Recombinant gene expression is the fastest growing area in the study of molecular biology. By the time the Human Genome Project is completed (~2002), several thousand sequences will be known, but the purpose of the resultant expression products will remain a mystery. Gene discovery requires efficient expression systems for determining the structure and function of gene products. Gene Expression Systems covers a variety of promoters and host organisms that researchers can tailor to their specific needs.
This detailed volume explores perspectives and methods using cell-free expression (CFE) to enable next-generation synthetic biology applications. The first section focuses on tools for CFE systems, including a primer on DNA handling and reproducibility, as well as methods for cell extract preparation from diverse organisms and enabling high-throughput cell-free experimentation. The second section provides an array of applications for CFE systems, such as metabolic engineering, membrane-based and encapsulated CFE, cell-free sensing and detection, and educational kits. 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, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Cell‐Free Gene Expression: Methods and Protocols serves as an ideal guide for researchers seeking technical methods to current aspects of CFE and related applications.
Experimental Manipulation of Gene Expression discusses a wide range of host systems in which to clone and express a gene of interest. The aims are for readers to quickly learn the versatility of the systems and obtain an overview of the technology involved in the manipulation of gene expression. Furthermore, it is hoped that the reader will learn enough from the various approaches to be able to develop systems and to arrange for a gene of particular interest to express in a particular system. The book opens with a chapter on the design and construction of a plasmid vector system used to achieve high-level expression of a particular phage regulatory protein normally found in minute amounts in a phage-infected bacterial cell. This is followed by separate chapters on topics such as high-level expression vectors that utilize efficient Escherichia coli lipoprotein promoter as well as various other portions of the lipoprotein gene Ipp; DNA cloning systems for streptomycetes; and the design and application of vectors for high-level, inducible synthesis of the product of a cloned gene in yeast.
With the advent of recombinant DNA technology, expressing heterologous proteins in microorganisms rapidly became the method of choice for their production at laboratory and industrial scale. Bacteria, yeasts and other hosts can be grown to high biomass levels efficiently and inexpensively. Obtaining high yields of recombinant proteins from this material was only feasible thanks to constant research on microbial genetics and physiology that led to novel strains, plasmids and cultivation strategies. Despite the spectacular expansion of the field, there is still much room for progress. Improving the levels of expression and the solubility of a recombinant protein can be quite challenging. Accumulation of the product in the cell can lead to stress responses which affect cell growth. Buildup of insoluble and biologically inactive aggregates (inclusion bodies) lowers the yield of production. This is particularly true for obtaining membrane proteins or high-molecular weight and multi-domain proteins. Also, obtaining eukaryotic proteins in a prokaryotic background (for example, plant or animal proteins in bacteria) results in a product that lack post-translational modifications, often required for functionality. Changing to a eukaryotic host (yeasts or filamentous fungi) may not be a proper solution since the pattern of sugar modifications is different than in higher eukaryotes. Still, many advances in the last couple of decades have provided to researchers a wide variety of strategies to maximize the production of their recombinant protein of choice. Everything starts with the careful selection of the host. Be it bacteria or yeast, a broad list of strains is available for overcoming codon use bias, incorrect disulfide bond formation, protein toxicity and lack of post-translational modifications. Also, a huge catalog of plasmids allows choosing for different fusion partners for improving solubility, protein secretion, chaperone co-expression, antibiotic resistance and promoter strength. Next, controlling culture conditions like temperature, inducer and media composition can bolster recombinant protein production. With this Research Topic, we aim to provide an encyclopedic account of the existing approaches to the expression of recombinant proteins in microorganisms, highlight recent discoveries and analyze the future prospects of this exciting and ever-growing field.
Protein expression in a heterologous host is a cornerstone of biomedical research and of the biotechnology industry. Despite the advanced state of protein expression technology improvements are still needed. For example, membrane proteins constitute a significant percentage of the total cellular proteins but as a class are very difficult to overexpress, especially in a heterologous host. The ideal host would have the ability to express any protein, with relevant post-translational modifications, and be as easy to work with as E. coli. In Heterologous Gene Expression in E. coli: Methods and Protocols, expert scientists intimately familiar with the relevant techniques offer chapters that greatly expand the utility of this expression host. The contributions in this detailed volume describe methods, for example, to successfully express proteins in E. coli that would otherwise form aggregates in this host, to add post-translational modifications, to incorporate non-standard amino acid residues or moieties into E. coli expressed proteins, to identify binding partners, and to express membrane proteins. Written in the highly successful Methods in Molecular BiologyTM format, chapters include introductions to their respective subjects, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Practical and cutting-edge, Heterologous Gene Expression in E. coli: Methods and Protocols seeks to familiarize the researcher with the myriad of E. coli expression strains available and move E. coli closer to that ideal of the perfect host.
