Methods in Biotechnology – serie

This authoritative collection of basic and advanced protocols permits biotechnologists to use plants rather than single cell fermentation systems for expressing recombinant proteins. The protocols provide clear step-by-step instructions for the cloning, expression, and analysis of a wide variety of recombinant proteins in plants, and for the cloning of genes into a number of different plant species. The book also describes promising experimental techniques that will become increasingly important in the future, and includes techniques for the characterization of medically important proteins expressed in plants.

A cutting-edge collection of detailed, step-by-step techniques and protocols for constructing, evaluating, and using enzyme- and microbial-based biosensors. Ideal for novices starting research in their field or experienced researchers wanting to use a biosensor for a specific analytical measurement, the methods detailed here allow biochemists, analytical chemists, microbiologists, and engineers to successfully apply biosensor technology to their specific problems. The techniques include classical enzyme biosensors based on potentiometric, amperometric, conductimetric, optical, and thermal transducers, as well cell-based biosensors based on amperometric and optical transducers. A companion volume, Affinity Biosensors: Protocols and Techniques, by Rogers & Mulchandani, concentrates on cell- and affinity-based biosensors.

The many thousands of human-made and other chemical compounds present in the environment offer a serious challenge to our btosphere. It is appropriate, therefore, that our response to these products of human kno- edge and ingenuity should draw on a body of mtenstve scientific endeavor that is no less impressive. Bioremediation offers the possibility of harnessing the diversity of the biosphere to degrade, remove, alter, or otherwise detoxify these various chemicals. It brings together scientists from a wide variety of disciplines and backgrounds, such as microbiology, molecular biology, a- lytical chemistry, and chemical and environmental engineering, among o- ers. These different fields, each with its own individual approach, have actively contributed to the development of bioremediation research tn recent years. The prmcipal objective of Bioremediation Protocols is to make the fruits of some of this research available in a different format to that of the textbook or journal article. It provides a selection of clearly written laboratory pro- cols presented as stepwise, easy-to-follow mstructions. In common with p- wous volumes in this and the companion Methods in Molecular Biology series, an extensive “Notes” section is provided with each chapter. This contains u- ful mformation (of a type often not normally included m a research paper) supplementmg the protocol. Reviews and case studies are also included to provide a deeper context to the methods chapters.

It was our intention and goal to bring together m Biopestzcides Use and Delavery the latest advances in the science and technology of the evolving field of biopesticides In the context of crop protectton, btopesttcides are a key component of integrated pest management (IPM) programs, m which biopesticides are delivered to crops m inundative quantities, vs the mocu- tive approach, which is charactertstic of classical biological control. Although there are several definitions of biopesttcides m the literature, we chose to define them as either microbials themselves or products derived from microbials, plants, and other biological entities. In the developed, industrial countries, primarily in Western Europe and the United States, biopesticides are receiving more practical attention, smce they are viewed as a means to reduce the load of synthetic chemical pes- tides m an effort to provide for safer foods and a cleaner envtronment.In the developing countries, biopestictdes are viewed as having the potential to - ploit nattve resources to produce crop protection agents that would replace imported chemical pesticides and conserve much-needed hard currencies These trends are well represented by the dynamic growth of engineered crops expressing the delta-endotoxm insecticidal protem crystals of Bacillus thuringzenszs (B. t ) m corn, cotton, and potatoes and the development of - combinant B. t.

A cutting-edge collection of detailed, step-by-step techniques and protocols for constructing, evaluating, and using affinity-based biosensors. Ideal for novices starting research in their field or experienced researchers wanting to use a biosensor for a specific analytical measurement, the methods detailed here allow biochemists, analytical chemists, microbiologists, and engineers to successfully apply biosensor technology to their specific problems. The techniques include the use of antibodies and membrane receptors to construct optical, thermal, acoustic, and electrochemically based biosensors. Additional techniques involve antibodies, receptors, nucleic acids, liposomes, and eukaryotic cells. A companion volume, Enzyme and Microbial Biosensors: Protocols and Techniques, by Mulchandani & Rogers, concentrates on enzyme-biosensors.

