MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'Springer protocols feed by Microbiology' source. Thu, 09 Feb 2012 21:35:10 +0100 http://www.medworm.com/index.php?rid=5664681&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-361-5_20 The detection and analysis of structural invariants in cellular reaction networks is of central importance to achieve a more comprehensive understanding of metabolism. In this work, we review different kinds of structural invariants in reaction networks and their Petri net-based representation. In particular, we discuss invariants that can be obtained from the left and right null spaces of the stoichiometric matrix which correspond to conserved moieties (P-invariants) and elementary flux modes (EFMs, minimal T-invariants). While conserved moieties can be used to detect stoichiometric inconsistencies in reaction networks, EFMs correspond to a mathematically rigorous definition of the concept of a biochemical pathway. As outlined here, EFMs allow to devise strategies for strain improvement, ... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5664681 Tue, 13 Dec 2011 05:00:00 +0100 5664681 http://www.medworm.com/index.php?rid=5664680&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-361-5_21 Using the example of phosphate regulation in enteric bacteria, we demonstrate the particular suitability of stochastic Petri nets to model biochemical phenomena and their simulative exploration by various features of the software tool Snoopy. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5664680 Tue, 13 Dec 2011 05:00:00 +0100 5664680 http://www.medworm.com/index.php?rid=5664679&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-361-5_22 We describe the use of this tool in the context of the modeling of bacterial regulatory networks, notably the network of global regulators controlling the adaptation of Escherichia coli to carbon starvation conditions. We show how the modeler, by means of GNA, can define a regulatory network, build a model of the network, determine the steady states of the system, perform a qualitative simulation of the network dynamics, and analyze the simulation results using model-checking tools. The example illustrates the interest of qualitative approaches for the analysis of the dynamics of bacterial regulatory networks. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5664679 Tue, 13 Dec 2011 05:00:00 +0100 5664679 http://www.medworm.com/index.php?rid=5664678&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-361-5_24 Mutualistic microbial symbioses are one of the key innovations in the evolution of biological diversity, enabling the expansion of species’ niches and the production of sophisticated structures such as the eukaryotic cell. For some of the best-studied cases, we are beginning to have network models of symbiotic metabolism, but this work is in its infancy and has not been developed with an evolutionary perspective. However, theoreticians have long been interested in how these symbioses arise and persist and have applied modelling approaches from economics, evolution, ecology, and sociobology to a number of fundamental questions. We provide an overview of these questions, followed by specific modelling examples. We cover economic game theory, including the Prisoner’s Dilemma, the ... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5664678 Tue, 13 Dec 2011 05:00:00 +0100 5664678 http://www.medworm.com/index.php?rid=5664677&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-361-5_25 Bacterial virulence is a multifactorial process. In this chapter, we review some known mechanisms used by bacteria to trigger their production of virulence factors. We develop the idea that although the onset of virulence shows up an abrupt transition, the modelling of this dynamics can be classified in two qualitatively distinct infectious transitions which are respectively called “shift” or “switch.” We review methods enabling one to determine the types of behaviour that can be exhibited by a given model and we consider applications in three cases of virulence factor regulation. We conclude that in most cases a “successful” infection would require that the onset of virulence follows an irreversible switch behaviour. (Source: Springer protocols feed by ... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5664677 Tue, 13 Dec 2011 05:00:00 +0100 5664677 http://www.medworm.com/index.php?rid=5664676&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-361-5_26 This chapter describes how to use Smoldyn, which is a computer program for modeling cellular systems with spatial and stochastic detail. Smoldyn represents each molecule of interest as an individual point-like particle. These simulated molecules diffuse, interact with surfaces (e.g., biological membranes), and undergo chemical reactions much as they would in real biochemical systems. Smoldyn has been used to model signal transduction within bacterial cells, pheromone signaling between yeast cells, bacterial carboxysome function, diffusion in crowded spaces, and many other systems. A new “rule-based modeling” feature automatically generates chemical species and reactions as they arise in simulations due to protein modifications and complexation. Smoldyn is easy to use, quantitat... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5664676 Tue, 13 Dec 2011 05:00:00 +0100 5664676 http://www.medworm.com/index.php?rid=5483990&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-361-5_4 We describe network analysis of evolutionary relationships among some MGE categories and sketch out possible developments of this type of approach to get more insight into the role of the mobilome in bacterial adaptation and evolution. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483990 Thu, 08 Dec 2011 15:39:09 +0100 5483990 http://www.medworm.com/index.php?rid=5483989&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-361-5_3 Metagenomics is revolutionizing the field of microbial ecology through techniques that eliminate the prerequisite of culturing. Metagenomic studies of microbial populations in different environments reveal the incredible diversity and adaptive capabilities of these organisms. With the advent of cheaper, high-throughput sequencing technologies, these studies are also producing vast amounts of sequence data. Here, we discuss the different components of a metagenomic study including sample collection, DNA extraction, sequencing, and informatics. We highlight their issues and challenges, and review the solutions that are currently in use. We conclude with examples of metagenomic studies conducted on environments of varying complexities. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483989 Thu, 08 Dec 2011 15:39:09 +0100 5483989 http://www.medworm.com/index.php?rid=5483988&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-361-5_2 In order to ensure their function(s) in the cell, proteins are organized in machineries, underlaid by a complex network of interactions. Identifying protein interactions is thus crucial to our understanding of cell functioning. Technical advances in molecular biology and genomic technology now allow for the systematic study of the interactions occurring in a given organism. This review first presents the techniques readily available to microbiologists for studying protein–protein interactions in bacteria, as well as their usability for high-throughput studies. Two types of techniques need to be considered: (1) the isolation of protein complexes from the organism of interest by affinity purification, and subsequent identification of the complex partners by mass spectrometry and (2) tw... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483988 Thu, 08 Dec 2011 15:39:09 +0100 5483988 http://www.medworm.com/index.php?rid=5483987&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-361-5_11 The BioCyc database collection at BioCyc.org integrates genome and cellular network information for more than 1,100 organisms. This method chapter describes Web-based tools for browsing metabolic and regulatory networks within BioCyc. These tools allow visualization of complete metabolic and regulatory networks, and allow the user to zoom-in on regions of the network of interest. The user can find objects of interest such as genes and metabolites within the networks, and can selectively examine the connectivity of the network. The EcoCyc database within the BioCyc collection has been extensively curated. The descriptions within EcoCyc of the Escherichia coli metabolic network and regulatory network were derived from thousands of publications. Other BioCyc databases received moderate levels... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483987 Thu, 08 Dec 2011 15:39:09 +0100 5483987 http://www.medworm.com/index.php?rid=5483986&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-361-5_10 RegulonDB contains the largest and currently best-known data set on transcriptional regulation in a single free-living organism, that of Escherichia coli K-12 (Gama-Castro et al. Nucleic Acids Res 36:D120–D124, 2008). This organized knowledge has been the gold standard for the implementation of bioinformatic predictive methods on gene regulation in bacteria (Collado-Vides et al. J Bacteriol 191:23–31, 2009). Given the complexity of different types of interactions, the difficulty of visualizing in a single figure of the whole network, and the different uses of this knowledge, we are making available different views of the genetic network. This chapter describes case studies about how to access these views, via precomputed files, web services and SQL, including sigma–gene r... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483986 Thu, 08 Dec 2011 15:39:09 +0100 5483986 http://www.medworm.com/index.php?rid=5483985&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-361-5_1 This introductory review synthesizes the contents of the volume Bacterial Molecular Networks of the series Methods in Molecular Biology. This volume gathers 9 reviews and 16 method chapters describing computational protocols for the analysis of metabolic pathways, protein interaction networks, and regulatory networks. Each protocol is documented by concrete case studies dedicated to model bacteria or interacting populations. Altogether, the chapters provide a representative overview of state-of-the-art methods for data integration and retrieval, network visualization, graph analysis, and dynamical modelling. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483985 Thu, 08 Dec 2011 15:39:09 +0100 5483985 http://www.medworm.com/index.php?rid=5483984&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-483-4_9 Classic strain development that combines random mutagenesis and selection has a long history of success in generation of biocatalysts with industrially designed traits. However, the genetic loci contributing to the phenotypic strain changes are difficult to identify prior to genome sequencing technology advancement. In this chapter, we present the approach using Roche 454 next-generation pyro-resequencing to identify the genotypic changes such as single nucleotide polymorphisms (SNP) associated with an ethanol-tolerant strain of Clostridium thermocellum. The parameters used to filter the pyro-resequencing output for SNP identification are also discussed. These can help researchers to identify the genotypic change of other biocatalysts for strain improvement through metabolic engineering. (... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483984 Thu, 08 Dec 2011 15:39:09 +0100 5483984 http://www.medworm.com/index.php?rid=5483983&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-483-4_8 In recent years there has been a growing interest in the precise and concerted assembly of multiple DNA fragments of diverse sizes, including chromosomes, and the fine tuning of gene expression levels and protein activity. Commercial DNA assembly solutions have not been conceived to support the cloning of very large or very small genetic elements or a combination of both. Here we summarize a series of protocols that allow the seamless, simultaneous, flexible, and highly efficient assembly of DNA elements of a wide range of sizes (up to hundred thousand base pairs). The protocols harness the power of homologous recombination and are performed either in vitro or within the living cells. The DNA fragments may or may not share homology at their ends. An efficient site-directed mutagenesis prot... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483983 Thu, 08 Dec 2011 15:39:09 +0100 5483983 http://www.medworm.com/index.php?rid=5483982&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-483-4_7 Discovery of promoter elements with previously unknown regulated responses is important for metabolic engineering. For example, promoters responsive to the end product can be useful to regulate expression with increasing levels of product. In addition, such promoters can be used as screens for production strain with increased titers. Use of reporter genes, such as a bioluminescent reporter luxCDABE, can facilitate promoter discovery. Here, protocols for analysis of genome-wide luxCDABE reporter gene collections in Escherichia coli are provided. Further, a protocol for using a selected para-hydroxycinnamic (pHCA)-responsive promoter as detection assay for bioproduced pHCA is provided. