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Applications of DNA-Stable Isotope Probing in Bioremediation StudiesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
DNA-stable isotope probing, a method to identify active microorganisms without the prerequisite of cultivation, has been widely applied in the study of microorganisms involved in the degradation of environmental pollutants. Recent advances and technique considerations in applying DNA-SIP in bioremediation are highlighted. A detailed protocol of a DNA-SIP experiment is provided. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - October 23, 2009 Category: Biotechnology Source Type: info

Ecophysiological Analysis of Microorganisms in Complex Microbial Systems by Combination of Fluorescence In Situ Hybridization with Extracellular Staining TechniquesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Ecophysiological analysis and functions of single cells in complex microbial systems can be examined by simple combinations of Fluorescence in situ hybridization (FISH) for identification with various staining techniques targeting functional phenotypes. In this chapter, we describe methods and protocols optimized for the study of extracellular enzymes, surface hydrophobicity and specific surface structures. Although primarily applied to the study of microbes in wastewater treatment (activated sludge and biofilms), the methods may also be used with minor modifications in several other ecosystems. (Source: Springer protocols...
Source: Springer protocols feed by Biotechnology - October 23, 2009 Category: Biotechnology Source Type: info

Combination of Fluorescence In Situ Hybridization with Staining Techniques for Cell Viability and Accumulation of PHA and polyP in Microorganisms in Complex Microbial SystemsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Fluorescence in situ hybridization (FISH) can be combined with a number of staining techniques to reveal the relationships between the microorganisms and their function in complex microbial systems with a single-cell resolution. In this chapter, we have focused on staining methods for intracellular storage compounds (polyhydroxyalkanoates, polyphosphate) and a measure for cell viability, reduction of the tetrazolium-based redox stain CTC. These protocols are optimized for the study of microorganisms in waste-water treatment (activated sludge and biofilms), but they may also be used with minor modifications in many other ec...
Source: Springer protocols feed by Biotechnology - October 23, 2009 Category: Biotechnology Source Type: info

Using Terminal Restriction Fragment Length Polymorphism (T-RFLP) Analysis to Assess Microbial Community Structure in Compost SystemsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in composting systems. This analysis is based on the restriction endonuclease digestion of fluorescently end-labeled PCR products. The digested product is mixed with a DNA size standard, itself labeled with a distinct fluorescent dye, and the fragments are then separated by capillary or gel electrophoresis using an automated sequencer. Upon analysis, only the terminal end-labeled restriction fragments are detected. An electropherogram is produced, which shows a profile of compost microbial co...
Source: Springer protocols feed by Biotechnology - October 23, 2009 Category: Biotechnology Source Type: info

Application of Fingerprinting Molecular Methods in Bioremediation StudiesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Bioremediation has been identified as a beneficial and effective strategy for the removal of recalcitrant environmental contaminants. Bioaugmentation of polluted environments with exogenous degrading microorganisms constitutes a major strategy of bioremediation. However, the ecological role of these strains and their impact on the endogenous microbial community of the micro-ecosystems where they are released should be known. Fingerprinting PCR-based methods, like denaturating gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (TRFLP), could be used in studies exploring the ecology of ...
Source: Springer protocols feed by Biotechnology - October 23, 2009 Category: Biotechnology Source Type: info

MPN- and Real-Time-Based PCR Methods for the Quantification of Alkane Monooxygenase Homologous Genes (alkB) in Environmental SamplesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Hydrocarbons are major contaminants of soil ecosystems as a result of uncontrolled oil spills and wastes disposal into the environment. Ecological risk assessment and remediation of affected sites is often constrained due to lack of suitable prognostic and diagnostic tools that provide information of abiotic–biotic interactions occurring between contaminants and biological targets. Therefore, the identification and quantification of genes involved in the degradation of hydrocarbons may play a crucial role for evaluating the natural attenuation potential of contaminated sites and the development of successful bioremed...
Source: Springer protocols feed by Biotechnology - October 23, 2009 Category: Biotechnology Source Type: info