A mixture of two polymers, or one polymer and a salt, in an aqueous medium separates into two phases: this phenomenon is useful in biotechn- ogy for product separations. Separation of biological molecules and particles in these aqueous two-phase systems (ATPS) was initiated over 40 years ago by P.-Å. Albertsson, and later proved to be of immense utility in biochemical and cell biological research. A boost in the application of ATPS was seen when problems of separations in biotechnology processes were encountered. Its simplicity, biocompatibility, and amenability to easy scaleup operations make the use of ATPS very attractive for large-scale bioseparations. Despite the advantages ATPS enjoys over other separation techniques, the application of two-phase systems has for a long time been confined to selected labora- ries. Recent years have, however, shown a trend in which increasing numbers of researchers employ two-phase partitioning techniques in both basic and applied research.

We are in a phase of the evolution of biotechnology in which the true and potential commercial importance of carbohydrates is becoming appre- ated more fully. Progress in providing hard facts to establish the commercial value ofpolysaccharides and oligosaccharides is limited, as always, by lack of funding and by a relative shortage of skilled practitioners in the production and analysis of those materials. Carbohydrate science has a reputation, not unmerited, for technical difficulty owing to the structural similarity of the many monosaccharide monomers and the potential, and real, complexity of oligosaccharides and polysaccharides, particularly heterosaccharides conta- ing many different monomers. Modem analytical and synthetic methods, in many cases using enzyme technology, are beginning to allow this complexity to be unraveled. Carbohydrate Biotechnology Protocols is aimed at those newcomers who have an interest in the production and use of carbohydrate materials, but have shied away from involvement for lack of detailed descriptions of appropriate methods, including the type of practical hints that may be provided by those skilled in those methods, but that are rarely described in research papers. The majority of the contributions to this book conform to the established format of the Methods in Biotechnology series. They begin with the theoretical and c- mercial background to the method or group of methods, provide a list of the reagents and equipment required for the procedure, then give a detailed st- by-step description of how to carry out the protocol.

Considerable effort and time is allocated to introducing cell culture and fermentation technology to undergraduate students in academia, generally through a range of courses in industrial biotechnology and related disciplines. Similarly, a large number of textbooks are available to describe the appli- tions of these technologies in industry. However, there has been a general lack of appreciation of the significant developments in downstream processing and isolation technology, the need for which is largely driven by the stringent re- latory requirements for purity and quality of injectable biopharmaceuticals. This is particularly reflected by the general absence of coverage of this s- ject in many biotechnology and related courses in educational institutions. For a considerable while I have felt that there is increasing need for an introductory text to various aspects of downstream processing, particularly with respect to the needs of the biopharmaceutical and biotechnology ind- try. Although there are numerous texts that cover various aspects of protein purification techniques in isolation, there is a need for a work that covers the broad range of isolation technology in an industrial setting. It is anticipated that Downstream Processing of Proteins: Methods and Protocols will play a small part in filling this gap and thus prove a useful contribution to the field. It is also designed to encourage educational strategists to broaden the coverage of these topics in industrial biotechnology courses by including accounts of this important and rapidly developing element of the industrial process.

Env1r0nmenta1 ha5 m1cr0610109y emer9ed an extreme1y act1ve, - and 1mp0rtant area 0f re5earch dur1n9 the few year5 and ha5 cha1- 1en9ed 50me 10n9-he1d 0f F0r examp1e, the num6er5 0f c010ny-f0rm1n9 un1t5 06ta1ned 0n a9ar med1a have way5 6een a55umed t0 ref1ect the num6er5 0f 6acter1a pre5ent any 91ven 5amp1e. H0wever, n0w kn0wn that many 6acter1a the 1a60rat0ry ad0pt unu5ua1 d0rmant 5tate5 when 5u6jected t0 the nutr1ent-1- c0nd1t10n5 1ted c0mm0n many ec05y5tem5, wh1ch, turn, make5 the5e 6- ter1a t0 cu1ture. 7h15 0ur current t0 cu1ture the va5t maj0r1ty 0f6acter1a kn0wn t0 re51de natura1 env1r0nment5. Kn0- ed9e 0f the pre5ence 0f 50me 5pec1e5 a natura1 env1r0nment can 6e 0f cru- 1mp0rtance, w1th part1cu1ar1y re5pect t0 detect10n 0f path09en1c 0r t0 m0n1t0r1n9 the fate and 0f man1pu1ated 9enet1ca11y 0r9an15m5 w1th1n ec05y5tem5. 7heref0re, there ha5 6een much eff0rt 9enerated t0 dev- new and n0ve1 meth0d5 f0r detect10n, and rec0very 0f cr00r9an15m5 fr0m natura1 ha61tat5. Centra1 t0 many 0f the5e meth0d5 ha5 6een the deve10pment and ap- 0f the techn14ue5 0f 610109y m01ecu1ar t0 env1r0nmenta1 09y. 7he5e have re5u1ted d1rect 0fm1cr061a1 6a5ed 0n DNA w1th0ut the need f0r any cu1ture.7here n0 d0u6t that w0u1d n0t have 6een w1th0ut the rev01ut10n c0mputer techn01- 09y that ha5 t0 the deve10pment 0f data6a5e5 acce55161e h19h1y 0f va5t am0unt5 0f 1nf0rmat10n. M01ecu1ar meth0d5 have 6een harne55ed and exp101ted a150 the deve10pment 0f meth0d5 that ena61e rap1d and aut0mated 0f m1cr00r9an15m5 fr0m natura1 env1r0nment5.

