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.

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.

This book describes advanced studies in cell-free synthetic biology, an emerging biotechnology that focuses on cell-free protein synthesis and cell-free systems for fundamental and industrial research in areas such as genetic circuit design, small-molecule synthesis, complicated-macromolecule synthesis, unnatural-macromolecule synthesis, high-throughput screening, artificial cells, and biomaterials. Cell-free synthetic biology is now an integral part of developing fields like nanotechnology, materials science, and personalized medicine. The book discusses the main research directions in the development of cell-free systems, as well as a number of applications of cell-free synthetic biology, ranging from structural biology to the human health industry. It is intended for students and researchers in life sciences, synthetic biology, bioengineering, and chemical engineering.

This is a unique book that describes the most recent achievements in the methodology of protein biosynthesis under cell-free conditions. Various versions of cell-free protein-synthesizing systems and their applications to production of individual proteins on a preparative scale are reviewed. The most recent, advanced methodologies, such as continuous-exchange and continuous-flow cell-free systems and novel effecting batch-format cell-free procedures, are considered. Special attention is drawn to the possibilities of structural (NMR; X-ray) analysis of various gene expression products with the use of a new generation of cell-free systems.

In the last ten years there has been a considerable increase of interest on the notion of the minimal cell. With this term we usually mean a cell-like structure containing the minimal and sufficient number of components to be defined as alive, or at least capable of displaying some of the fundamental functions of a living cell. In fact, when we look at extant living cells we realize that thousands of molecules are organized spatially and functionally in order to realize what we call cellular life. This fact elicits the question whether such huge complexity is a necessary condition for life, or a simpler molecular system can also be defined as alive. Obviously, the concept of minimal cell encompasses entire families of cells, from totally synthetic cells, to semi-synthetic ones, to primitive cell models, to simple biomimetic cellular systems. Typically, in the experimental approach to the construction of minimal the main ingredient is the compartment. Lipid vesicles (liposomes) are used to host simple and complex molecular transformations, from single or multiple enzymic reactions, to polymerase chain reactions, to gene expression. Today this research is seen as part of the broader scenario of synthetic biology but it is rooted in origins of life studies, because the construction of a minimal cell might provide biophysical insights into the origins of primitive cells, and the emergence of life on earth. The volume provides an overview of physical, biochemical and functional studies on minimal cells, with emphasis to experimental approaches. 15 International experts report on their innovative contributions to the construction of minimal cells.

The latest volume in the Advanced Biotechnology series provides an overview of the main production hosts and platform organisms used today as well as promising future cell factories in a two volume book. Alongside describing tools for genetic and metabolic engineering for strain improvement, the authors also impart topical information on computational tools, safety aspects and industrial-scale production. Following an introduction to general concepts, historical developments and future technologies, the text goes on to cover multi-purpose bacterial cell factories, including those organisms that exploit anaerobic biosynthetic power. Further chapters deal with microbes used for the production of high-value natural compounds and those obtained from alternative raw material sources, concluding with eukaryotic workhorses.

Since newly created beings are often perceived as either wholly good or bad, the genetic alteration of living cells impacts directly on a symbolic meaning deeply imbedded in every culture. During the earlier years of gene expression research, te- nological applications were confined mainly to academic and industrial laboratories, and were perceived as highly beneficial since molecules that were previously unable to be separated or synthesized became accessible as therapeutic agents. Such were the success stories of hormones, antibodies, and vaccines produced in the bacterium Escherichia coli. Originally this bacterium gained fame among humans for being an unwanted host in the intestine, or worse yet, for being occasionally dangerous and pathogenic. H- ever, it was easily identified in contaminated waters during the 19th century, thus becoming a clear indicator of water pollution by human feces. Tamed, cultivated, and easily maintained in laboratories, its fast growth rate and metabolic capacity to adjust to changing environments fascinated the minds of scientists who studied and modeled such complex phenomena as growth, evolution, genetic exchange, infection, survival, adaptation, and further on—gene expression. Although at the lower end of the complexity scale, this microbe became a very successful model system and a key player in the fantastic revolution kindled by the birth of recombinant DNA technology.

During the past decade as the data on gene sequences and expression patterns rapidly accumulated, cell-free protein synthesis technology has also experienced a revolution, becoming a powerful tool for the preparation of proteins for their functional and structural analysis. In Cell-Free Protein Production: Methods and Protocols, experts in the field contribute detailed techniques, the uses of which expand deep into the studies of biochemistry, molecular biology, and biotechnology. Beginning briefly with basic methods and historical aspects, the book continues with thorough coverage of protein preparation methods, the preparation of proteins that are generally difficult to prepare in their functional forms, applications of the cell-free technologies to protein engineering, as well as some methods that are expected to constitute a part of future technologies. Written in the highly successful Methods in Molecular BiologyTM series format, the chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Cell-Free Protein Production: Methods and Protocols aims to help researchers continue the growth of the vital exploration of cell-free sciences and technologies in order to better understand the dynamic lives of cells.

With its detailed description of membrane protein expression, high-throughput and genomic-scale expression studies, both on the analytical and the preparative scale, this book covers the latest advances in the field. The step-by-step protocols and practical examples given for each method constitute practical advice for beginners and experts alike.