Addressing PCR Biases in Environmental Microbiology StudiesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Each step of a molecular environmental microbiology study is prone to errors, though the qualitative and quantitative biases of PCR amplification could result in the most serious biases. One has to be aware of this fact, and well-characterized PCR biases have to be avoided by using target-optimized PCR protocols. The most important tasks are primer and thermal profile optimization. We have shown that primer mismatches, even in the case of universal primers, can cause almost complete missing of common taxa from clone libraries, for example. Similarly high annealing temperatures can drastically distort community composition ...
Source: Springer protocols feed by Biotechnology - October 23, 2009 Category: Biotechnology Source Type: info

Heavy Metal Bioavailability and Bioaccessibility in SoilEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This chapter considers the use of a variety of approaches to assess either the bioavailability or the bioaccessibility of metals in soil. The bioavailability of metals from soils is considered with respect to a series of single-extraction methods, including the use of ethylenediaminetetraacetic acid (EDTA), acetic acid, diethylenetriaminepentaacetic acid (DTPA), ammonium nitrate, calcium chloride and sodium nitrate. Then, a procedure for the recovery of metals using a three-stage sequential extraction protocol is described. Two alternate approaches for assessing the environmental health risk to humans by undertaking in vit...
Source: Springer protocols feed by Biotechnology - October 23, 2009 Category: Biotechnology Source Type: info

Measurement of Bioaccessibility of Organic Pollutants in SoilEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present a rapid and easily reproducible HPCD shake extraction technique that has been experimentally demonstrated to directly predict microbial availability and degradation in soil. This method can provide practitioners with both an indication of bioremediation end-points and may be valuable in the risk assessment of contaminated land. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - October 23, 2009 Category: Biotechnology Source Type: info

Community-Level Physiological ProfilingEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Community-level physiological profiling (CLPP) is a technique which offers an easily applied protocol yielding information regarding mixed microbial community function and functional adaptations over space and time. Different communities can be compared and classified based on sole carbon source utilization patterns (CSUPs) gathered using BIOLOG™ microplates. One of the most challenging aspects associated with the CLPP method is in the data analysis. This chapter describes the relatively simple CLPP laboratory protocol and provides a detailed description of different data analysis techniques. (Source: Springer protoc...
Source: Springer protocols feed by Biotechnology - October 23, 2009 Category: Biotechnology Source Type: info

Application of Bioassays for the Ecotoxicity Assessment of Contaminated SoilsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The use of bioassays for soil characterization is receiving significant attention as a complementary tool to chemical analysis. Bioassays consist of direct toxicity assays of environmental samples that are transferred to the laboratory and analyzed for toxicity against selected organisms. Such soil samples contain the combination of the different pollutants present in situ and enable factors such as the bioavailability of contaminants or the interactions (synergic and antagonic) between them to be simultaneously studied. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - October 23, 2009 Category: Biotechnology Source Type: info

Monitored Natural AttenuationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Monitored natural attenuation (MNA) is an in situ remediation technology that relies on naturally occurring and demonstrable processes in soil and groundwater which reduce the mass and concentration of the contaminants. Natural attenuation (NA) involves both aerobic and anaerobic degradation of the contaminants due to the fact that oxygen is used up near the core of the contaminant plume. The aerobic and anaerobic microbial processes can be assessed by microbial activity measurements and molecular biology methods in combination with chemical analyses. The sampling and knowledge on the site conditions are of major importanc...
Source: Springer protocols feed by Biotechnology - October 23, 2009 Category: Biotechnology Source Type: info

The Role of Decision Support for Bioremediation Strategies, Exemplified by Hydrocarbons for In Site and Ex Situ ProceduresEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Despite the widespread availability of state-of-the-art biological techniques, remediation practitioners have been slow to adopt these technologies to assist in designing or indeed monitoring remediation strategies. In part, this is because practitioners are driven by cost and fail to see the benefit of emerging technologies, and in part because most companies have only a small portfolio of procedures available to them. Here, we review the component parts required to design a decision support tool, appraise one that the authors have developed and critically evaluate its application to case studies. If bioremediation is to ...
Source: Springer protocols feed by Biotechnology - October 23, 2009 Category: Biotechnology Source Type: info