Experienced practitioners present detailed accounts of a wide variety of techniques using supercritical fluids. These range from the supercritical fluid extraction methods for numerous compounds to the ninhydrin staining of fingerprints on checks and banknotes - and from the detection of impurities in pharmaceuticals to a wide variety of applications throughout environmental and food science, and across analytical, clinical, and medicinal chemistry. Detailed step-by-step instructions enable users to apply these essential techniques successfully the first time and include modifications that permit their effective adaptation to novel experimental or process conditions. Versatile and comprehensive, Supercritical Fluid Methods and Protocols offers both novice and experienced investigators and laboratory analysts powerful tools that enable successful biological and bioprocess analyses and optimization today.

Two of the recent books in the Methods in Molecular Biology series, Yeast Protocols and Pichia Protocols, have been narrowly focused on yeasts and, in the latter case, particular species of yeasts. Food Microbiology Pro- cols, of necessity, covers a very wide range of microorganisms. Our book treats four categories of microorganisms affecting foods: (1) Spoilage organisms; (2) pathogens; (3) microorganisms in fermented foods; and (4) microorganisms p- ducing metabolites that affect the flavor or nutritive value of foods. Detailed information is given on each of these categories. There are several chapters devoted to the microorganisms associated with fermented foods: these are of increasing importance in food microbiology, and include one bacteriophage that kills the lactic acid bacteria involved in the manufacture of different foods—cottage cheese, yogurt, sauerkraut, and many others. The other nine chapters give procedures for the maintenance of lactic acid bacteria, the isolation of plasmid and genomic DNA from species of Lac- bacillus, determination of the proteolytic activity of lactic acid bacteria, det- mination of bacteriocins, and other important topics.

Enzymatic catalysis has gained considerable attention in recent years as an efficient tool in the preparation of natural products, pharmaceuticals, fine chemicals, and food ingredients. The high selectivity and mild reaction con- tions associated with enzymatic transformations have made this approach an attractive alternative in the synthesis of complex bioactive compounds, which are often difficult to obtain by standard chemical routes. However, the maj- ity of organic compounds are not very soluble in water, which was traditi- ally perceived as the only suitable reaction medium for the application of biocatalysts. The realization that most enzymes can function perfectly well under nearly anhydrous conditions and, in addition, display a number of useful properties, e. g. , highly enhanced stability and different selectivity, has d- matically widened the scope of their application to the organic synthesis. Another great attraction of using organic solvents rather than water as a reaction solvent is the ability to perform synthetic transformations with re- tively inexpensive hydrolytic enzymes. It is worth reminding the reader that in vivo, the synthetic and hydrolytic pathways are catalyzed by different enzymes. However, elimination of water from the reaction mixture enables the “reversal” of hydrolytic enzymes and thus avoids the use of the expensive cofactors or activated substrates that are required for their synthetic count- parts.

The methods included in Environmental Microbiology: Methods and Pro- cols can be placed in the categories “Communities and Biofilms,” “Fermented Milks,” “Recovery and Determination of Nucleic Acids,” and the review s- tion, containing chapters on the endophytic bacterium, Bacillus mojavensis, the engineering of bacteria to enhance their ability to carry out bioremediation of aromatic compounds, using the hemoglobin gene from a strain of Vitreoscilla 23 spp., and the use of chemical shift reagents and Na NMR to study sodium gradients in microorganisms, all of which should be of interest to investigators in these fields. The subjects treated within the different categories also cover a wide range, with methods ranging from those for the study of marine organisms, through those for the investigation of microorganisms occurring in ground waters, including subsurface ground waters, to other types of environmental waters, to as varied subjects as the biodiversity of yeasts found in northwest Argentina. The range of topics described in the Fermented Milks section is smaller, but significant for investigators in areas concerned with milk as an item of foods for infants, small children, and even adults.

