Electrotransformation of Bacteria


Book Description

In this manual, protocols for the transformation of about 40 strains of bacteria are described, with the emphasis placed on the individual critical procedural steps, since the practical details mainly depend on the bacterial strain under investigation. This presentation together with the theoretical introductionary chapters, allows users to modify and adapt each protocol to their own experiments. Bacterial strains with relevance in the food industry, biotechnology, medical and veterinary fields, agroindustry and environmental sciences are covered.




Electroporation Protocols for Microorganisms


Book Description

Electroporation is one of the most widespread techniques used in modem molecular genetics. It is most commonly used to introduce DNA into cells for investigations of gene structure and function, and in this regard, electroporation is both highly versatile, being effective with nearly all species and cell types, and highly efficient. For many cell types, electroporation is either the most efficient or the only means known to effect gene transfer. However, exposure of cells to brief, hi- intensity electric fields has found broad application in other aspects of biological research, and is now routinely used to introduce other types of biological and analytic molecules into cells, to induce cell-cell fusion, and to transfer DNA directly between different species. The first seven chapters of Electroporation Protocols for Micro organisms describe the underlying theory of electroporation, the com mercially available instrumentation, and a number of specialized electroporation applications, such as cDNA library construction and interspecies DNA electrotransfer. Each of the remaining chapters pre sents a well developed method for electrotransformation of a particular bacterial, fungal, or protist species. These chapters also serve to intro duce those new to the field the important research questions that are currently being addressed with particular organisms, highlighting both the major advantages and limitations of each species as a model organ ism, and explaining the roles that electroporation has played in the development of the molecular genetic systems currently in use.




Handbook of Electroporation


Book Description

This major reference work is a one-shot knowledge base on electroporation and the use of pulsed electric fields of high intensity and their use in biology, medicine, biotechnology, and food and environmental technologies. The Handbook offers a widespread and well-structured compilation of 156 chapters ranging from the foundations to applications in industry and hospital. It is edited and written by most prominent researchers in the field. With regular updates and growing in its volume it is suitable for academic readers and researchers regardless of their disciplinary expertise, and will also be accessible to students and serious general readers. The Handbook's 276 authors have established scholarly credentials and come from a wide range of disciplines. This is crucially important in a highly interdisciplinary field of electroporation and the use of pulsed electric fields of high intensity and its applications in different fields from medicine, biology, food processing, agriculture, process engineering, energy and environment. An Editorial Board of distinguished scholars from across the world has selected and reviewed the various chapters to ensure the highest quality of this Handbook. The book was edited by an international team of Section Editors: P. Thomas Vernier, Boris Rubinsky, Juergen Kolb, Damijan Miklavcic, Marie-Pierre Rols, Javier Raso, Richard Heller, Gregor Serša, Dietrich Knorr, and Eugene Vorobiev.




Guide to Electroporation and Electrofusion


Book Description

Guide to Electroporation and Electrofusion is designed to cover all relevant topics pertaining to both electroporation and electrofusion. Divided into four major parts, the book covers fundamental aspects, as well as more advance aspects of the electroporation-electrofusion relationship. The book first covers the basic principles and fundamentals by presenting the most recent theoretical and experimental studies from various fields, such as physics, chemistry, and biology. Next, the book tackles the applications of electroporation and electrofusion in biology, such as transferring, manipulating, and transforming genetic materials. In the third section, the book discusses experimental protocols to serve as a guide when performing experiments using electroporation and electrofusion. The final section discusses the instruments needed to effectively perform an experiment that involves electroporation and electrofusion. This book will be of great used to both novice and advanced researchers whose work involves electroporation and electrofusion, as it provides comprehensive information regarding these topics.