Plasmid-Mediated Bioaugmentation of Wastewater Microbial Communities in a Laboratory-Scale BioreactorEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Xenobiotic degradation during biological wastewater treatment can be established or enhanced by bioaugmentation − the addition of biological agents carrying biodegradation genes required to perform the task. Whereas the addition of microbial cells carrying chromosomally encoded catabolic genes can be impaired by limited survival of the added microorganisms, the addition of donor organisms carrying a transmissible catabolic plasmid is a promising alternative. This plasmid can spread within the indigenous microbial community of the system, circumventing the need for extended survival of the introduced bacterial strain....
Source: Springer protocols feed by Biotechnology - October 23, 2009 Category: Biotechnology Source Type: info

The Application of Molecular Techniques to the Study of Wastewater Treatment SystemsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Wastewater treatment systems tend to be engineered to select for a few functional microbial groups that may be organized in various spatial structures such as activated sludge flocs, biofilm or granules and represented by single coherent phylogenic groups such as ammonia-oxidizing bacteria (AOB) and polyphosphate-accumulating organisms (PAO). In order to monitor and control engineered microbial structure in wastewater treatment systems, it is necessary to understand the relationships between the microbial community structure and the process performance. This review focuses on bacterial communities in wastewater treatment p...
Source: Springer protocols feed by Biotechnology - October 23, 2009 Category: Biotechnology Source Type: info

Two-Dimensional Gel Electrophoresis: Discovering Biomolecules for Environmental BioremediationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Environmental contamination has been viewed as an ecological malaise for which bioremediation can be prescribed as a “perfect medicine.” The solution to the problems with bioremediation lies in analyzing to what extent the microbes’ physiological machinery contributes to the degradation process and which biomolecules and their mechanisms are responsible for regulatory factors within the degradation system, such as protein, metabolite, and enzymatic chemical transformation. In the post-genomic era, recent advances in proteomics have allowed us to elucidate many complex biological mechanisms. Two-dimensiona...
Source: Springer protocols feed by Biotechnology - October 23, 2009 Category: Biotechnology Source Type: info

Lime PretreatmentEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Lime pretreatment has proven to be a useful method for selectively reducing the lignin content of lignocellulosic biomass without significant loss in carbohydrates, thus realizing an important increase in biodigestibility. In lime pretreatment, the biomass is pretreated with calcium hydroxide and water under different conditions of temperature and pressure. It can be accomplished in one of three fashions: (1) short-term pretreatment that lasts up to 6 h, requires temperatures of 100–160°C, and can be applied with or without oxygen (pressure ~200 psig); (2) long-term pretreatment taking up to 8 weeks, requiring on...
Source: Springer protocols feed by Biotechnology - September 23, 2009 Category: Biotechnology Source Type: info

Dilute Acid and Autohydrolysis PretreatmentEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Exposure of cellulosic biomass to temperatures of about 120–210°C can remove most of the hemicellulose and produce cellulose-rich solids from which high glucose yields are possible with cellulase enzymes. Furthermore, the use of dilute sulfuric acid in this pretreatment operation can increase recovery of hemicellulose sugars substantially to about 85–95% of the maximum possible versus only about 65% if no acid is employed. The use of small-diameter tubes makes it possible to employ high solids concentrations similar to those preferred for commercial operations, with rapid heat-up, good temperature control, ...
Source: Springer protocols feed by Biotechnology - September 23, 2009 Category: Biotechnology Source Type: info

Liquid Hot Water Pretreatment of Cellulosic BiomassEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Lignocellulosic biomass is an abundant and renewable resource for fuel ethanol production. However, the lignocellulose is recalcitrant to enzymatic hydrolysis because of its structural complexity. Controlled-pH liquid hot water (LHW) pretreatment of cellulosic feedstock improves its enzymatic digestibility by removing hemicellulose and making the cellulose more accessible to cellulase enzymes. The removed hemicellulose is solubilized in the liquid phase of the pretreated feedstock as oligosaccharides. Formation of monomeric sugars during the LHW pretreatment is minimal. The LHW pretreatment is carried out by cooking the fe...
Source: Springer protocols feed by Biotechnology - September 23, 2009 Category: Biotechnology Source Type: info