Leading experts in enzyme manipulation describe in detail their cutting-edge techniques for the screening, evolution, production, immobilization, and application of enzymes. These readily reproducible methods can be used to improve enzyme function by directed evolution, to covalently immobilize enzymes, to microencapsulate enzymes and cells, and to manufacture enzymes for human health, nutrition, and environmental protection. Overview chapters on microorganisms as a source of metabolic and enzymatic diversity, and on the fast-moving field of enzyme biosensors are presented. Microbial Enzymes and Biotransformations offers laboratory and industrial scientists a wealth of proven enzymatic protocols that show clearly how to go from laboratory results to successful industrial applications.

Enzymes and whole cells are able to catalyze the most complex chemical processes under the most benign experimental and environmental conditions. In this way, enzymes and cells could be excellent catalysts for a much more sustainable chemical industry. However, enzymes and cells also have some limitations for nonbiological applications: fine chemistry, food chemistry, analysis, therapeutics, and so on. Enzymes and cells may be unstable, difficult to handle under nonconventional conditions, poorly selective toward synthetic substrates, and so forth. From this point of view, the transformation—from the laboratory to industry—of chemical processes catalyzed by enzymes and cells may be one of the most complex and exciting goals in biotechnology. For many industrial applications, enzymes and cells have to be immobilized, via very simple and cost-effective protocols, in order to be re-used over very long periods of time. From this point of view, immobilization, simplicity, and stabilization have to be strongly related concepts. Over the last 30 years, a number of protocols for the immobilization of cells and enzymes have been reported in scientific literature. However, only very few protocols are simple and useful enough to greatly improve the functional properties of enzymes and cells, activity, stability, selectivity, and related properties.

A comprehensive collection of robust methods for the detection of pesticide compounds or their metabolites useful in food, environmental, and biological monitoring, and in studies of exposure via food, water, air, and the skin or lungs. The readily reproducible methods range from gas and liquid chromatography coupled to mass spectrometry detection and other classic detectors, to capillary electrophoresis and immunochemical or radioimmunoassay methods. The authors have focused on extraction and cleanup procedures, in order to develop and optimize more fullyautomated and miniaturized methods, including solid-phase extraction, solid-phase microextraction, microwave-assisted extraction, and on-line tandem liquid chromatography (LC/LC) trace enrichment, among others. The protocols offer step-by-step laboratory instructions, an introduction outlining the principles behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.

A collection of readily reproducible classic and emerging molecular methods for the laboratory isolation and identification of the pathogens, viruses, and parasites that cause food-borne disease. Among the pathogens covered are specific bacteria, including Salmonella spp, Campylobacter spp., Listeria spp., and Bacillus spp.; viruses, including noroviruses and enteroviruses; and parasites, including Cryptosporidium and seafood nematode worms. The protocols follow the successful Methods in Biotechnology™ series format, each offering step-by-step laboratory instructions, an introduction outlining the principles behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.

Phytoremediation: Methods and Reviews presents the most innovative recent methodological developments in phytoremediation research, and outlines a variety of the contexts in which phytoremediation has begun to be applied. A significant portion of this volume is devoted to groundbreaking methods for the production of plants that are able to degrade, take up, or tolerate the effects of pollutants. Phytoremediation: Methods and Reviews adopts a multidisciplinary approach to the examination of principles and practices of phytoremediation, from molecular manipulation to field application.Parts I and II discuss detailed protocols for achieving several different goals of phytoremediation, including enhancing contaminant degradation, uptake, and tolerance by plants; exploiting plant diversity for phytoremedation; modifying contaminant availability; and experimentally analyzing phytoremediation potential. Parts III and IV examine a variety of progressive techniques for phytoremediation and explore their implementation and success on a global scale. This cutting-edge volume highlights the myriad of contexts in which phytoremediation can be applied, and energizes new research by describing ways in which barriers to success have been recently overcome.