Electroporation and Electrofusion in Cell Biology


Book Description

Cells can be funny. Try to grow them with a slightly wrong recipe, and they turn over and die. But hit them with an electric field strong enough to knock over a horse, and they do enough things to justify international meetings, to fill a sizable book, and to lead one to speak of an entirely new technology for cell manipulation. The very improbability of these events not only raises questions about why things happen but also leads to a long list of practical systems in which the application of strong electric fields might enable the merger of cell contents or the introduction of alien but vital material. Inevitably, the basic questions and the practical applications will not keep in step. The questions are intrinsically tough. It is hard enough to analyze the action of the relatively weak fields that rotate or align cells, but it is nearly impossible to predict responses to the cell-shredding bursts of electricity that cause them to fuse or to open up to very large molecular assemblies. Even so, theoretical studies and systematic examination of model systems have produced some creditable results, ideas which should ultimately provide hints of what to try next.




Critical Electric Field Quantification for Inducing Bacterial Electroporation


Book Description

Electroporation is one of the most popular methods for rapid and efficient intracellular transport of foreign molecules, such as peptides, drugs, and DNA, and is crucial for applications in genetic engineering. However, there is still a lack of mechanistic understanding of electroporation, and the most common method to optimize electroporation parameters for bacterial genetic transformation is still based on trial and error. In this work, to alleviate this issue we use microfluidic techniques to achieve rapid screening of electric fields for electroporation in live bacteria. To determine the critical electric field required for inducing bacterial electroporation, we extend upon a previously developed microfluidic assay. By quantifying the critical electric field values of gram-positive bacteria Corynebacterium glutamicum and Bacillus subtilis 168, and the gram-negative bacteria Escherichia coli BL21 (DE3) and Pseudomonas syringae ESC 10 in 10% glycerol and 0.01 x PBS, we study the effects of using different buffer solutions for different bacteria in the electroporation process. We also investigate the relationship between cell polarizability and critical electric field for these cells in the aforementioned buffer solutions. Next, we discuss the accuracy of the measurement by illuminating how undesired phenomena such as electroosmosis and electrophoresis contribute to the measurement error. Results of this study will enhance our understanding of the electroporation process and provide insights in discovering and optimizing electroporation protocols.




Electroporation of Bacteria and Yeast: An Experimental Study of Molecular Transport


Book Description

This reseaech involves a basic study of electroporation with an emphasis on molecular transport across cell membranes. We have explored a new mechanism for drug delivery, the use of electroporation to move molecules into, across and out of tissues. Electroporation of human skin may provide the basis for greatly enhanced transdermal drug delivery, and for non-invasive "sensing" of biochemicals within the body. Another advance is a new general method for rapidly determinmg the clonal growth of individual cells. For more details, see attached.




Pseudomonas Methods and Protocols


Book Description

In Pseudomonas aeruginosa, expert researchers in the field detail many of the methods which are now commonly used to study this fascinating microorganism. Chapters include microbiological methods to high-throughput molecular techniques that have been developed over the last decade. 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 key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Pseudomonas aeruginosa aids in the continuing study of new and cutting edge findings.




Laboratory Methods in Enzymology: DNA


Book Description

Methods in Enzymology volumes provide an indispensable tool for the researcher. Each volume is carefully written and edited by experts to contain state-of-the-art reviews and step-by-step protocols. In this volume, we have brought together a number of core protocols concentrating on DNA, complementing the traditional content that is found in past, present and future Methods in Enzymology volumes. Indispensable tool for the researcher Carefully written and edited by experts to contain step-by-step protocols In this volume we have brought together a number of core protocols concentrating on DNA




Bacterial Chemosensing


Book Description

This volume covers a wide range of up-to-date technologies that have been successfully applied to study the chemosensing behavior of the traditional model species, such as Escherichia coli and Salmonella typhimurium, while being also applicable to a wide spectrum of other species. Beginning with an introduction, the sections of the book explore methods for studying bacterial chemotaxis at the population and whole-cell levels, in vivo analysis of receptor function, cryo-EM methods for studying chemoreceptor structure, as well as intracellular movement of chemosensory proteins, high-throughput methods to screen for novel chemoeffectors, and chemical tools and computer simulations for analyzing chemotaxis. Written for the highly successful Methods in Molecular Biology series, chapters include brief introductions to their topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips for troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Bacterial Chemosensing: Methods and Protocols provides an extensive repertoire of approaches that can be extended to understanding chemotaxis, in particular, and chemosensing, in general, in the context of the enormously varied lifestyles adopted in the larger bacterial world.