Pretreatment of Biomass by Aqueous Ammonia for Bioethanol ProductionEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The methods of pretreatment of lignocellulosic biomass using aqueous ammonia are described. The main effect of ammonia treatment of biomass is delignification without significantly affecting the carbohydrate contents. It is a very effective pretreatment method especially for substrates that have low lignin contents such as agricultural residues and herbaceous feedstock. The ammonia-based pretreatment is well suited for simultaneous saccharification and co-fermentation (SSCF) because the treated biomass retains cellulose as well as hemicellulose. It has been demonstrated that overall ethanol yield above 75% of the theoretic...
Source: Springer protocols feed by Biotechnology - September 23, 2009 Category: Biotechnology Source Type: info

Lignocellulosic Biomass Pretreatment Using AFEXEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Although cellulose is the most abundant organic molecule, its susceptibility to hydrolysis is restricted due to the rigid lignin and hemicellulose protection surrounding the cellulose micro fibrils. Therefore, an effective pretreatment is necessary to liberate the cellulose from the lignin–hemicellulose seal and also reduce cellulosic crystallinity. Some of the available pretreatment techniques include acid hydrolysis, steam explosion, ammonia fiber expansion (AFEX), alkaline wet oxidation, and hot water pretreatment. Besides reducing lignocellulosic recalcitrance, an ideal pretreatment must also minimize formation o...
Source: Springer protocols feed by Biotechnology - September 23, 2009 Category: Biotechnology Source Type: info

Genetic Transformation of SwitchgrassEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Switchgrass (Panicum virgatum L.) is a highly productive warm-season C4 species that is being developed into a dedicated biofuel crop. This chapter describes a protocol that allows the generation of transgenic switchgrass plants by Agrobacterium tumefaciens-mediated transformation. Embryogenic calluses induced from caryopses or inflorescences were used as explants for inoculation with A. tumefaciens strain EHA105. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Calluses resistant to hygromycin were obtained after 5–6 weeks of selection. Soil-g...
Source: Springer protocols feed by Biotechnology - September 23, 2009 Category: Biotechnology Source Type: info

Agronomic Experiences with Miscanthus x giganteus in Illinois, USAEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Since 2002, researchers at the University of Illinois, Urbana-Champaign, Illinois, have been studying the perennial warm-season grass Miscanthus × giganteus (M. × g.) to determine its potential as a biomass feedstock. M. × g. originated in Japan and is a hybrid believed to have M. sinensis and M. sacchariflorus as its parents. Until recently, it was used as a landscape plant in the United States, but it is now the subject of research interest because of its potentially great biomass production. In central Illinois, M. × g. begins growth in April, typically reaches 2 m by the end of May, and is norma...
Source: Springer protocols feed by Biotechnology - September 23, 2009 Category: Biotechnology Source Type: info

Selecting, Establishing, and Managing Switchgrass (Panicum virgatum) for BiofuelsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Switchgrass is being widely considered as a feedstock for biofuel production. Much remains to be learned about ideal feedstock characteristics, but switchgrass offers many advantages already and can perhaps be manipulated to offer more. When planning to grow switchgrass, select a cultivar that is well adapted to the location – generally a lowland cultivar for the southern United States and an upland cultivar at higher latitudes. Plant non-dormant seed after soils are well warmed, preferably with no-till methods and always with good weed control. Except for weeds, few pests appear to be widespread; but disease and ins...
Source: Springer protocols feed by Biotechnology - September 23, 2009 Category: Biotechnology Source Type: info

Biomass Supply Logistics and InfrastructureEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Feedstock supply system encompasses numerous unit operations necessary to move lignocellulosic feedstock from the place where it is produced (in the field or on the stump) to the start of the conversion process (reactor throat) of the biorefinery. These unit operations, which include collection, storage, preprocessing, handling, and transportation, represent one of the largest technical and logistics challenges to the emerging lignocellulosic biorefining industry. This chapter briefly reviews the methods of estimating the quantities of biomass, followed by harvesting and collection processes based on current practices on h...
Source: Springer protocols feed by Biotechnology - September 23, 2009 Category: Biotechnology Source Type: info