The development of biotechnology over the last 20 years, and particularly the use of recombinant DNA techniques, has rapidly expanded the opportu- ties for human benefits from living resources. Efforts to reduce pollution, p- vent environmental damage, combat microbial infection, improve food production, and so on can each involve fermentation or the environmental - lease of microorganisms. Many products of fermentation technology, such as alcoholic beverages, bread, antibiotics, amino acids, vitamins, enzymes, and others, have been influenced by the progress of recombinant DNA techniques. The development of new products or the more efficient manufacturing of those already being produced often involve the use of microorganisms as cell fac- ries for many productions and biotransformations. Microbial Processes and Products is intended to provide practical expe- mental laboratory procedures for a wide range of processes and products me- ated by microorganisms. Although not an exhaustive treatise, it provides a detailed “step-by-step” description of the most recent developments in such applied biotechnological processes. The detailed protocols we provide are cross-referenced in the Notes section, contain critical details, lists of problems and their troubleshooting, as well as safety recommendations that may not n- mally appear in journal articles and can be particularly useful for those un- miliar with specific techniques.

Animal Cell Biotechnology: Methods and Protocols, Second Edition constitutes a comprehensive manual of state-of-the-art and new techniques for setting up mammalian cell lines for production of biopharmaceuticals, and for optimizing critical parameters for cell culture considering the whole cascade from lab to final production. The volume is divided into five parts that reflect the processes required for different stages of production. In Part I, basic techniques for establishment of production cell lines are addressed, especially transduction techniques, cells for gene therapy and antibody production. Part II addresses basic cultivation techniques, such as microcarrier culture and encapsulation.Part III covers cell characterization and analysis, including flow cytometric applications, NMR-based techniques, and biochemical and cytometric techniques. Part IV details cultivation techniques, such as disposable bioreactors, hollow fiber cell culture, fixed bed reactors, and configuration of bioreactors. Part V covers downstream techniques such as membrane filtration techniques, while Part VI describes special applications, including retroviral vectors.Animal Cell Biotechnology: Methods and Protocols, Second Edition provides a compendium of techniques for scientists in industrial and research laboratories that use mammalian cells for biotechnology purposes.

Recombinant Proteins from Plants is one of the most exciting and fastest developing areas in biology. The latest molecular techniques are being applied to the exploitation of plants as novel expression systems for the p- duction and overproduction of heterologous and native proteins. Transgenic plant technology is currently used in three broad areas: the expression of - combinant proteins to improve crop quality by increasing disease/pest res- tance or increasing tolerance to stress, optimizing plant productivity and yield by the genetic manipulation of metabolic pathways, and the large-scale co- effective production of recombinant proteins for use as specialist industrial or therapeutic biomolecules. The intention of Recombinant Proteins from Plants is to provide c- prehensive and detailed protocols covering all the latest molecular approaches. Because the production oftransgenic plants has become routine in many la- ratories, coverage is also given to some of the more "classical" approaches to the separation, analysis, and characterization of recombinant proteins. The book also includes areas of research that we believe will become increasingly important in the near future: efficient transformation of monocots with Agrobacterium optimizing the stability of recombinant proteins, and a section highlighting the immunotherapeutic potential of plant-expressed proteins.

In 1962 Clark and Lyons pioneered the concept of a biosensor. They p- posed immobilizing enzymes at electrochemical detectors to form “enzyme el- trodes” in order to expand the analyte range of ther base sensor. Smce then, the field of blosensors has greatly expanded. Some of the reasons for the expansion include both advances in signal transduction technologies and the incorporation of different biological sensing elements (Table 1). As a consequence, there are now a bewildering array of permutations of the biological sensing element and signal transducers that can be used to c- struct a biosensor. The purpose of the two volumes of Protocols and Te- niques in Biosensors is to provide a basic reference tool and starting point for use by graduate students, postdoctoral and senior researchers, and technicians m academics, industry, and government research establishments, to enable rapid entry into the field of biosensors. There are a variety of approaches that researchers employ to select a combination of bioaffinity elements and signal transducers. One commonly used approach is to identtfy the compound or compounds of interest; identify the biological molecule that yields an appropriate recognitionlselectivtty and dynamic concentration range for the assay; and choose an assay format and signal transduction technology that will meet the analytical requirements for the proposed application, This volume, Enzyme and Microbial Biosensors: Techniques and Protocols, describes a variety of transduction technologies that have been interfaced to enzymes and microorganisms.

