The critically acclaimed laboratory standard for more than fifty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 400 volumes (all of them still in print), the series contains much material still relevant today—truly an essential publication for researchers in all fields of life sciences. This new volume presents methods related to the use of bacterial genetics for genomic engineering. The book includes sections on strain collections and genetic nomenclature; transposons; and phage.

Viruses have been studied since 1892, and for many years the emphasis has been on the mechanism of viral infection and disease. By the 1950s, researchers had begun to use enzymes and genomes of viruses of bacteria (bacteriophages or phages) as tools in developing molecular biology. Therefore, phage biology was biased on a few models like Lambda, p22, and T4 among other families that are extremely abundant, diverse, and unexplored. Yet even in the well-studied phage models, new pieces of evidences suggest that structural proteins with unknown functions may be involved in trilateral interactions between phage, host, and environments. Recently, there has been renewed interest in phage therapy due to the development of antibiotic resistance and microbiome-related diseases require precision treatments. Nevertheless, critical features regarding the discovery of new phages, characterizing their unknown functions, and therapeutic applications remain unaddressed. This dissertation examines the issues listed above with various techniques on cultured and uncultured environmental phages. In chapter 2, unknown function open reading frames (ORFs) in marine virome were predicted and experimentally validated as a workflow to investigate the rapid growing Next Generation Sequencing (NGS) data. Machine-learning algorithms trained by protein features were utilized to detect and categorize phage structural genes. The selected candidates were cloned, over-expressed, and purified for reconstitutions of structures in vitro. Chapter 3 is dedicated to phage-glycan interactions in mucosal surfaces. Immunoglobulin-like (Ig-like) domains have been found in many phage structural proteins and they were proposed to bind glycans displayed on mucus subunits. In chapter 3, methods of phage engineering were tested for editing Ig-like domains. In addition, various assays were accessed for characterizing the phenotypes of phage interacting with mucus. Chapter 4 is a pioneer study of phage therapy targeting immature microbiota in the gastrointestinal systems. In collaboration with Center for Phage Technology at Texas A&M University, a toxin-free, non-replicating, non-lytic Bacillus prophage-like element, PBSX, was selected and engineered as "phagocin" aiming to kill pathogenic strains. In chapter 4, the DNA packaging pattern in PBSX was further investigated for better understanding of its life cycle and future applications.

CRISPR/Cas is a recently described defense system that protects bacteria and archaea against invasion by mobile genetic elements such as viruses and plasmids. A wide spectrum of distinct CRISPR/Cas systems has been identified in at least half of the available prokaryotic genomes. On-going structural and functional analyses have resulted in a far greater insight into the functions and possible applications of these systems, although many secrets remain to be discovered. In this book, experts summarize the state of the art in this exciting field.

Historically, the first observation of a transmissible lytic agent that is specifically active against a bacterium (Bacillus anthracis) was by a Russian microbiologist Nikolay Gamaleya in 1898. At that time, however, it was too early to make a connection to another discovery made by Dmitri Ivanovsky in 1892 and Martinus Beijerinck in 1898 on a non-bacterial pathogen infecting tobacco plants. Thus the viral world was discovered in two of the three domains of life, and our current understanding is that viruses represent the most abundant biological entities on the planet. The potential of bacteriophages for infection treatment have been recognized after the discoveries by Frederick Twort and Felix d’Hérelle in 1915 and 1917. Subsequent phage therapy developments, however, have been overshadowed by the remarkable success of antibiotics in infection control and treatment, and phage therapy research and development persisted mostly in the former Soviet Union countries, Russia and Georgia, as well as in France and Poland. The dramatic rise of antibiotic resistance and especially of multi-drug resistance among human and animal bacterial pathogens, however, challenged the position of antibiotics as a single most important pillar for infection control and treatment. Thus there is a renewed interest in phage therapy as a possible additive/alternative therapy, especially for the infections that resist routine antibiotic treatment. The basis for the revival of phage therapy is affected by a number of issues that need to be resolved before it can enter the arena, which is traditionally reserved for antibiotics. Probably the most important is the regulatory issue: How should phage therapy be regulated? Similarly to drugs? Then the co-evolving nature of phage-bacterial host relationship will be a major hurdle for the production of consistent phage formulae. Or should we resort to the phage products such as lysins and the corresponding engineered versions in order to have accurate and consistent delivery doses? We still have very limited knowledge about the pharmacodynamics of phage therapy. More data, obtained in animal models, are necessary to evaluate the phage therapy efficiency compared, for example, to antibiotics. Another aspect is the safety of phage therapy. How do phages interact with the immune system and to what costs, or benefits? What are the risks, in the course of phage therapy, of transduction of undesirable properties such as virulence or antibiotic resistance genes? How frequent is the development of bacterial host resistance during phage therapy? Understanding these and many other aspects of phage therapy, basic and applied, is the main subject of this Topic.