Biodiesel: Small Scale Production and Quality RequirementsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Biodiesel is produced by reacting vegetable oils or animal fats with alcohol in the presence of an alkaline catalyst. The resulting methyl esters, which are the biodiesel fuel, are separated from the by-product glycerin, and then washed with water and dehydrated to produce fuel that must meet standardized specifications. Degraded oils containing high levels of free fatty acids can also be converted to biodiesel, but pretreatment with acid-catalyzed esterification is required. The resulting fuel is suitable for use as a neat fuel in diesel engines or blended with conventional diesel fuel. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - September 23, 2009 Category: Biotechnology Source Type: info

Simultaneous Saccharification and Fermentation and Partial Saccharification and Co-Fermentation of Lignocellulosic Biomass for Ethanol ProductionEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Ethanol production by fermentation of lignocellulosic biomass-derived sugars involves a fairly ancient art and an ever-evolving science. Production of ethanol from lignocellulosic biomass is not avant-garde, and wood ethanol plants have been in existence since at least 1915. Most current ethanol production relies on starch- and sugar-based crops as the substrate; however, limitations of these materials and competing value for human and animal feeds is renewing interest in lignocellulose conversion. Herein, we describe methods for both simultaneous saccharification and fermentation (SSF) and a similar but separate process f...
Source: Springer protocols feed by Biotechnology - September 23, 2009 Category: Biotechnology Source Type: info

Basic Laboratory Culture Methods for Anaerobic BacteriaEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Oxygen is either limiting or absent in many ecosystems. Anaerobic bacteria are often key players in such environments and these organisms have important roles in geo-elemental cycling, agriculture, and medicine. The metabolic versatility of anaerobes is exploited in a variety of industrial processes including fermented food production, biochemical synthesis, and bioremediation. There has been recent considerable interest in developing and enhancing technologies that employ anaerobes as biocatalysts. The study of anaerobic bacteria requires specialized techniques, and specific methods are described for the culture and manip...
Source: Springer protocols feed by Biotechnology - September 23, 2009 Category: Biotechnology Source Type: info

Assessing Cellulase Performance on Pretreated Lignocellulosic Biomass Using Saccharification and Fermentation-Based ProtocolsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe two procedures that use washed pretreated cellulosic material to measure the efficacy of cellulase enzymes. First, a saccharification assay that measures glucose yield as a function of the amount of cellulase used in the process. And second, the simultaneous saccharification and fermentation (SSF) assay measures cellulase performance by the amount of ethanol produced from enzymatic hydrolysis of the cellulosic material. You can use both assays to screen cellulases under a variety of substrate types, loadings, and process conditions. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - September 23, 2009 Category: Biotechnology Source Type: info

Cellulase AssaysEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Cellulose is a heterogeneous polysaccharide, and its enzymatic hydrolysis requires endoglucanase, exoglucanase (cellobiohydrolase), and β-glucosidase to work together. We summarize the most commonly used assays for individual enzymes and cellulase mixture. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - September 23, 2009 Category: Biotechnology Source Type: info

Preparation and Analysis of Biomass LigninsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Lignin, comprised primarily of three randomly polymerized phenylpropenyl monomers, is, arguably, the second most common organic molecule on earth. In current biorefinery applications, lignin is burned, usually in concentrated pulping or hydrolysis liquor, as a source of process steam and both internal and exported electricity. The aromatic content of lignin makes it a potentially attractive feedstock for high-value aromatic chemicals, polymers, and carbon products (graphite, activated carbon, and carbon fiber). Revenue from production of lignin-based chemicals could play a major role in biorefinery profitability if cost-ef...
Source: Springer protocols feed by Biotechnology - September 23, 2009 Category: Biotechnology Source Type: info