The many thousands of human-made and other chemical compounds present in the environment offer a serious challenge to our btosphere. It is appropriate, therefore, that our response to these products of human kno- edge and ingenuity should draw on a body of mtenstve scientific endeavor that is no less impressive. Bioremediation offers the possibility of harnessing the diversity of the biosphere to degrade, remove, alter, or otherwise detoxify these various chemicals. It brings together scientists from a wide variety of disciplines and backgrounds, such as microbiology, molecular biology, a- lytical chemistry, and chemical and environmental engineering, among o- ers. These different fields, each with its own individual approach, have actively contributed to the development of bioremediation research tn recent years. The prmcipal objective of Bioremediation Protocols is to make the fruits of some of this research available in a different format to that of the textbook or journal article. It provides a selection of clearly written laboratory pro- cols presented as stepwise, easy-to-follow mstructions. In common with p- wous volumes in this and the companion Methods in Molecular Biology series, an extensive “Notes” section is provided with each chapter. This contains u- ful mformation (of a type often not normally included m a research paper) supplementmg the protocol. Reviews and case studies are also included to provide a deeper context to the methods chapters.

It was our intention and goal to bring together m Biopestzcides Use and Delavery the latest advances in the science and technology of the evolving field of biopesticides In the context of crop protectton, btopesttcides are a key component of integrated pest management (IPM) programs, m which biopesticides are delivered to crops m inundative quantities, vs the mocu- tive approach, which is charactertstic of classical biological control. Although there are several definitions of biopesttcides m the literature, we chose to define them as either microbials themselves or products derived from microbials, plants, and other biological entities. In the developed, industrial countries, primarily in Western Europe and the United States, biopesticides are receiving more practical attention, smce they are viewed as a means to reduce the load of synthetic chemical pes- tides m an effort to provide for safer foods and a cleaner envtronment.In the developing countries, biopestictdes are viewed as having the potential to - ploit nattve resources to produce crop protection agents that would replace imported chemical pesticides and conserve much-needed hard currencies These trends are well represented by the dynamic growth of engineered crops expressing the delta-endotoxm insecticidal protem crystals of Bacillus thuringzenszs (B. t ) m corn, cotton, and potatoes and the development of - combinant B. t.

The frequency of reports concemmg the interface of biological reco- tion elements to signal transduction technologies has risen dramatically over the last decade. Because any one of a wide variety of biological recognition elements (e. g. , antibodies, receptors, DNA, microorganisms, or enzymes) can theoretically be interfaced with any one of a wide variety of signal transducers (e. g. , optical, electrochemical, thermal, or acoustic), the potential range of devices and techniques can be bewildering. The purpose of this volume and the previous volume in this series is to provide a basic reference and startmg point for investigators in academics, mdustry, and government to begin or expand their biosensors research. This volume, Methods in Biotechnology vol. 7: Affinity Biosensors: Techniques and Protocols, describes a variety of classical and emerging transduction technologies that have been interfaced to bioaffinity elements (e. g. , antibodies and receptors). Some of the reasons for the expansion in the use of affinity-based biosensors include both advances in signal transduction technologies (e. g. , fiber optics, microelectromcs, and microfabrication) and the availability of bioafflmty elements. More specifically, with respect to biological recognttion elements, commercially and noncommercially produced antibodies directed toward a variety of analytes have become widely available. In addition, te- niques for the purification and stabilization of receptors have also significantly improved. As a result of these recent advances in the field, biosensors research and development projects are being pursued by mvestigators from a wide range of disciplines.

A mixture of two polymers, or one polymer and a salt, in an aqueous medium separates into two phases: this phenomenon is useful in biotechn- ogy for product separations. Separation of biological molecules and particles in these aqueous two-phase systems (ATPS) was initiated over 40 years ago by P.-Å. Albertsson, and later proved to be of immense utility in biochemical and cell biological research. A boost in the application of ATPS was seen when problems of separations in biotechnology processes were encountered. Its simplicity, biocompatibility, and amenability to easy scaleup operations make the use of ATPS very attractive for large-scale bioseparations. Despite the advantages ATPS enjoys over other separation techniques, the application of two-phase systems has for a long time been confined to selected labora- ries. Recent years have, however, shown a trend in which increasing numbers of researchers employ two-phase partitioning techniques in both basic and applied research.