Phage-display technology has begun to make critical contributions to the study of molecular recognition. DNA sequences are cloned into phage, which then present on their surface the proteins encoded by the DNA. Individual phage are rescued through interaction of the displayed protein with a ligand, and the specific phage is amplified by infection of bacteria. Phage-display technology is powerful but challenging and the aim of this manual is to provide comprehensive instruction in its theoretical and applied so that any scientist with even modest molecular biology experience can effectively employ it. The manual reflects nearly a decade of experience with students of greatly varying technical expertise andexperience who attended a course on the technology at Cold Spring Harbor Laboratory. Phage-display technology is growing in importance and power. This manual is an unrivalled source of expertise in its execution and application.

The 21st century has witnessed a complete revolution in the understanding and description of bacteria in eco- systems and microbial assemblages, and how they are regulated by complex interactions among microbes, hosts, and environments. The human organism is no longer considered a monolithic assembly of tissues, but is instead a true ecosystem composed of human cells, bacteria, fungi, algae, and viruses. As such, humans are not unlike other complex ecosystems containing microbial assemblages observed in the marine and earth environments. They all share a basic functional principle: Chemical communication is the universal language that allows such groups to properly function together. These chemical networks regulate interactions like metabolic exchange, antibiosis and symbiosis, and communication. The National Academies of Sciences, Engineering, and Medicine's Chemical Sciences Roundtable organized a series of four seminars in the autumn of 2016 to explore the current advances, opportunities, and challenges toward unveiling this "chemical dark matter" and its role in the regulation and function of different ecosystems. The first three focused on specific ecosystemsâ€"earth, marine, and humanâ€"and the last on all microbiome systems. This publication summarizes the presentations and discussions from the seminars.

This text presents the modeling, analysis, and design methods for systems biology. It discusses how to examine experimental data to learn about mathematical models, develop efficient abstraction and simulation methods to analyze these models, and use analytical methods to design new circuits. The author reviews basic molecular biology and biochemistry principles, covers several methods for modeling and analyzing genetic circuits, and uses phage lambda as an example throughout to help illustrate the methods. He also explores the emerging area of synthetic biology. iBioSim software, lecture slides, and a password-protected solutions manual are available on the author's website.

This authoritative, timely, and comprehensively referenced compendium on the bacteriophages explores current views of how viruses infect bacteria. In combination with classical phage molecular genetics, new structural, genomic, and single-molecule technologies have rendered an explosion in our knowledge of phages. Bacteriophages, the most abundant and genetically diverse type of organism in the biosphere, were discovered at the beginning of the 20th century and enjoyed decades of used as anti-bacterial agents before being eclipsed by the antibiotic era. Since 1988, phages have come back into the spotlight as major factors in pathogenesis, bacterial evolution, and ecology. This book reveals their compelling elegence of function and their almost inconceivable diversity.Much of the founding work in molecular biology and structural biology was done on bacteriophages. These are widely used in molecular biology research and in biotechnology, as probes and markers, and in the popular method of assesing gene expression.