High-Throughput Screening of Plant Cell-Wall Composition Using Pyrolysis Molecular Beam Mass SpectroscopyEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe a high-throughput method for estimating cell-wall chemistry traits using analytical pyrolysis. The instrument used to perform the high-throughput cell-wall chemistry analysis consists of a commercially available pyrolysis unit and autosampler coupled to a custom-built molecular beam mass spectrometer. The system is capable of analyzing approximately 42 biomass samples per hour. Lignin content and syringyl to guaiacol (S/G) ratios can be estimated directly from the spectra and differences in cell wall chemistry in large groups of samples can easily be identified using multivariate statistical data analysis metho...
Source: Springer protocols feed by Biotechnology - September 23, 2009 Category: Biotechnology Source Type: info

Biomass Compositional Analysis for Energy ApplicationsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In its broadest definition, biomass can be described as all material that was or is a part of a living organism. For renewable energy applications, however, the definition of biomass is usually limited to include only materials that are plant-derived such as agricultural residues (e.g., wheat straw, corn stover) by-products of industrial processes (e.g., sawdust, sugar cane bagasse, pulp residues, distillers grains), or dedicated energy crops (e.g., switchgrass, sorghum, Miscanthus, short-rotation woody crops). This chapter describes analytical methods developed to measure plant components with an emphasis on the measureme...
Source: Springer protocols feed by Biotechnology - September 23, 2009 Category: Biotechnology Source Type: info

Analytical Characterization of Fermentation Inhibitors in Biomass Pretreatment Samples Using Liquid Chromatography, UV-Visible Spectroscopy, and Tandem Mass SpectrometryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A variety of chemicals are produced upon pretreatment of lignocellulosic biomass. Aliphatic acids, aromatic acids, aldehydes, and phenolic compounds are of particular interest due to their presumed inhibitory influence on downstream enzymatic or microbial steps in biomass-to-ethanol conversion. Herein, we describe a series of analytical protocols that collectively enable quantitative monitoring of 40 potential fermentation inhibitors in biomass pretreatment samples. Solid samples are accommodated by first employing pressurized fluid extraction to generate an aqueous “wash stream.” Sample preparation for liquids...
Source: Springer protocols feed by Biotechnology - September 23, 2009 Category: Biotechnology Source Type: info

Analysis of Signaling Pathways in Zebrafish Development by MicroinjectionEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The objective of this chapter is to describe methods developed in this and other laboratories for microinjection and calcium imaging in the unfertilized zebrafish egg. Methods of immobilizing the oocyte include a holding chamber and a holding pipette. The holding chamber allows imaging of three or four oocytes simultaneously, while the holding pipette facilitates imaging of localized regions in the oocyte. Injection of calcium green dextran via holding chambers allowed detection of global changes in Ca2+ release following fertilization and development through early blastula stages. Injection and imaging with the holding pi...
Source: Springer protocols feed by Biotechnology - December 1, 2008 Category: Biotechnology Source Type: info

Protein Inhibition by Microinjection and RNA-Mediated Interference in Tissue Culture Cells: Complementary Approaches to Study Protein FunctionEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A major goal in cell biology is to understand the molecular mechanisms of the biological process under study, which requires functional information about the roles of individual proteins in the cell. For many non-genetic model organisms researchers have relied on the use of inhibitory reagents, such as antibodies that can be microinjected into cells. More recently, the advent of RNA-mediated interference (RNAi) has allowed scientists to knockdown individual proteins and to examine the consequences of the knockdown. In this chapter we present a comparison between microinjection of inhibitory reagents and RNAi for the analys...
Source: Springer protocols feed by Biotechnology - December 1, 2008 Category: Biotechnology Source Type: info

Quantitative Microinjection of Mouse Oocytes and EggsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Quantitative microinjection is used to introduce known quantities of molecules or probes into single cells to examine cellular function. The relatively large mammalian oocyte or egg is easily manipulated and can be injected with impermeant reagents including a variety of signaling molecules and fluorescent probes. Techniques have been developed to inject picoliter quantities of solution into oocytes and eggs with precision and reliability. The methods described here outline the quantitative injection procedures as they are used to inject mouse oocytes and eggs in a culture dish on the stage on an inverted microscope. The t...
Source: Springer protocols feed by Biotechnology - December 1, 2008 Category: Biotechnology Source Type: info