We are in a phase of the evolution of biotechnology in which the true and potential commercial importance of carbohydrates is becoming appre- ated more fully. Progress in providing hard facts to establish the commercial value ofpolysaccharides and oligosaccharides is limited, as always, by lack of funding and by a relative shortage of skilled practitioners in the production and analysis of those materials. Carbohydrate science has a reputation, not unmerited, for technical difficulty owing to the structural similarity of the many monosaccharide monomers and the potential, and real, complexity of oligosaccharides and polysaccharides, particularly heterosaccharides conta- ing many different monomers. Modem analytical and synthetic methods, in many cases using enzyme technology, are beginning to allow this complexity to be unraveled. Carbohydrate Biotechnology Protocols is aimed at those newcomers who have an interest in the production and use of carbohydrate materials, but have shied away from involvement for lack of detailed descriptions of appropriate methods, including the type of practical hints that may be provided by those skilled in those methods, but that are rarely described in research papers. The majority of the contributions to this book conform to the established format of the Methods in Biotechnology series. They begin with the theoretical and c- mercial background to the method or group of methods, provide a list of the reagents and equipment required for the procedure, then give a detailed st- by-step description of how to carry out the protocol.

Considerable effort and time is allocated to introducing cell culture and fermentation technology to undergraduate students in academia, generally through a range of courses in industrial biotechnology and related disciplines. Similarly, a large number of textbooks are available to describe the appli- tions of these technologies in industry. However, there has been a general lack of appreciation of the significant developments in downstream processing and isolation technology, the need for which is largely driven by the stringent re- latory requirements for purity and quality of injectable biopharmaceuticals. This is particularly reflected by the general absence of coverage of this s- ject in many biotechnology and related courses in educational institutions. For a considerable while I have felt that there is increasing need for an introductory text to various aspects of downstream processing, particularly with respect to the needs of the biopharmaceutical and biotechnology ind- try. Although there are numerous texts that cover various aspects of protein purification techniques in isolation, there is a need for a work that covers the broad range of isolation technology in an industrial setting. It is anticipated that Downstream Processing of Proteins: Methods and Protocols will play a small part in filling this gap and thus prove a useful contribution to the field. It is also designed to encourage educational strategists to broaden the coverage of these topics in industrial biotechnology courses by including accounts of this important and rapidly developing element of the industrial process.

Env1r0nmenta1 ha5 m1cr0610109y emer9ed an extreme1y act1ve, - and 1mp0rtant area 0f re5earch dur1n9 the few year5 and ha5 cha1- 1en9ed 50me 10n9-he1d 0f F0r examp1e, the num6er5 0f c010ny-f0rm1n9 un1t5 06ta1ned 0n a9ar med1a have way5 6een a55umed t0 ref1ect the num6er5 0f 6acter1a pre5ent any 91ven 5amp1e. H0wever, n0w kn0wn that many 6acter1a the 1a60rat0ry ad0pt unu5ua1 d0rmant 5tate5 when 5u6jected t0 the nutr1ent-1- c0nd1t10n5 1ted c0mm0n many ec05y5tem5, wh1ch, turn, make5 the5e 6- ter1a t0 cu1ture. 7h15 0ur current t0 cu1ture the va5t maj0r1ty 0f6acter1a kn0wn t0 re51de natura1 env1r0nment5. Kn0- ed9e 0f the pre5ence 0f 50me 5pec1e5 a natura1 env1r0nment can 6e 0f cru- 1mp0rtance, w1th part1cu1ar1y re5pect t0 detect10n 0f path09en1c 0r t0 m0n1t0r1n9 the fate and 0f man1pu1ated 9enet1ca11y 0r9an15m5 w1th1n ec05y5tem5. 7heref0re, there ha5 6een much eff0rt 9enerated t0 dev- new and n0ve1 meth0d5 f0r detect10n, and rec0very 0f cr00r9an15m5 fr0m natura1 ha61tat5. Centra1 t0 many 0f the5e meth0d5 ha5 6een the deve10pment and ap- 0f the techn14ue5 0f 610109y m01ecu1ar t0 env1r0nmenta1 09y. 7he5e have re5u1ted d1rect 0fm1cr061a1 6a5ed 0n DNA w1th0ut the need f0r any cu1ture.7here n0 d0u6t that w0u1d n0t have 6een w1th0ut the rev01ut10n c0mputer techn01- 09y that ha5 t0 the deve10pment 0f data6a5e5 acce55161e h19h1y 0f va5t am0unt5 0f 1nf0rmat10n. M01ecu1ar meth0d5 have 6een harne55ed and exp101ted a150 the deve10pment 0f meth0d5 that ena61e rap1d and aut0mated 0f m1cr00r9an15m5 fr0m natura1 env1r0nment5.