High-throughput measurements of gene expression and genetic marker data facilitate systems biologic and systems genetic data analysis strategies. Gene co-expression networks have been used to study a variety of biological systems, bridging the gap from individual genes to biologically or clinically important emergent phenotypes.
Cell-free protein synthesis is coming of age! Motivated by an escalating need for efficient protein synthesis and empowered by readily accessible cell-free protein synthesis kits, the technology is expanding both in the range of feasible proteins and in the ways that proteins can be labeled and modified. This volume follows "Cell-Free Translation Systems", edited by Professor Alexander S. Spirin in 2002. Since then, an impressive collection of new work has emerged that demonstrates a substantial expansion of capability. In this volume, we show that proteins now can be efficiently produced using PCR products as DNA templates and that even membrane proteins and proteins with multiple disulfide proteins are obtained at high yields. Many additional advances are also presented. It is an exciting time for protein synthesis technology.
Bacteria in various habitats are subject to continuously changing environmental conditions, such as nutrient deprivation, heat and cold stress, UV radiation, oxidative stress, dessication, acid stress, nitrosative stress, cell envelope stress, heavy metal exposure, osmotic stress, and others. In order to survive, they have to respond to these conditions by adapting their physiology through sometimes drastic changes in gene expression. In addition they may adapt by changing their morphology, forming biofilms, fruiting bodies or spores, filaments, Viable But Not Culturable (VBNC) cells or moving away from stress compounds via chemotaxis. Changes in gene expression constitute the main component of the bacterial response to stress and environmental changes, and involve a myriad of different mechanisms, including (alternative) sigma factors, bi- or tri-component regulatory systems, small non-coding RNA’s, chaperones, CHRIS-Cas systems, DNA repair, toxin-antitoxin systems, the stringent response, efflux pumps, alarmones, and modulation of the cell envelope or membranes, to name a few. Many regulatory elements are conserved in different bacteria; however there are endless variations on the theme and novel elements of gene regulation in bacteria inhabiting particular environments are constantly being discovered. Especially in (pathogenic) bacteria colonizing the human body a plethora of bacterial responses to innate stresses such as pH, reactive nitrogen and oxygen species and antibiotic stress are being described. An attempt is made to not only cover model systems but give a broad overview of the stress-responsive regulatory systems in a variety of bacteria, including medically important bacteria, where elucidation of certain aspects of these systems could lead to treatment strategies of the pathogens. Many of the regulatory systems being uncovered are specific, but there is also considerable “cross-talk” between different circuits. Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria is a comprehensive two-volume work bringing together both review and original research articles on key topics in stress and environmental control of gene expression in bacteria. Volume One contains key overview chapters, as well as content on one/two/three component regulatory systems and stress responses, sigma factors and stress responses, small non-coding RNAs and stress responses, toxin-antitoxin systems and stress responses, stringent response to stress, responses to UV irradiation, SOS and double stranded systems repair systems and stress, adaptation to both oxidative and osmotic stress, and desiccation tolerance and drought stress. Volume Two covers heat shock responses, chaperonins and stress, cold shock responses, adaptation to acid stress, nitrosative stress, and envelope stress, as well as iron homeostasis, metal resistance, quorum sensing, chemotaxis and biofilm formation, and viable but not culturable (VBNC) cells. Covering the full breadth of current stress and environmental control of gene expression studies and expanding it towards future advances in the field, these two volumes are a one-stop reference for (non) medical molecular geneticists interested in gene regulation under stress.
After genomic sequencing, microarray technology has emerged as a widely used platform for genomic studies in the life sciences. Microarray technology provides a systematic way to survey DNA and RNA variation. With the abundance of data produced from microarray studies, however, the ultimate impact of the studies on biology will depend heavily on data mining and statistical analysis. The contribution of this book is to provide readers with an integrated presentation of various topics on analyzing microarray data.