Germline Transformation of Caenorhabditis elegans by InjectionEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Microinjection is a commonly used technique for DNA transformation in Caenorhabditis elegans. It is a powerful tool that links genetic and molecular analysis to phenotypic analysis. In this chapter we shall provide an overview of microinjection for germline transformation in worms. Our discussion will emphasize C. elegans reproductive biology, applications and protocols for carrying out microinjection in order to successfully obtain transgenic worms. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - December 1, 2008 Category: Biotechnology Source Type: info

Microinjection of Follicle-Enclosed Mouse OocytesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The mammalian oocyte develops within a complex of somatic cells known as a follicle, within which signals from the somatic cells regulate the oocyte, and signals from the oocyte regulate the somatic cells. Because isolation of the oocyte from the follicle disrupts these communication pathways, oocyte physiology is best studied within an intact follicle. Here we describe methods for quantitative microinjection of follicle-enclosed mouse oocytes, thus allowing the introduction of signaling molecules as well as optical probes into the oocyte within its physiological environment. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - December 1, 2008 Category: Biotechnology Source Type: info

Exploring the Cytoskeleton During Intracytoplasmic Sperm Injection in HumansEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Understanding the cellular events during fertilization in mammals is a major challenge that can contribute to the improvement of future infertility treatments in humans and reproductive performance in farm animals. Of special interest is the role of the oocyte and sperm cytoskeleton during the initial interaction between gametes. The aim of this chapter is to describe methods for studying cytoskeletal features during in vitro fertilization after intracytoplasmic sperm injection (ICSI) in humans. The following protocols will provide a detailed description of how to perform immunodetection and imaging of human eggs, zygotes,...
Source: Springer protocols feed by Biotechnology - December 1, 2008 Category: Biotechnology Source Type: info

Functional Studies of Regulatory Genes in the Sea Urchin EmbryoEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Sea urchin embryos are characterized by an extremely simple mode of development, rapid cleavage, high transparency, and well-defined cell lineage. Although they are not suitable for genetic studies, other approaches are successfully used to unravel mechanisms and molecules involved in cell fate specification and morphogenesis. Microinjection is the elective method to study gene function in sea urchin embryos. It is used to deliver precise amounts of DNA, RNA, oligonucleotides, peptides, or antibodies into the eggs or even into blastomeres. Here we describe microinjection as it is currently applied in our laboratory and sho...
Source: Springer protocols feed by Biotechnology - December 1, 2008 Category: Biotechnology Source Type: info

Somatic Cell Nuclear Transfer in the MouseEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals since “Dolly,” the first animal cloned from an adult cell was reported in 1997. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, this technique has been proven to work in more than 10 mammalian species. Among them, the mouse provides the best model for both basic and applied research of somatic cloning because of its abounding genetic resources, rapid sexual maturity and propagation, minim...
Source: Springer protocols feed by Biotechnology - December 1, 2008 Category: Biotechnology Source Type: info

Expression of Exogenous mRNA in Xenopus laevis Embryos for the Study of Cell Cycle RegulationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The microinjection of mRNA that is transcribed and capped in vitro into fertilized eggs and embryos of Xenopus laevis provides a powerful means for discovering the function of proteins during early development. Proteins may be overexpressed for a gain-of-function effect or exogenous protein function may be compromised by the microinjection of mRNA encoding “dominant-negative” proteins. This methodology is particularly suited for the investigation of the regulation of the cell cycle, checkpoints, and apoptosis in early development. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - December 1, 2008 Category: Biotechnology Source Type: info

Use of Luciferase Chimaera to Monitor PLCζ Expression in Mouse EggsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The microinjection of cRNA encoding phospholipase Cζ (PLC zeta) causes Ca2+ oscillations and the activation of development in mouse eggs. The PLCζ protein that is expressed in eggs after injection of cRNA is effective in causing Ca2+ oscillations at very low concentrations. In order to measure the amount and timecourse of protein expression we have tagged PLCζ with firefly luciferase. The expression of the luciferase protein tag in eggs is then measured by incubation in luciferin combined with luminescence imaging, or by the lysis of eggs in the presence of Mg-ATP and luciferin in a luminometer. The use of l...
Source: Springer protocols feed by Biotechnology - December 1, 2008 Category: Biotechnology Source Type: info

