
Release : 1998-09-07
Category : Science
ISBN :

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Green Fluorescent Protein: Properties, Applications, and Protocols is the first book devoted to this rapidly evolving reporter system. This collection of essays from pioneers in the field tackles both theory and practice, offering numerous case studies, examples, illustrations, and troubleshooting tips. The book clearly demonstrates how to tailor GFP to specific systems, maximize expression and enhance detection in a variety of organisms and cell types, generate variants with altered properties, explore new ways to use GFP, and more. Topics covered include: * The discovery of GFP * Bioluminescence and biofluorescence in nature * Biochemical and physical properties of GFP * The three-dimensional structure of GFP and its implications for function and design * Molecular biology and modification of GFP * Uses of GFP in various organisms, including prokaryotes, yeast, C. elegans, drosophila, plants, transgenic vertebrates, and mammalian cells * Detailed protocols for the expression of GFP, specimen preparation, visualization and recording of GFP fluorescence, and GFP websites

Author : Robert Hoffman
Publisher : Humana Press
Page : 269 pages
File Size : 44,82 MB
Release : 2012-06-15
Category : Medical
ISBN : 9781617797989

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The discovery and genetic engineering of fluorescent proteins has revolutionized cell biology. What was previously invisible in the cell often can be made visible with the use of fluorescent proteins. In Vivo Cellular Imaging Using Fluorescent Proteins: Methods and Protocols presents state-of-the-art research that has contributed to the fluorescent protein revolution to visualize biological processes in the live animal. This volume covers an array of topics from the employment of the chick CAM model using fluorescent proteins and other fluorescent probes, to intravital fluorescent imaging, as well as 3-dimensional imaging, and design instructions on how to create new and improved far-red and infrared fluorescent proteins, to name a few. Written in the successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, In Vivo Cellular Imaging Using Fluorescent Proteins: Methods and Protocols is the first volume in the new field of in vivo cell biology and it serves both professionals and novices with its well-honed methodologies.

Author : Gregor Jung
Publisher : Springer Science & Business Media
Page : 287 pages
File Size : 35,21 MB
Release : 2012-01-05
Category : Science
ISBN : 3642233775

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Fluorescent proteins are intimately connected to research in the life sciences. Tagging of gene products with fluorescent proteins has revolutionized all areas of biosciences, ranging from fundamental biochemistry to clinical oncology, to environmental research. The discovery of the Green Fluorescent Protein, its first, seminal application and the ingenious development of a broad palette of fluorescence proteins of other colours, was consequently recognised with the Nobel Prize for Chemistry in 2008. Fluorescent Proteins II highlights the physicochemical and biophysical aspects of fluorescent protein technology beyond imaging. It is tailored to meet the needs of physicists, chemists and biologists who are interested in the fundamental properties of fluorescent proteins, while also focussing on specific applications. The implementations described are cutting-edge studies and exemplify how the physical and chemical properties of fluorescent proteins can stimulate novel findings in life sciences.

Author : Toonika Rinken
Publisher : BoD – Books on Demand
Page : 364 pages
File Size : 40,73 MB
Release : 2013-03-13
Category : Medical
ISBN : 9535110047

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As biosensors comprise a prospective alternative to traditional chemical analyses, enabling fast on- and in-line measurements with sufficient selectivity, the field is expanding rapidly and is offering new ideas and developments every day. This book aims to cover the present state of the art in the biosensor technology and introduce the general aspects of biosensor- based techniques and methods. The book consists of 13 chapters by 44 authors and is divided into 3 sections, focused on bio-recognition techniques, signal transduction methods and signal analysis.