Analysis of 14-3-3 Family Member Function in Xenopus Embryos by Microinjection of Antisense Morpholino OligosEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The 14-3-3 intracellular phosphoserine/threonine-binding proteins are adapter molecules that regulate signal transduction, cell cycle, nutrient sensing, apoptotic, and cytoskeletal pathways. There are seven 14-3-3 family members, encoded by separate genes, in vertebrate organisms. To evaluate the role of individual 14-3-3 proteins in vertebrate embryonic development, we utilized an antisense morpholino oligo microinjection technique in Xenopus laevis embryos. By use of this method, we showed that embryos lacking specific 14-3-3 proteins displayed unique phenotypic abnormalities. Specifically, embryos lacking 14-3-3 τ e...
Source: Springer protocols feed by Biotechnology - December 1, 2008 Category: Biotechnology Source Type: info

A Microinjectable Biological System, the Xenopus Oocyte, as an Approach to Understanding Signal Transduction Protein FunctionEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe how to microinject proteins or peptides in this model and we study, as an example, the Grb2 transduction cascade. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - December 1, 2008 Category: Biotechnology Source Type: info

Combining Microinjection and Immunoblotting to Analyze MAP Kinase Phosphorylation in Single Starfish Oocytes and EggsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The starfish oocyte has proven useful for studies involving microinjection because it is relatively large (190 μm) and optically clear. These oocytes are easily obtained from the ovary arrested at prophase of meiosis I, making them useful as a model system for the study of cell cycle-related events. In this chapter, a method for combining microinjection with immunoblotting of single cells is described. Individual starfish oocytes are injected, removed from the microinjection chamber, and analyzed by immunoblotting for the dual-phosphorylated form of mitogen-activated protein kinase (MAPK). This method will allow fo...
Source: Springer protocols feed by Biotechnology - December 1, 2008 Category: Biotechnology Source Type: info

Bacteriophage φC31 Integrase Mediated Transgenesis in Xenopus laevis for Protein Expression at Endogenous LevelsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Bacteriophage φC31 inserts its genome into that of its host bacterium via the integrase enzyme which catalyzes recombination between a phage attachment site (attP) and a bacterial attachment site (attB). Integrase requires no accessory factors, has a high efficiency of recombination, and does not need perfect sequence fidelity for recognition and recombination between these attachment sites. These imperfect attachment sites, or pseudo-attachment sites, are present in many organisms and have been used to insert transgenes in a variety of species. Here we describe the φC31 integrase approach to make transgenic Xenopu...
Source: Springer protocols feed by Biotechnology - December 1, 2008 Category: Biotechnology Source Type: info

DNA Delivery by Microinjection for the Generation of Recombinant Mammalian Cell LinesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Gene transfer methods for producing recombinant cell lines are often not very efficient. One reason is that the recombinant DNA is delivered into the cell cytoplasm and only a small fraction reaches the nucleus. This chapter describes a method for microinjecting DNA directly into the nucleus. Direct injection has several advantages including the ability to deliver a defined copy number into the nucleus, the avoidance of DNAses that are present in the cell cytoplasm, and the lack of a need for extensive subcloning to find the recombinant cells. The procedure is described for two cell lines, CHO DG44 and BHK-21, using green ...
Source: Springer protocols feed by Biotechnology - December 1, 2008 Category: Biotechnology Source Type: info

Computational Methods in Nanostructure DesignEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Self-assembling peptides can serve as building blocks for novel biomaterials. Replica exchange molecular dynamics simulations are a powerful means to probe the conformational space of these peptides. We discuss the theoretical foundations of this enhanced sampling method and its use in biomolecular simulations. We then apply this method to determine the monomeric conformations of the Alzheimer amyloid-β(12–28) peptide that can serve as initiation sites for aggregation. (Source: Springer protocols feed by Biotechnology)
Source: Springer protocols feed by Biotechnology - September 1, 2008 Category: Biotechnology Source Type: info