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483982 Thu, 08 Dec 2011 15:39:09 +0100 5483982 http://www.medworm.com/index.php?rid=5483981&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-483-4_6 A method for in vivo evolution of metabolic pathways in bacteria is described. This method is a powerful tool for synthetic biology type of metabolic design and can lead to the creation of new metabolic pathways or the improvement of existing metabolic enzymes. The proposed strategy also permits to relate the evolved phenotype to the genotype and to analyze evolution phenomenon at the genetic, biochemical, and metabolic levels. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483981 Thu, 08 Dec 2011 15:39:09 +0100 5483981 http://www.medworm.com/index.php?rid=5483980&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-483-4_5 MIRAGE is a unique in vivo genome editing technique that exploits the inherent instability of inverted repeats (palindromes) in the Saccharomyces cerevisiae chromosome. As a technique able to quickly create deletions as well as precise point mutations, it is valuable in applications that require creation of designer strains of this yeast. In particular, it has various potential applications in metabolic engineering, systems biology, synthetic biology, and molecular genetics. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483980 Thu, 08 Dec 2011 15:39:09 +0100 5483980 http://www.medworm.com/index.php?rid=5483979&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-483-4_4 In this study, the high isoprenoid flux E. coli was transformed with a plasmid carrying the β-carotene biosynthetic genes from Pantoea stewartii for β-carotene production. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483979 Thu, 08 Dec 2011 15:39:09 +0100 5483979 http://www.medworm.com/index.php?rid=5483978&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-483-4_3 Our laboratory specializes in directed protein evolution, i.e., evolution of proteins under defined selective pressures in the laboratory. Our target genes are encoded in ColE1 plasmids to facilitate the generation of libraries in vivo. We have observed that when random mutations are not restricted to the coding sequence of the target genes, directed evolution results in a strong positive selection of plasmid origin of replication (ori) mutations. Surprisingly, this is true even during evolution of new biochemical activities, when the activity that is being selected was not originally present. The selected plasmid ori mutations are diverse and produce a range of plasmid copy numbers, suggesting a complex interplay between ori and coding mutations rather than a simple enhancement of level o... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483978 Thu, 08 Dec 2011 15:39:09 +0100 5483978 http://www.medworm.com/index.php?rid=5483977&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-483-4_2 There is increasing interest in utilization of engineered microorganisms for the production of renewable chemicals and next-generation biofuels. However, imbalances between the cofactor consumption of the engineered production pathway and the reducing equivalents provided by the cell have been shown to limit yields. This imbalance can be overcome by adjusting the cofactor dependencies of the pathway enzymes to match the available cofactors in the cell. We show how cofactor preference can be reversed by structure-guided directed evolution of the target enzyme. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483977 Thu, 08 Dec 2011 15:39:09 +0100 5483977 http://www.medworm.com/index.php?rid=5483976&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-483-4_1 Cellulose is an easily renewable and highly occurring resource. To take advantage of this great potential, there is a constant need of new cellulose degrading enzymes. In industrial applications enzymes have to function under extreme conditions like high temperature, very acidic or basic pH and different solvents. Cellulases have a huge area of application, for example the textile and food industry as well as the generation of bioethanol as an alternative energy source. They have the ability to yield a great energetic potential, but there is still a lack of economical technologies to conquer the stability of the cellulose structure. Via metagenomic research and well-directed screening, it is possible to detect new cellulases, which are active under tough industrial conditions. (Source: Spr... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483976 Thu, 08 Dec 2011 15:39:09 +0100 5483976 http://www.medworm.com/index.php?rid=5483975&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-483-4_19 Phage infections in bacterial bioprocesses constitute one of the most devastating threats to the productivity of the biotechnology facilities. There are several factors, which can decide if an infection would occur, and if it would turn into an outbreak and heavy contamination of the production facility. This issue is discussed on the basis of literature survey and experience of Phage Consultants. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483975 Thu, 08 Dec 2011 15:39:09 +0100 5483975 http://www.medworm.com/index.php?rid=5483974&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-483-4_18 Escherichia coli is the most commonly used microorganism for production of recombinant proteins for different applications. Acetate accumulation during aerobic growth on glucose has significant negative impact on recombinant protein production in Escherichia coli. Various strategies, such as process and genetic approaches have been developed to limit acetate formation to increase the productivity of recombinant proteins. We developed a strategy to combine inactivation of pyruvate oxidase (poxB) and over-expression of acety-CoA synthetase (acs) in E. coli K strain for controlling acetate accumulation. A recombinant peptide was expressed and produced in the engineered strains with a very low acetate ­formation in a 10-L fermentation process. (Source: Springer protocols feed by Microbiolo... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483974 Thu, 08 Dec 2011 15:39:09 +0100 5483974 http://www.medworm.com/index.php?rid=5483973&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-483-4_17 A cornerstone of Synthetic Biology is the engineering of gene regulatory networks. Construction of such biological circuits has been used not only to elucidate the dynamics of gene expression but also for designing whole-cell biosensors that translate environmental signals into quantifiable outputs. To this end, distinct components of given regulatory systems are rationally rewired in a way that translates an external stimulus (for instance, the presence of one chemical species) into a measurable readout typically fluorescence or luminescence. Various biosensors for BTEX (a mixture of benzene, toluene, ethylbenzene and xylenes) are based on XylR, the main transcriptional regulator of the TOL pathway of Pseudomonas putida mt-2. In the presence of its natural effectors (e.g., m-xylene, tolue... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483973 Thu, 08 Dec 2011 15:39:09 +0100 5483973 http://www.medworm.com/index.php?rid=5483972&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-483-4_16 Lignocellulosic biomass is a potential feedstock for bioethanol production. Biomass hydrolysates, prepared with a procedure including pretreatment and hydrolysis, are considered to be used as fermentation media for microorganisms, such as yeast. During the hydrolysate preparation procedure, toxic compounds are released or formed which may inhibit the growth of the microorganism and thus the product formation. To study the effects of these compounds on fermentation performance, the production of various hydrolysates with diverse inhibitory effects is of importance. A platform of methods that generates hydrolysates through four different ways and tests their inhibitory effects using Bioscreen C Analyzer growth tests is described here. The four methods, based on concentrated acid, dilute acid... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483972 Thu, 08 Dec 2011 15:39:09 +0100 5483972 http://www.medworm.com/index.php?rid=5483971&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-483-4_15 While large amount of strains can be quickly generated via metabolic engineering, the speed/efficiency of evaluating each strain becomes the bottleneck in the process from strain development to final production. In this chapter, a method is introduced to rapidly evaluate strain performance in fed-batch fermentation mode by using dynamic dissolved oxygen stat feed back control with no additional advanced online measurement. In addition, a scale-down feature is integrated in the method to mimic the limitation of oxygen transfer in large-scale vessels, so that strains can be evaluated under the conditions close to that in large-scale bioreactors. The method has been implemented in several commercial standard benchtop scale fermentation systems with different fermentation control software. (So... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483971 Thu, 08 Dec 2011 15:39:09 +0100 5483971 http://www.medworm.com/index.php?rid=5483970&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-483-4_14 Industrial biotechnology employs microorganisms (strains) for manufacture of certain food or industrial products to meet the increasing need of the world. To develop a bioproduction process, the first step is to screen out a production strain from isolated, mutated, or genetically engineered strain candidates. To maximize the bioproduction of a selected strain, bioreaction (fermentation) conditions need to be optimized. Fermentation experiments in shake flasks, bench-scale fermentors, or a combination of both are the conventional methods for both strain screening and fermentation optimization. Shake-flask experiments are easy to handle and cost-effective compared to experiments in fermentors, but the lower controllability makes the shake-flask data less informative for fermentation scale-u... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483970 Thu, 08 Dec 2011 15:39:09 +0100 5483970 http://www.medworm.com/index.php?rid=5483969&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-483-4_13 Metabolic engineers modify biological systems through the use of modern molecular biology tools in order to obtain desired phenotypes. However, due to the extreme complexity and interconnectedness of metabolism in all organisms, it is often difficult to a priori predict which changes will yield the optimal results. Flux balance analysis (FBA) is a mathematical approach that uses a genomic-scale metabolic network models to afford in silico prediction and optimization of metabolic changes. In particular, a genome-scale approach can help select gene targets for knockout and overexpression. This approach can be used to help expedite the strain engineering process. Here, we give an introduction to the use of FBA and provide details for its implementation in a microbial metabolic engineering con... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483969 Thu, 08 Dec 2011 15:39:09 +0100 5483969 http://www.medworm.com/index.php?rid=5483968&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-483-4_12 Many proteins and peptides have been used in therapeutic or industrial applications. They are often produced as recombinant forms by microbial fermentation. Targeted metabolic engineering of the production strains has usually been the approach taken to increase protein production, and this approach requires sufficient knowledge about cell metabolism and regulation. Random screening is an alternative approach that could circumvent the knowledge requirement, but is hampered by lack of suitable high-throughput screening methods. We developed a novel fluorescence-activated cell sorting (FACS) method to screen for cells with increased peptide production. Using a model peptide rich in certain amino acids, we showed that increased fluorescence clones sorted from a plasmid expression library conta... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483968 Thu, 08 Dec 2011 15:39:09 +0100 5483968 http://www.medworm.com/index.php?rid=5483967&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-483-4_11 A program of mutation and screening, with stepwise reverse engineering or “decoding” of the improved strain, is a way to better understand the genetics and physiology of the strain improvement process. As more is learned about the genetics of strain improvement, it is hoped that more fundamental principles will emerge about the types of mutations and genetic manipulations that reliably lead to higher producing strains. This will accelerate the construction of higher producing strains by metabolic engineering in the future. In this chapter, a detailed tagged mutagenesis approach is described using in vitro transposon mutagenesis which allowed the successful identification of key genes involved in macrolide (erythromycin) antibiotic biosynthesis. (Source: Springer protocols feed ... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483967 Thu, 08 Dec 2011 15:39:09 +0100 5483967 http://www.medworm.com/index.php?rid=5483966&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-483-4_10 The production of biofuels from renewable sources by microbial engineering has gained increased attention due to energy and environmental concerns. Butanol is one of the important gasoline-substitute fuels and can be produced by native microorganism Clostridium acetobutylicum. To develop a fundamental tool to understand C. acetobutylicum, a high resolution proteome reference map for this species has been established. To better understand the relationship between butanol tolerance and butanol yield, we performed a comparative proteomic analysis between the wild-type strain DSM 1731 and its mutant Rh8 at acidogenic and solventogenic phases, respectively. The 102 differentially expressed proteins that are mainly involved in protein folding, solvent formation, amino acid metabolism, protein sy... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5483966 Thu, 08 Dec 2011 15:39:09 +0100 5483966 http://www.medworm.com/index.php?rid=5169244&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_25 Systematic analysis of Saccharomyces cerevisiae metabolic functions and pathways has been the subject of extensive studies and established in many aspects. With the reconstruction of the yeast genome-scale metabolic (GSM) network and in silico simulation of the GSM model, the nature of the underlying cellular processes can be tested and validated with the increasing metabolic knowledge. GSM models are also being exploited in fundamental research studies and industrial applications. In this chapter, the principle concepts for construction, simulation and validation of GSM models, progressive applications of the yeast GSM models, and future perspectives are described. This will support and encourage researchers who are interested in systemic analysis of yeast metabolism and systems biology. ... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169244 Tue, 19 Jul 2011 23:00:00 +0100 5169244 http://www.medworm.com/index.php?rid=5169243&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_24 Building a dynamic model of a complete biological cell is one of the great challenges of the 21st century. While this objective could appear unrealistic until recently, considerable improvements in high-throughput data collection techniques, computational performance, data integration, and modeling approaches now allow us to consider it within reach in the near future. In this chapter, we review recent developments that pave the way toward the construction of genome-scale dynamic models. We first describe methodologies for the integration of heterogeneous “omics” datasets, which enable the interpretation of cellular activity at the genome scale and in fluctuating conditions, providing the necessary input to models. We subsequently discuss principles of such models and describe ... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169243 Tue, 19 Jul 2011 23:00:00 +0100 5169243 http://www.medworm.com/index.php?rid=5169242&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_23 Genetic regulatory circuits are often regarded as precise machines that accurately determine the level of expression of each protein. Most experimental technologies used to measure gene expression levels are incapable of testing and challenging this notion, as they often measure levels averaged over entire populations of cells. Yet, when expression levels are measured at the single cell level of even genetically identical cells, substantial cell-to-cell variation (or “noise”) may be observed. Sometimes different genes in a given genome may display different levels of noise; even the same gene, expressed under different environmental conditions, may display greater cell-to-cell variability in specific conditions and more tight control in other situations. While at first glance n... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169242 Tue, 19 Jul 2011 23:00:00 +0100 5169242 http://www.medworm.com/index.php?rid=5169241&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_22 The yeast Saccharomyces cerevisiae is the model organism in which protein interactions have been most extensively analyzed. The vast majority of these interactions have been characterized by a variety of sophisticated high-throughput techniques probing different aspects of protein association. This chapter summarizes the major techniques, highlights their complementary nature, discusses the data they produce, and highlights some of the biases from which they suffer. A main focus is the key role played by computational methods for processing, analyzing, and validating the large body of noisy data produced by the experimental procedures. It also describes how computational methods are used to extend the coverage and reliability of protein interaction data by integrating information from hete... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169241 Tue, 19 Jul 2011 23:00:00 +0100 5169241 http://www.medworm.com/index.php?rid=5169240&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_21 The rapidly increasing availability of DNA sequence data from modern high-throughput experimental techniques has created the need for computational algorithms to aid in motif discovery in genomic DNA. Such algorithms are typically used to find a statistical representation of the nucleotide sequence of the target site of a DNA-binding protein within a collection of DNA sequences that are thought to contain segments to which the protein is bound. A major assumption of these algorithms is that the protein recognizes the primary order of nucleotides in the sequence. However, proteins can also recognize the three-dimensional shape and structure of DNA. To account for this, we developed a computational method to predict the local structural profiles of any set of DNA sequences and then to search... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169240 Tue, 19 Jul 2011 23:00:00 +0100 5169240 http://www.medworm.com/index.php?rid=5169239&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_20 One of the major objectives of systems biology is the development of mathematical models for the quantitative description of complex biological systems, such as living cells. Biological data and software tools for the design, analysis, and simulation of models are two basic ingredients for the new field of systems biology. In this chapter we give an overview of databases and repositories that provide valuable information for the integrative analysis and modeling of data generated by the different omics techniques. We also provide a review of the most popular software tools currently used in computational systems biology studies. Standards for the annotation of biological data and for the analysis and exchange of models are fundamental for the success of systems biology and provide the glue... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169239 Tue, 19 Jul 2011 23:00:00 +0100 5169239 http://www.medworm.com/index.php?rid=5169238&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_19 Cellular networks and processes can be mathematically described and analyzed in various ways. Here, the case example of a MAP kinase (MAPK) cascade is used to detail steps in the formulation of a system of ordinary differential equations governing the temporal behavior of a signal transduction pathway after stimulation. Different analysis methods for the model are explained and demonstrated, such as stoichiometric analysis, sensitivity analysis, or studying the effect of deletions and protein overexpression. Finally, a perspective on standards concerning modeling in systems biology is given. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169238 Tue, 19 Jul 2011 23:00:00 +0100 5169238 http://www.medworm.com/index.php?rid=5169237&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_9 Nearly all eukaryotic mRNAs terminate in a poly(A) tail that serves important roles in mRNA utilization. In the cytoplasm, the poly(A) tail promotes both mRNA stability and translation, and these functions are frequently regulated through changes in tail length. To identify the scope of poly(A) tail length control in a transcriptome, we developed the polyadenylation state microarray (PASTA) method. It involves the purification of mRNA based on poly(A) tail length using thermal elution from poly(U) sepharose, followed by microarray analysis of the resulting fractions. In this chapter we detail our PASTA approach and describe some methods for bulk and mRNA-specific poly(A) tail length measurements of use to monitor the procedure and independently verify the microarray data. (Source: Springer... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169237 Tue, 19 Jul 2011 23:00:00 +0100 5169237 http://www.medworm.com/index.php?rid=5169236&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_8 This chapter describes the RNA sequencing (RNA-Seq) protocol, whereby RNA from yeast cells is prepared for sequencing on an Illumina Genome Analyzer. The protocol can easily be altered to use RNA from a different organism. This chapter covers RNA extraction, cDNA synthesis, cDNA fragmentation, and Illumina cDNA library generation and contains some brief remarks on bioinformatic analysis. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169236 Tue, 19 Jul 2011 23:00:00 +0100 5169236 http://www.medworm.com/index.php?rid=5169235&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_7 In the last decade, it became clear that transcription goes far beyond that of protein-coding genes. Most RNA molecules are transcribed from intergenic regions or introns and exhibit much variability in size, expression level, secondary structure, and evolutionary conservation. While for several types of non-coding RNAs some cellular functions have been reported, like for micro-RNAs and small nucleolar RNAs, for most others no indications of function or regulation have so far been found. Therefore, the RNA population inside a cell is diverse and cryptic and, thus, demands powerful methods to study its composition, abundance, and structure. DNA oligonucleotide microarrays have proven to be of great utility to study transcription of genes in various organisms. Recently, due to advancement in... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169235 Tue, 19 Jul 2011 23:00:00 +0100 5169235 http://www.medworm.com/index.php?rid=5169234&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_6 Cryptic unstable transcripts (CUTs) have been recently described as a major class of non-coding RNAs. These transcripts are, however, extremely unstable in normal cells and their analyzes pose specific technical problems. In this chapter, after a brief introduction discussing general aspects associated with the analysis of non-coding RNAs, we provide details of methods to enrich, map, and quantify this unconventional class of transcripts. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169234 Tue, 19 Jul 2011 23:00:00 +0100 5169234 http://www.medworm.com/index.php?rid=5169233&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_5 In this protocol, we describe a pipeline for transcript analysis in yeast via the quantification of mRNA expression levels. In the first section, we consider the well-established, proprietary Affymetrix GeneChip® approach to generating transcriptomics data. In the next section, we concentrate on providing a detailed protocol for the validation of these data using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The protocol provides suggested examples of hardware, software, and consumables/reagents required to perform these experiments. There are of course many other options available using alternative approaches (or indeed suppliers), but this protocol is intended to provide an approach that is flexible, inexpensive, sensitive, and easy to use. (Source: Springer... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169233 Tue, 19 Jul 2011 23:00:00 +0100 5169233 http://www.medworm.com/index.php?rid=5169232&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_4 The genomes of eukaryotic organisms are packaged into nuclei by wrapping DNA around proteins in a structure known as chromatin. The most basic unit of chromatin, the nucleosome, consists of approximately 146 bp of DNA wrapped around an octamer of histone proteins. The placement of nucleosomes relative to a gene can influence the regulation of the transcription of this gene. Furthermore, the N-terminal tails of histone proteins are subjected to numerous post-translational modifications that are also known to influence gene regulation. In recent years, a number of genome-scale approaches to identify modifications to chromatin have been developed. Techniques combining chromatin immunoprecipitation (ChIP) with microarrays (ChIP-chip) and second-generation sequencing (ChIP-Seq) have led to... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169232 Tue, 19 Jul 2011 23:00:00 +0100 5169232 http://www.medworm.com/index.php?rid=5169231&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_3 Chromatin plays critical roles in processes governed in different timescales – responses to environmental changes require rapid plasticity, while long-term stability through multiple cell generations requires epigenetically heritable chromatin. Understanding the dynamic behavior of chromatin is of great interest for fields ranging from transcriptional regulation through meiosis and gametogenesis. Here, we describe a protocol for measuring histone replacement rates genome wide in the budding yeast Saccharomyces cerevisiae. With suitable modifications, this protocol could be applied to other organisms, or to replacement dynamics of other DNA-associated proteins. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169231 Tue, 19 Jul 2011 23:00:00 +0100 5169231 http://www.medworm.com/index.php?rid=5169230&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_2 In the early days of the yeast genome sequencing project, gene annotation was in its infancy and suffered the problem of many false positive annotations as well as missed genes. The lack of other sequences for comparison also prevented the annotation of conserved, functional sequences that were not coding. We are now in an era of comparative genomics where many closely related as well as more distantly related genomes are available for direct sequence and synteny comparisons allowing for more probable predictions of genes and other functional sequences due to conservation. We also have a plethora of functional genomics data which helps inform gene annotation for previously uncharacterised open reading frames (ORFs)/genes. For Saccharomyces cerevisiae this has resulted in a continuous updat... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169230 Tue, 19 Jul 2011 23:00:00 +0100 5169230 http://www.medworm.com/index.php?rid=5169229&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_18 Cell growth is highly regulated and its deregulation is related to many human diseases such as cancer. Nutritional cues stimulate cell growth through modulation of TOR (target of rapamycin) signaling pathway. At the center of this pathway is a large serine/threonine protein kinase TOR, which forms two distinct functional complexes TORC1 and TORC2 in a cell. TORC1 senses the environmental nutrient quality/quantity and transmits the growth signals to multiple effectors to regulate a broad spectrum of biological processes including translation initiation, ribosome biogenesis, autophagy, nutrient uptake, and metabolism. By using budding yeast as a model, recent studies began to elucidate the complexity of the TOR signaling pathway. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169229 Tue, 19 Jul 2011 23:00:00 +0100 5169229 http://www.medworm.com/index.php?rid=5169228&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_17 Protein interactions are inherently dynamic. In no system is this more true and important than in signaling pathways, where spatial and temporal control of specific protein interactions is key to signaling specificity and timing. While genetic and biochemical interactions form a necessary and important starting point for deciphering interactions among signaling components, they struggle to provide precise information of where and when interactions occur in a live cell setting. In contrast, live cell fluorescence studies such as those outlined below are able to provide quantitative information on the strength, nature, timing, and location of homotypic and heterotypic protein interactions. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169228 Tue, 19 Jul 2011 23:00:00 +0100 5169228 http://www.medworm.com/index.php?rid=5169227&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_16 Competition experiments are an effective way to provide a measurement of the fitness of yeast strains. The availability of the Saccharomyces cerevisiae yeast knock-out (YKO) deletion collection allows scientists to retrieve fitness data for the ~6,000 S. cerevisiae genes at the same time in a given environment. The molecular barcodes, characterizing each yeast mutant, serve as strain identifiers, which can be detected in a single microarray analysis. Competition experiments in continuous culture using chemically defined media allow a more specific discrimination of the strains based on their fitness profile. With this high-throughput approach, a series of genes that, when one allele is missing, result in either defective (haplo-insufficient) or favored (haplo-proficient) growth phenotype h... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169227 Tue, 19 Jul 2011 23:00:00 +0100 5169227 http://www.medworm.com/index.php?rid=5169226&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_15 The automated cell, compound and environment screening system (ACCESS) was developed as an automated platform for chemogenomic research. In the yeast Saccharomyces cerevisiae, a number of genomic screens rely on the modulation of gene dose to determine the mode of action of bioactive compounds or the effects of environmental/compound perturbations. These and other phenotypic experiments have been shown to benefit from high-resolution growth curves and a highly automated controlled environment system that enables a wide range of multi-well assays that can be run over many days without any manual intervention. Furthermore, precise control of drug dosing, timing of drug exposure, and precise timing of cell harvesting at specific generation times are important for optimal results. Some of thes... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169226 Tue, 19 Jul 2011 23:00:00 +0100 5169226 http://www.medworm.com/index.php?rid=5169225&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_14 Metabolomics involves the investigation of the intracellular (endometabolome) and extracellular (exometabolome) pools of metabolites in biological systems. Methods to sample the exometabolome and to quench metabolism and extract intracellular metabolites for the model eukaryote Saccharomyces cerevisiae are presented here. These methods have been developed and validated to provide a fit-for-purpose protocol for global analyses of the S. cerevisiae metabolome. The protocol allows the extraction of a wide variety of metabolite classes and provides reproducible results to allow relative and semi-quantitative comparisons between samples of different origin. For exometabolome studies, fast sampling and separation of cells by syringe filtration is recommended. For endometabolome studies, fast que... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169225 Tue, 19 Jul 2011 23:00:00 +0100 5169225 http://www.medworm.com/index.php?rid=5169224&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_13 Ubiquitin-protein ligases (E3s) are responsible for target recognition and subsequent modification of selected substrates within the ubiquitin proteasomal system (UPS). Substrates of this pathway are covalently modified by the attachment of ubiquitin usually onto Lys residues. As a result, these modified proteins can be targeted for degradation, endocytosis, protein sorting, subnuclear trafficking, or other fates. Despite the advancements in understanding the underlying mechanisms of the ubiquitin system, the substrates of most E3 enzymes remain largely unknown. Here, we describe the development of a high-throughput method to identify in vitro substrates for E3 ligases on a global proteomic scale. The enzymatic activity (ubiquitylation) and binding of ubiquitin ligases to their substrates ... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169224 Tue, 19 Jul 2011 23:00:00 +0100 5169224 http://www.medworm.com/index.php?rid=5169223&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_12 Phenotypic variations of an organism may arise from alterations of cellular networks, ranging from the complete loss of a gene product to the specific perturbation of a single molecular interaction. In interactome networks that are modeled as nodes (macromolecules) connected by edges (interactions), these alterations can be thought of as node removal and edge-specific or “edgetic” perturbations, respectively. Here we present two complementary strategies, forward and reverse edgetics, to investigate the phenotypic outcomes of edgetic perturbations of binary protein–protein interaction networks. Both approaches are based on the yeast two-hybrid system (Y2H). The first allows the determination of the interaction profile of proteins encoded by alleles with known phenotypes to... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169223 Tue, 19 Jul 2011 23:00:00 +0100 5169223 http://www.medworm.com/index.php?rid=5169222&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_11 Early achievements in proteomics were qualitative, typified by the identification of very small quantities of proteins. However, as the subject has developed, there has been a pressure to develop approaches to define the amounts of each protein – whether in a relative or an absolute sense. A further dimension to quantitative proteomics embeds the behavior of each protein in terms of its turnover. Virtually every protein in the cell is in a dynamic state, subject to continuous synthesis and degradation, the relative rates of which control the expansion or the contraction of the protein pool, and the absolute values of which dictate the temporal responsiveness of the protein pool. Strategies must therefore be developed to assess the turnover of individual proteins in the proteome. Beca... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169222 Tue, 19 Jul 2011 23:00:00 +0100 5169222 http://www.medworm.com/index.php?rid=5169221&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_10 Whilst the study of yeast genomes and transcriptomes is in an advanced state, there is still much to learn about the resulting proteins in terms of cataloging, characterization of post-translational modifications, turnover, and the dynamics of sub-cellular localization and interactions. Analysis of the transcripts gives little insight into function or diversity as changes in RNA levels do not always correlate with the resulting protein abundance. A number of global and targeted attempts have been made to catalog and characterize the yeast proteome and we describe here the methods used to gain a greater understanding of the yeast proteome. This comprehensive review also describes future approaches that will aid completion in identifying and characterizing the remaining 20% of the undetermin... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169221 Tue, 19 Jul 2011 23:00:00 +0100 5169221 http://www.medworm.com/index.php?rid=5169220&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-173-4_1 In this chapter, we present an up-to-date view of the optimal characteristics of the yeast Saccharomyces cerevisiae as a model eukaryote for systems biology studies, with main molecular mechanisms, biological networks, and sub-cellular organization essentially conserved in all eukaryotes, derived from a complex common ancestor. The existence of advanced tools for molecular studies together with high-throughput experimental and computational methods, most of them being implemented and validated in yeast, with new ones being developed, is opening the way to the characterization of the core modular architecture and complex networks essential to all eukaryotes. Selected examples of the latest discoveries in eukaryote complexity and systems biology studies using yeast as a reference model and t... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=5169220 Tue, 19 Jul 2011 23:00:00 +0100 5169220 http://www.medworm.com/index.php?rid=4820072&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_20 This study shows a new approach to detect aflatoxins based on enzyme inhibition with several advantages, such as the easiness of use, the rapidity, and the cost-effectiveness, demonstrating a possible use as screening method for this type of mycotoxins. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820072 Fri, 13 May 2011 22:51:51 +0100 4820072 http://www.medworm.com/index.php?rid=4820071&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_19 Aspergillus flavus is an important fungal species that frequently contaminates food commodities with diverse toxins, with aflatoxins being the most relevant in food safety. In addition, this is one of the major pathogenic Aspergillus species. In this work, specific PCR-based protocol for this species is described which allows the discrimination of other closely related species from the Aspergillus section Flavi, particularly Aspergillus parasiticus. The specific primers were designed on the multicopy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA). (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820071 Fri, 13 May 2011 22:51:51 +0100 4820071 http://www.medworm.com/index.php?rid=4820070&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_18 A high-performance liquid chromatographic method with on-line post-column photochemical derivatization and fluorimetric detection for the simultaneous separation and quantitative determination of aflatoxin (AF) B1, B2, G1, and G2 in foodstuffs and feed materials is reported. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820070 Fri, 13 May 2011 22:51:51 +0100 4820070 http://www.medworm.com/index.php?rid=4820069&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_17 A sensitive and reliable analytical method based on immunoaffinity chromatography cleanup followed by HPLC separation and fluorimetric detection is described for the quantitative determination of aflatoxin M1 in milk. The chromatographic separation is accomplished by using a C18 column and a gradient elution with methanol, acetonitrile, and water. No extraction solvent process is required and a minimal milk sample cleanup is performed by a direct loading of the immunoaffinity columns and elution with methanol. The method has been successfully validated according to Decision EC No 657/2002 by using the conventional validation approach. The results of the validation process demonstrate the agreement of the method with the provisions of Regulation EC No 401/2006. (Source: Springer protocols f... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820069 Fri, 13 May 2011 22:51:51 +0100 4820069 http://www.medworm.com/index.php?rid=4820068&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_16 A sensitive and selective analytical method for the quantitative determination of fumonisins B1 (FB1) and B2 (FB2) in maize-based foods for direct human consumption is described. The method, based on high-performance liquid chromatography and fluorescence detection, presents a rapid and automated online post-column derivatization, performed with o-phthalaldehyde and N,N-dimethyl-2-mercaptoethylamine (Thiofluor™). A complete separation of fumonisins is achieved in less than 13 min by using a C18 column and a gradient elution. Fumonisins are extracted from the sample with a mixture of water, acetonitrile, and methanol. The filtered extract is purified by immunoaffinity column and FB1 and FB2 are eluted with methanol. The method has been successfully validated, and performances comply w... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820068 Fri, 13 May 2011 22:51:51 +0100 4820068 http://www.medworm.com/index.php?rid=4820067&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_15 Fumonisin mycotoxins are common contaminants in many grains, often at very low levels. Maize is ­particularly problematic as one of the organisms that commonly produce fumonisins, the fungus Fusarium verticillioides, often exists as an endophyte of maize. Fumonisin is a potent inhibitor of the enzyme ceramide synthase, and this inhibition results in the accumulation of a variety of upstream compounds, most notably, the sphingoid bases sphingosine, sphinganine, 1-deoxysphinganine and, in plants, phytosphingosine. Fumonisin exposure results in a wide variety of species, sex, and strain-specific responses. This method provides a relatively fast means of extracting fumonisins, sphingoid bases, and sphingoid base 1-phosphates from tissues and cells, as well as the subsequent analyses and qu... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820067 Fri, 13 May 2011 22:51:51 +0100 4820067 http://www.medworm.com/index.php?rid=4820066&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_9 Polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate or sodium deoxycholate (SDS- or DOC-slab-PAGE) is a powerful technique for the separation of smooth(S)-type bacterial lipopolysaccharides (LPS). In order to recover the individual LPS species from the polyacrylamide gel for subsequent analyses, a sensitive, nondestructive reverse staining of slab-PAGE-separated LPS has been developed. The individual reverse-stained LPS bands can be rapidly and efficiently recovered into an aqueous 5% triethylamine solution when they are extruded to produce fine gel microparticles. Based on these principles, an isolation methodology that combines preparative slab-PAGE, reverse staining, extrusion, and passive elution can be used to isolate, to electrophoretic homogeneity, micr... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820066 Fri, 13 May 2011 22:51:51 +0100 4820066 http://www.medworm.com/index.php?rid=4820065&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_8 Endotoxins (lipopolysaccharides, LPSs) are components of the envelope of Gram-negative bacteria. These molecules, responsible for both advantageous and harmful biological activities of these microorganisms, are highly immunogenic and directly involved in numerous bacterial diseases in humans such as Gram-negative sepsis. The characterization of endotoxins is of importance, since their physiological and pathophysiological effects depend on their chemical structure. The differences among LPSs from different bacterial serotypes and their mutants include variations mainly within the composition and length of their O-specific polysaccharide chains. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820065 Fri, 13 May 2011 22:51:50 +0100 4820065 http://www.medworm.com/index.php?rid=4820064&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_14 This chapter presents a conjugation method for coupling probes bearing hydrazine or primary amino groups to a lipopolysaccharide (LPS). LPS is modified by the activation of the hydroxyl groups present in its O-antigen moiety with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP). The method yields conjugates with good labeling ratios, preserving the endotoxic activity of the lipid A moiety. Conjugation of smooth-form LPS from Salmonella enterica sv. Minnesota with dansyl hydrazine and horseradish peroxidase yields labeling ratios above 110 nmol dansyl/mg LPS, with nearly no loss of the original endotoxic activity. In the case of horseradish peroxidase, introducing a spacer, a ratio of 29 nmol HRP/mg LPS was obtained, preserving 65% of the original endotoxic activity and an enzymat... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820064 Fri, 13 May 2011 22:51:50 +0100 4820064 http://www.medworm.com/index.php?rid=4820063&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_13 This chapter presents a conjugation method for coupling probes bearing hydrazine or primary amino groups to a smooth(S)-form lipopolysaccharide (LPS). LPS is modified by the activation of the hydroxyl groups present in its O-antigen moiety with cyanogen bromide in aqueous acetone. The method yields conjugates with good labeling ratios, preserving the endotoxic activity of the lipid A moiety. Conjugation of smooth-form LPS from Salmonella enterica sv. Minnesota with dansyl hydrazine and horseradish ­peroxidase yields labeling ratios above 300 nmol dansyl per mg LPS, with nearly no loss of the original endotoxin activity. In the case of horseradish peroxidase, introducing a spacer, a ratio of 28 nmol HRP per mg LPS is obtained, preserving 65% of the original endotoxic activity. (Source: ... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820063 Fri, 13 May 2011 22:51:50 +0100 4820063 http://www.medworm.com/index.php?rid=4820062&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_12 Lipopolysaccharides (LPSs) are major components of the external membrane of Gram-negative bacteria, and act as an effective permeability barrier. They are essentially composed of a hydrophilic polysaccharide region linked to an hydrophobic one, termed lipid A. Depending on their individual variable fine structures, they may be potent immunomodulators. Because of the structural importance and role of lipid A in bacterial pathogenesis, herein we describe two rapid practical micromethods for structural analysis. The first method allows the direct isolation of lipid A from whole bacteria cell mass; the second describes conditions for the sequential release of fatty acids, enabling the determination of their substitution position in the lipid A structure to be determined by matrix-assisted lase... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820062 Fri, 13 May 2011 22:51:50 +0100 4820062 http://www.medworm.com/index.php?rid=4820061&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_11 Treatment of infections caused by Gram-negative bacteria is difficult due in large part to problems arising from innate and acquired drug resistance, resulting in a limited number of effective antibiotics. Consequently, antibiotics that can circumvent mechanisms of drug resistance are needed. Lipid A is a glucosamine phospholipid that acts as an anchor for lipopolysaccharides (LPS) that comprise the outer membranes of Gram-negative bacteria, a barrier for small molecule entry into the cell, and is also the portion of LPS that stimulates the immune system in septic shock. Consequently, inhibitors of lipid A biosynthesis have the potential to function as antibiotics and/or anti-endotoxins in the treatment of Gram-negative bacterial infections. Current efforts in the development of antibiotic... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820061 Fri, 13 May 2011 22:51:50 +0100 4820061 http://www.medworm.com/index.php?rid=4820060&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_10 Bacterial endotoxins or lipopolysaccharides (LPS) are among the most potent activators of innate immune system, yet mechanisms of their action and, in particular, the role of the glycans remain elusive. Efficient noninvasive labeling strategies are necessary for studying interactions of LPS glycans with biological systems. Here, we describe a new method for labeling LPS and other lipoglycans with luminescent quantum dots (QDots). The labeling is achieved by the partitioning of hydrophobic quantum dots into the core of various LPS aggregates without disturbing the native LPS structure. The biofunctionality of the LPS–QDot conjugates is demonstrated by labeling of mouse monocytes. This simple method will find broad applicability in studies concerned with visualization of LPS biodistrib... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820060 Fri, 13 May 2011 22:51:50 +0100 4820060 http://www.medworm.com/index.php?rid=4820059&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_7 Cyanobacterial mass occurrences are widespread and often contain hepatotoxic, i.e. microcystin- and nodularin-producing, species. Nowadays, detection of microcystin (mcy) and nodularin synthetase (nda) genes is widely used for the recognition of toxic cyanobacterial strains in environmental water samples. Chip assay presented here combines ligation detection reaction and hybridization on a universal microarray to detect and identify the mcyE/ndaF genes of five cyanobacterial genera specifically and sensitively. Thus, one chip assay can reveal the co-occurrence of several hepatotoxin producers. The presented quantitative real-time PCR method is used for the detection of either microcystin-producing Anabaena or Microcystis. Determination of the mcyE-gene copy numbers allows the identificatio... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820059 Fri, 13 May 2011 22:51:50 +0100 4820059 http://www.medworm.com/index.php?rid=4820058&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_6 Shiga toxin-producing Escherichia coli is a leading cause of human gastroenteritis from food and waterborne sources worldwide. Shiga toxins 1 and 2 are important virulence factors linked to severe human illness. In particular, Shiga toxin 2 is composed of a diverse and heterogeneous group of subtypes with differential cytotoxicities in mammalian cells. In this chapter, we describe the use of the Vero-d2EGFP fluorescent assay to examine the relative toxicities of Stx2 and Stx2 subtypes expressed by strains of Shiga toxin-producing E. coli. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820058 Fri, 13 May 2011 22:51:50 +0100 4820058 http://www.medworm.com/index.php?rid=4820057&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_5 Escherichia coli O157:H7, a major cause of food-borne illness, produces Shiga toxins (Stxs) that block protein synthesis by inactivating the ribosome. In this chapter, we describe a simple cell-based fluorescent assay to detect Stxs and inhibitors of toxin activity. The assay can also be used to detect other plant and bacterial toxins that arrest protein synthesis. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820057 Fri, 13 May 2011 22:51:50 +0100 4820057 http://www.medworm.com/index.php?rid=4820056&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_4 The ability to rapidly differentiate Bacillus anthracis spores from spores belonging to other Bacillus spp. is potentially useful for combating the intentional release of this biothreat agent. Furthermore, not all B. anthracis strains are fully virulent and the ability to determine the potential virulence of the endospore is also important. In this chapter, we describe a two-color flow cytometric assay capable of simultaneously identifying B. anthracis spores and the presence of spore-associated protective antigen, a virulence marker for strains harboring the pXO1 plasmid. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820056 Fri, 13 May 2011 22:51:50 +0100 4820056 http://www.medworm.com/index.php?rid=4820055&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_3 Both botulinum neurotoxins (BoNTs) and anthrax lethal factor, a component of anthrax toxin, exhibit zinc metalloprotease activity. The assay detailed here is capable of quantitatively detecting these proteins by measuring their enzymatic functions with high sensitivity. The detection method encompasses two steps: (1) specific target capture and enrichment and (2) cleavage of a fluorogenic substrate by the immobilized active target, the extent of which is quantitatively determined by differential fluorometry. Because a critical ingredient for the target enrichment is an immobilization matrix made out of hundreds of thousands of microscopic, antibody-coated beads, we have termed this detection method an assay with a large immuno-sorbent surface area (ALISSA). The binding and reaction surface... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820055 Fri, 13 May 2011 22:51:49 +0100 4820055 http://www.medworm.com/index.php?rid=4820054&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_2 Loop-mediated isothermal amplification (LAMP) is an established nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. The LAMP assay requires 12–18 min for amplification with a single colony on selective agar from cholera toxin (CT)-producing Vibrio cholerae strains and less than 60 min with human feces and seafood samples. The assay requires less than 35 and 80 min for the detection of CT-producing V. cholerae with a colony on selective agar and with human feces and seafood samples from the beginning of DNA extraction to final determination. The LAMP amplification can be judged by both turbidimetric analysis and visual assessment with the unaided eye. The sensitivity of the LAMP assay is tenfold higher than that of the PCR a... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820054 Fri, 13 May 2011 22:51:49 +0100 4820054 http://www.medworm.com/index.php?rid=4820053&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-102-4_1 Bacterial protein toxins are involved in a number of infectious and foodborne diseases and are considered as potential biological warfare agents as well. Their sensitive multiplex detection in complex environmental, food, and biological samples are an important although challenging task. Solid-phase immunoaffinity capture provides an efficient way to enrich and purify a wide range of proteins from complex mixtures. We have shown that staphylococcal enterotoxins, for example, can be efficiently enriched by means of magnetic immunocapture using antibody functionalized paramagnetic beads. The method was successfully interfaced by the on-beads and off-beads detection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry at the protein level and by the off-beads nan... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4820053 Fri, 13 May 2011 22:51:49 +0100 4820053 http://www.medworm.com/index.php?rid=4683423&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-086-7_16 Methods and procedures in molecular biology used to study fungal pathogenesis have significantly improved during the last decade. In this chapter, we provide step-by-step procedures for performing genetics and biochemical studies in the human pathogenic fungal microorganism Cryptococcus neoformans (Cn). These methods are employed for studying the pathobiology of Cn and for experimental validation of theoretical models of fungal pathogenicity. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4683423 Thu, 07 Apr 2011 16:14:44 +0100 4683423 http://www.medworm.com/index.php?rid=4683422&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-086-7_15 Candida albicans is a pleiomorphic fungal pathogen whose morphogenetic plasticity has long been considered as a major virulence factor. In addition to the yeast-filament transition, C. albicans cells also have the unique ability to switch between two epigenetic phases referred to as white and opaque. White and opaque cells harbor identical genomes yet they differ in cellular morphologies, gene expression profiles, mating abilities, and virulence properties. The switching process is regulated by a small network of transcription factors and is suggested to be driven by stochastic fluctuations of the regulatory components, which correlates with altered switching frequencies. Traditionally, phase variants have been identified based on cellular morphologies and expression levels of a few marker... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4683422 Thu, 07 Apr 2011 16:14:44 +0100 4683422 http://www.medworm.com/index.php?rid=4683421&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-086-7_14 Analysis of gene function often involves detailed studies of when a given gene is expressed or silenced. Transposon mutagenesis is a powerful tool to generate insertional mutations that provide with a selectable marker and a reporter gene that can be used to analyze the transcriptional activity of a specific locus in a variety of microorganisms to study gene regulation. Then the reporter gene expression can be easily measured under different conditions to gain insight into the regulation of the particular locus of interest. We have used transposon mutagenesis as a tool to generate insertional mutations with a modified Tn7 transposon containing the reporter gene URA3 (Tn7-URA3) to study subtelomeric silencing in the opportunistic fungal pathogen Candida glabrata. This method consists of two... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4683421 Thu, 07 Apr 2011 16:14:44 +0100 4683421 http://www.medworm.com/index.php?rid=4683420&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-086-7_13 We present methods to quantify relevant protein–protein interactions in that network and to visualize such differences in simple plate assays allowing for genetic approaches in further studies. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4683420 Thu, 07 Apr 2011 16:14:44 +0100 4683420 http://www.medworm.com/index.php?rid=4683419&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-086-7_12 Genetic networks underlying many biological processes, such as vertebrate somitogenesis, cell cycle, hormonal signaling, and circadian rhythms, are characterized by oscillations in gene expression. It has been recognized that the frequency and amplitude of gene expression oscillations vary among individuals and can be controlled by specific expression quantitative trait loci (eQTLs). In this chapter, we develop a dynamic model for mapping and identifying such eQTLs by integrating mathematical aspects of oscillatory dynamics into the functional mapping framework. The model can determine whether and how eQTLs regulate individual genes’ activation kinetics and expression dynamics by estimating and testing Fourier series parameters for different eQTL genotypes. We incorporate a general a... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4683419 Thu, 07 Apr 2011 16:14:44 +0100 4683419 http://www.medworm.com/index.php?rid=4683418&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-086-7_11 We describe here how one can use this resource to investigate the molecular sources of stochasticity in a gene regulatory network. The approach is general enough to be applied to any network of interest, as long as the experimental read-out offers robust statistics. For a given network, a typical study first identifies two backgrounds A and B displaying different levels of stochasticity and then study the network in A × B progeny. Taking advantage of microarrays or resequencing technologies, genotyping of appropriate segregants can then lead to the genomic regions housing modulators of stochasticity. The powerful toolbox available to manipulate the yeast genome offers several ways to narrow these regions further and to unambiguously demonstrate the regulatory consequenc... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4683418 Thu, 07 Apr 2011 16:14:44 +0100 4683418 http://www.medworm.com/index.php?rid=4683417&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-086-7_9 Over the past decade, researchers have recognized the need to study biological systems as integrated systems. While the reductionist approaches of the past century have made remarkable advances of our understanding of life, the next phase of understanding comes from systems-level investigations. Additionally, biology has become a data-intensive field of research. The introduction of high throughput sequencing, microarrays, high throughput proteomics, metabolomics, and now lipidomics are producing significantly more data than can be interpreted using existing methods. The field of systems biology brings together methods from computer science, modeling, statistics, engineering, and biology to explore the volumes of data now being produced and to develop mathematical representations of metabo... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4683417 Thu, 07 Apr 2011 16:14:43 +0100 4683417 http://www.medworm.com/index.php?rid=4683416&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-086-7_8 Organisms have the ability to counteract environmental perturbations and keep certain components within a cell homeostatically regulated. Closely related to homeostasis is the behavior of perfect adaptation where an organism responds to a step-wise perturbation by regulating some of its components, after a transient period, to their original pre-perturbation values. A particular interesting type of model relates to the so-called robust behavior where the homeostatic or perfect adaptation property is independent of the magnitude of the applied step-wise perturbation. It has been shown that this type of behavior is related to the control-theoretic concept of integral feedback (or integral control). Using downloadable MATLAB examples, we demonstrate how robust perfect adaptation sites can be ... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4683416 Thu, 07 Apr 2011 16:14:43 +0100 4683416 http://www.medworm.com/index.php?rid=4683415&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-086-7_7 In this chapter, stochasticity in gene expression is investigated using $$ \Omega $$ -expansion technique. Two theoretical models are considered here, one concern the stochastic fluctuations in a single-gene network with negative feedback regulation, and the other the additivity of noise propagation in a protein cascade. All of these theoretical analyses may provide a basic framework for understanding stochastic gene expression. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4683415 Thu, 07 Apr 2011 16:14:43 +0100 4683415 http://www.medworm.com/index.php?rid=4683414&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-086-7_10 In this study, the problem of finding similar change patterns is induced to clustering with the derivative Fourier coefficients. This work is aimed at discovering gene groups with similar change patterns which share similar biological properties. We developed a statistical model using derivative Fourier coefficients to identify similar change patterns of gene expression. We used a model-based method to cluster the Fourier series estimation of derivatives. We applied our model to cluster change patterns of yeast cell cycle microarray expression data with alpha-factor synchronization. It showed that, as the method clusters with the probability-neighboring data, the model-based clustering with our proposed model yielded biologically interpretable results. We expect that our proposed Fourier a... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4683414 Thu, 07 Apr 2011 16:14:43 +0100 4683414 http://www.medworm.com/index.php?rid=4683413&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-086-7_6 Determining the in vivo kinetics of a signaling pathway is a challenging task. We can measure a property we termed pathway bandwidth to put in vivo bounds on the kinetics of the mitogen-activated protein kinase (MAPk) signaling cascade in Saccharomyces cerevisiae that responds to hyperosmotic stress [the High Osmolarity Glycerol (HOG) pathway]. Our method requires stimulating cells with square waves of oscillatory hyperosmotic input (1 M sorbitol) over a range of frequencies and measuring the activity of the HOG pathway in response to this oscillatory input. The input frequency at which the pathway’s steady-state activity drops precipitously because the stimulus is changing too rapidly for the pathway to respond faithfully is defined as the pathway bandwidth. In this chapter, we... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4683413 Thu, 07 Apr 2011 16:14:43 +0100 4683413 http://www.medworm.com/index.php?rid=4683412&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-086-7_5 Gene functional studies consist of phenotyping cells with altered gene expression. Improving the precision of current gene expression control techniques would enable more detailed studies of gene function. Here, we provide protocols for building synthetic gene constructs for tuning the expression of a gene in all the cells of a population precisely and uniformly, achieving expression levels proportional to the extracellular inducer concentration. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4683412 Thu, 07 Apr 2011 16:14:43 +0100 4683412 http://www.medworm.com/index.php?rid=4683411&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-086-7_4 This chapter describes a method for generating yeast respiratory oscillations in continuous culture and monitoring rhythmic promoter activity of the culture by automated real-time recording of luminescence. These techniques chiefly require the use of a strain of Saccharomyces cerevisiae that has been genetically modified to express firefly luciferase under the control of a promoter of interest and a continuous culture bioreactor that incorporates a photomultiplier apparatus for detecting light emission. Additionally, this chapter describes a method for observing rhythmic (cell cycle-related) promoter activity in small batch cultures of yeast through luminescence monitoring. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4683411 Thu, 07 Apr 2011 16:14:43 +0100 4683411 http://www.medworm.com/index.php?rid=4683410&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-086-7_3 Promoters contain a large number of binding sites for transcriptional factors transmitting signals from a variety of cellular pathways. The promoter processes these input signals and sets the level of gene expression, the output of the gene. Here, we describe how to design genetic constructs and measure gene expression to deliver data suitable for quantitative analysis. Synthetic genetic constructs are well suited to precisely control and measure gene expression to construct cis-regulatory input functions. These functions can be used to predict gene expression based on signal intensities transmitted to activators and repressors in the gene regulatory region. Simple models of gene expression are presented for competitive and noncompetitive repressions. Complex phenomena, exemplified by syne... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4683410 Thu, 07 Apr 2011 16:14:43 +0100 4683410 http://www.medworm.com/index.php?rid=4683409&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-086-7_2 Gene transcription is a dynamic process in which the desired amount of an mRNA is obtained by the equilibrium between its transcription (TR) and degradation (DR) rates. The control mechanism at the RNA polymerase level primarily causes changes in TR. Despite their importance, TRs have been rarely measured. In the yeast Saccharomyces cerevisiae, we have implemented two techniques to evaluate TRs: run-on and chromatin immunoprecipitation of RNA polymerase II. These techniques allow the discrimination of the relative importance of TR and DR in gene regulation for the first time in a eukaryote. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4683409 Thu, 07 Apr 2011 16:14:43 +0100 4683409 http://www.medworm.com/index.php?rid=4683408&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-086-7_1 We describe and compare two complementary approaches to estimating global transcript stability: (1) direct measurement of decay rates; (2) indirect estimation of turnover from determination of mRNA synthesis rates and steady-state levels. Since the two approaches have distinct strengths yet confer different cellular perturbations, it is valuable to consider results obtained with both methods. The practical aspects of the chapter are written from a yeast perspective; the general considerations hold true for all eukaryotes, however. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=4683408 Thu, 07 Apr 2011 16:14:43 +0100 4683408 http://www.medworm.com/index.php?rid=3729657&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-365-7_15 Mouse models have been developed to study the pathogenic process of Clostridium difficile infections, first the intestinal colonization and second the toxin production. These models have also been used to test the role of environmental conditions that modulate infection. Different mouse models have been used successfully to study C. difficile infections such as conventional mice, gnotobiotic mouse models including the monoxenic C. difficile mouse model, and the human microbiota-associated mouse model. The advantages and disadvantages of these models are discussed. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=3729657 Fri, 31 Jul 2009 23:00:00 +0100 3729657 http://www.medworm.com/index.php?rid=3729656&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-365-7_14 The Golden Syrian hamster is widely regarded as the most relevant small animal model of Clostridium difficile disease as oral infection of animals pre-treated with antibiotics reproduces many of the symptoms observed in man. These include diarrhoea, histological damage, colonisation of the large bowel and sporulation of the organism at the terminal stage of the disease. However, infection results in a fatal outcome, which in the past has been used as an experimental endpoint. More recently, attempts have been made to refine the model to maximise the scientific data generated whilst minimising animal suffering. This has been achieved using a combination of qualitative and quantitative measurements taken during the course of the infection and at post-mortem. This has allowed timing of experi... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=3729656 Fri, 31 Jul 2009 23:00:00 +0100 3729656 http://www.medworm.com/index.php?rid=3729655&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-365-7_13 Genetic manipulation of Clostridium difficile is notoriously difficult, currently there is only one reliable method for generating random mutations in the organism and that is to use the conjugative transposon Tn916. Tn916 enters the genome of most strains of C. difficile with no obvious target site preference. In order to use the genome strain C. difficile 630 for transposon mutagenesis a erythromycin-sensitive derivative C. difficile 630Δerm was constructed and the Tn916 derivative, Tn916ΔE, was shown to enter the genome at multiple sites enabling the construction of a Tn916 insertion library. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=3729655 Fri, 31 Jul 2009 23:00:00 +0100 3729655 http://www.medworm.com/index.php?rid=3729654&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-365-7_12 Clostridium difficile is the causative agent of a range of intestinal diseases, collectively referred to as Clostridium difficile-associated disease (CDAD). The recent emergence of “hypervirulent” strains associated with increased rates of mortality and severity of disease in humans has highlighted the need to study this organism at the molecular level. These studies will increase our knowledge of the mechanisms by which C. difficile causes disease and facilitate the rational design of new and improved therapeutics. The study of C. difficile has long been hampered by difficulties in genetically manipulating the organism. It has been only recently (within the last decade) that methods have been developed to introduce plasmid DNA into C. difficile and most importantly to enable t... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=3729654 Fri, 31 Jul 2009 23:00:00 +0100 3729654 http://www.medworm.com/index.php?rid=3729653&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-365-7_11 Members of the genus Clostridium have long been recognised as important to humankind and its animals, both in terms of the diseases they cause and the useful biological processes they undertake. This has led to increasing efforts directed at deriving greater information on their basic biology, most notably through genome sequence. Accordingly, annotated sequences of all of the most important species are now available. However, full exploitation of the data generated has been hindered by the lack of mutational tools that may be used in functional genomic studies. Thus, the number of clostridial mutants generated has until recently been disappointingly small. In particular, the construction of directed mutants using classical homologous recombination-based methods has met with only limited s... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=3729653 Fri, 31 Jul 2009 23:00:00 +0100 3729653 http://www.medworm.com/index.php?rid=3729652&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-365-7_10 Clostridium difficile is a pathogen on the move, as evidenced by the rapid transcontinental spread of the so-called hypervirulent 027 strains, followed by the emergence of further PCR ribotypes such as 017, 078 and 106. This provides a rare opportunity to study the evolution of virulence in action. However, to fully exploit this opportunity, robust phylogenetic methods on a diverse set of characterised strains are required to provide a reference evolutionary framework to study C. difficile epidemiology, ecology and virulence. Traditional phylogenetic classification of bacteria to study evolutionary relatedness is based on the characterisation of a limited number of genes, rRNA or signature sequences. However, due to the acquisition of DNA through lateral gene transfer, the differences betw... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=3729652 Fri, 31 Jul 2009 23:00:00 +0100 3729652 http://www.medworm.com/index.php?rid=3729651&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-365-7_9 Clostridium difficile is a gram-positive, spore-forming, toxin-producing anaerobic bacillus that is being increasingly implicated as the leading cause of diarrhea and colitis, particularly in hospitalized, elderly patients. Studies to date suggest that C. difficile toxins A and B play a major role in the observed colonic inflammation and associated disease pathogenesis; however, the role of other potential bacterial factors at present remains unknown. Early effects of C. difficile on host intestinal epithelia include modest induction of innate immune responses with progressive loss of intestinal epithelial cell barrier function and cell death. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=3729651 Fri, 31 Jul 2009 23:00:00 +0100 3729651 http://www.medworm.com/index.php?rid=3729650&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-365-7_8 The bacterial cell surface is an important structure as it mediates interactions with the external environment. In the case of pathogens like Clostridium difficile, the cell wall and its components also have to mediate interactions with the host cells and their products. In this chapter we discuss the various methods used for dissecting the cell surface and the biochemical and immunological procedures that are commonly used to analyse the properties of the proteins within the cell wall. A major consideration is the S-layer which in C. difficile shows considerable variation in sequence and between strains, a property which is also reflected in its antigenic properties. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=3729650 Fri, 31 Jul 2009 23:00:00 +0100 3729650 http://www.medworm.com/index.php?rid=3729649&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-365-7_7 Toxin A (TcdA) and Toxin B (TcdB) are the major virulence factors that contribute to the pathogenesis of Clostridium difficile-associated diarrhoea (CDAD). These enterotoxins act by glucosylation of members of the Rho protein family of small GTP-binding proteins. This leads to the disorganization of the host cell actin cytoskeleton (cytopathic effect) and apoptosis (cytotoxic effect). Due to their glucosyltransferase activity, they are referred as “clostridial glucosylating toxins”. The severe form of CDAD has been recently correlated to the levels of toxin production. This reinforces the idea that regulation of toxin production is an important part of the C. difficile infection. Genes encoding TcdA (tcdA) and TcdB (tcdB) are present in a pathogenicity locus (PaLoc) that also i... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=3729649 Fri, 31 Jul 2009 23:00:00 +0100 3729649 http://www.medworm.com/index.php?rid=3729648&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-365-7_6 Multilocus sequence typing (MLST), a nucleotide sequence-based characterization of allelic polymorphism of housekeeping genes, has been proposed as a new approach for population and evolutionary genetics and global epidemiology of bacterial pathogens. MLST provides unambiguous sequence data that can be generated from various laboratories and should be shared in a common web database. Here are presented most of materials, methods, and programs or software necessary to perform MLST on Clostridium difficile. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=3729648 Fri, 31 Jul 2009 23:00:00 +0100 3729648 http://www.medworm.com/index.php?rid=3729647&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-365-7_5 Clostridium difficile shows considerable variability in the PaLoc region encoding two main virulence factors, toxins TcdA and TcdB. Strains with changes in PaLoc are defined as variant toxinotypes and currently 27 such groups are recognized (I to XXVII). Toxinotype 0 includes strains with PaLoc identical to the reference laboratory strain VPI 10463. Toxinotyping is a RFLP-PCR-based method using a combination of restriction patterns of part of tcdB and tcdA genes for determination of toxinotype. Variations in PaLoc can affect the toxin production or could result in production of toxins with altered properties. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=3729647 Fri, 31 Jul 2009 23:00:00 +0100 3729647 http://www.medworm.com/index.php?rid=3729646&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-365-7_4 Molecular typing methods for Clostridium difficile are based on gel electrophoresis of restriction fragments (endonuclease restriction analysis, REA; pulsed field gel electrophoresis PFGE; toxinotyping), PCR amplification (PCR ribotyping, arbitrarily primed PCR, multilocus variable-number tandem-repeat analysis MLVA), and sequence analysis (multilocus sequence typing MLST; slpA typing, tandem repeat sequence typing). We will describe two standard methods (PCR ribotyping predominantly used throughout Europe and PFGE which is predominantly used in North America) and will discuss the difficulties of inter-laboratory comparability and unification of typing nomenclature. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=3729646 Fri, 31 Jul 2009 23:00:00 +0100 3729646 http://www.medworm.com/index.php?rid=3729645&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-365-7_3 Clostridium difficile infection (CDI) occurs as a disease with a spectrum of severity ranging from mild, self-limiting diarrhoea to a severe colitis, pseudomembraneous colitis or toxic megacolon. The disease arises as a major complication of antibiotic therapy and is most commonly acquired in hospital. The laboratory investigation of faecal samples is supportive of a clinical suspicion that a patient has the disease. Currently the mainstay of diagnosis is the demonstration of C. difficile toxins in a diarrhoeal sample; only a few laboratories set up cultures for the organism. However, toxin tests should not be used as stand alone tests since some patients with disease do not have detectable levels of toxin in their faeces. Furthermore, other patients may have large amounts of toxin in the ... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=3729645 Fri, 31 Jul 2009 23:00:00 +0100 3729645 http://www.medworm.com/index.php?rid=3729644&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-365-7_2 Clostridium difficile is a spore-forming, toxin-producing, anaerobic bacterium abundant in soils and water. Frequent and early colonization of the human intestinal flora is common and often asymptomatic. Antimicrobials given commonly disrupt the intestinal microflora and through proliferation in colon and production of toxin A and B it precipitates C. difficile infection (CDI). The enterocytic detachment and bowel inflammation provoke C. difficile-associated diarrhoea (CDAD) sometimes developing into severe pseudomembranous colitis (PMC) and paralytic ileus. Infection is acquired from an endogenous source or from spores in the environment, most easily facilitated during hospital stay. In the elderly, comorbidity, hospitalization and antimicrobial treatment present as major risk factors and... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=3729644 Fri, 31 Jul 2009 23:00:00 +0100 3729644 http://www.medworm.com/index.php?rid=3729643&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-365-7_1 Never before has there been a more timely opportunity to investigate the molecular genetics of Clostridium difficile. Over the last few years the perception of C. difficile has changed from an obscure, and often under-researched, bacterium to one of major clinical importance, at least in industrialized nations. Coupled with the increased interest in this organism researchers now have a greater understanding of its genetic content and molecular epidemiology; a direct consequence of the multiple C. difficile genomes which have been, and currently are being, sequenced. Concurrent with the sequencing efforts have been the development of tools to genetically manipulate the organism. We are now in a position to answer fundamental questions about the biology and pathogenicity of the organism. The... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=3729643 Fri, 31 Jul 2009 23:00:00 +0100 3729643 http://www.medworm.com/index.php?rid=2517004&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_9 The need for inhibitors for enzymes linked with microbial infection, specifically the NS3 protease of hepatitis C virus (HCV), inspired us to develop a unique, rapid and easy color-based method described herein. The NS3 serine protease of HCV has a role in processing viral polyprotein and it has been implicated in interactions with various cell constituents, resulting in phenotypic changes including malignant transformation. NS3 is currently regarded a prime target for antiviral drugs. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2517004 Sun, 01 Mar 2009 00:00:00 +0100 2517004 http://www.medworm.com/index.php?rid=2517003&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_8 We describe a human cell display strategy to isolate high-affinity single-chain antibody fragments (scFvs) specific for CD22 for the treatment of B-cell malignancies. Our strategy uses flow cytometry and human embryonic kidney 293T (HEK-293T) cells that are widely used for transient protein expression. Flow cytometry enhances the screen’s sensitivity thereby allowing us to isolate high-affinity scFvs. Using human cell display, one could isolate and engineer scFvs, single domains, Fabs, or whole IgGs for increased affinity and other biological functions. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2517003 Sun, 01 Mar 2009 00:00:00 +0100 2517003 http://www.medworm.com/index.php?rid=2517002&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_7 Phage display has the capacity to rapidly isolate recombinant antibodies against protein targets and other molecules of significant size. However, there is no obvious lower limit to the power of the selection methods: this chapter describes how the techniques of phage display can be adapted to allow the isolation of antibodies against very small compounds. Antibodies generated in this way have many uses including the detection and quantitative analysis of the target chemical moiety in samples such as foods, water, and body fluids. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2517002 Sun, 01 Mar 2009 00:00:00 +0100 2517002 http://www.medworm.com/index.php?rid=2517001&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_6 The cartilaginous fish (sharks, skates, and rays) are the oldest phylogenetic group in which a human-type adaptive immune system and immunoglobulins (Igs) have been found. In addition to their conventional (heavy-light chain heterodimeric) isotypes, IgM and IgW, sharks produce the novel isotype, IgNAR, a heavy chain homodimer that does not associate with light chains. Instead, its variable (V) regions act as independent, soluble units in order to bind antigen. In this chapter, we detail our immunization protocol in order to raise a humoral IgNAR response in the nurse shark (Ginglymostoma cirratum) and the subsequent cloning of the single-domain V regions from this isotype in order to select antigen-specific binders by phage display. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2517001 Sun, 01 Mar 2009 00:00:00 +0100 2517001 http://www.medworm.com/index.php?rid=2517000&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_5 This chapter describes the construction and screening of a library of single-chain variable fragments (svFv) derived from patients with autoimmune disease. The methods cover the isolation of mononuclear cells from peripheral blood, preparation of RNA, and recovery of immunoglobulin-coding sequences by polymerase chain reaction (PCR). Cloning into a phage display vector and screening of the scFv display library by a simple panning procedure are described. These methods are applicable to library construction from any patient group or (with alternative primer sets) any mammalian species. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2517000 Sun, 01 Mar 2009 00:00:00 +0100 2517000 http://www.medworm.com/index.php?rid=2516999&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_4 A method for the construction of West Nile virus immune donor antibody repertoires is described. B cells are harvested from a suitable donor and the antibody variable genes are amplified using polymerase chain reaction (PCR). The PCR fragments are cloned in a phage display vector to construct a repertoire that can be used in panning procedures to identify many unique monoclonal antibodies. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2516999 Sun, 01 Mar 2009 00:00:00 +0100 2516999 http://www.medworm.com/index.php?rid=2516998&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_3 A method is described for affinity isolation of antigen-specific circulating B cells of interest for subsequent generation of immune antibody phage display libraries. This approach should overcome the problem of low yields of monoclonal antibodies of interest in the libraries generated from peripheral blood lymphocytes caused by the low abundance of antigen-specific B cells in the circulation. The preselection of B cells is based on the specificity of the surface Ig receptor and is accomplished using the antigen of interest conjugated to magnetic beads. This method should significantly increase the frequency of antibody phage particles of interest in the library and allow for efficient isolation monoclonal antibodies with the predefined specificity. (Source: Springer protocols feed by Micr... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2516998 Sun, 01 Mar 2009 00:00:00 +0100 2516998 http://www.medworm.com/index.php?rid=2516997&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_2 Diversity—the variability carried by the amino acid sequences of a synthetic antibody library—can be generated by synthetic degenerate oligonucleotides. One can experiment with different diversity designs in the variable domains of light and heavy chains (VH and VL) to generate antibody libraries with different properties. The ability to precisely define the final diversity of a library facilitates the process of isolating, characterizing, and optimizing an antibody lead. Here we describe detailed protocols for the design and construction of phage-displayed synthetic antibody libraries in which diversity is generated in the complementarity determining regions (CDRs) of the VH of a single humanized bivalent Fab scaffold. The example used in the protocol provides a general method... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2516997 Sun, 01 Mar 2009 00:00:00 +0100 2516997 http://www.medworm.com/index.php?rid=2516996&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_1 Antibody-based immunologic reagents are useful for identifying, isolating, or eliminating cells with particular characteristics related to different diseases. Phage display is a highly valuable technique for antibody selection related to this purpose. In brief, a diverse group of antibody genes prepared from a patient or generated in vitro are inserted into a phagemid vector or the phage genome so that when the protein is expressed, it becomes anchored on the surface of the phage by fusion to a coat protein. A diverse library of recombinant antibodies is generated in this way and can then be exposed or panned on the antigen of interest, typically, this being a molecule associated with a particular pathological condition. Phage that carry proteins or peptides bind preferentially to the targ... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2516996 Sun, 01 Mar 2009 00:00:00 +0100 2516996 http://www.medworm.com/index.php?rid=2516995&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_18 Numerous techniques are available for investigating protein-ligand interactions. The phage display technique is one such method routinely used to identify antibody-antigen interactions and has the benefit of being easily adaptable to high-throughput screening platforms. Once identified, antigen-binding domains on fragment antibodies or single-chain fragment antibodies (scFv) can be expressed and purified for further studies. In this chapter, we describe a method for high-level expression of a phage display-derived scFv in Pichia pastoris. The phage display-derived antibody A33scFv recognizes a cell surface glycoprotein (designated A33) expressed in colon cancer that serves as a target antigen for radioimmunoimaging and/or immunotherapy of human colon cancer. The expression and purification... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2516995 Sun, 01 Mar 2009 00:00:00 +0100 2516995 http://www.medworm.com/index.php?rid=2516994&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_17 We present here an alternative procedure that uses cytoplasmic expression of soluble active scFv. This can be accomplished by using either specially engineered E. coli strains or hyperstable scFvs. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2516994 Sun, 01 Mar 2009 00:00:00 +0100 2516994 http://www.medworm.com/index.php?rid=2516993&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_16 The isolation of recombinant antibodies by phage display naturally leads to experiments to evaluate their biological and immunological properties. Although crude preparations may have their value in initial studies, the need often exists for highly purified protein that can be tested in vivo. This chapter describes methods to generate high yields of scFv from bacterial cultures and to purify protein to the degree of homogeneity required for the most exacting analysis. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2516993 Sun, 01 Mar 2009 00:00:00 +0100 2516993 http://www.medworm.com/index.php?rid=2516992&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_15 We describe procedures for intracellular expression of scFv in eukaryotic cells. Starting from a scFv gene cloned in a phage-display vector, we describe the cloning step into a mammalian expression vector, the transient transfection of a HeLa cell line, and the monitoring of intrabody expression by immunofluorescence staining and FACS analysis. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2516992 Sun, 01 Mar 2009 00:00:00 +0100 2516992 http://www.medworm.com/index.php?rid=2516991&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_14 Conventionally, antibody phage display has been used to isolate recombinant antibodies that are monovalent in their interaction with target antigens. These antibodies can be reengineered for expression in mammalian cell culture as full-length, monospecific immunoglobulins. An emerging branch of research has sought to generate bivalent recombinant antibodies by manipulating the length of the linker separating heavy- and light-chain variable domains in single-chain Fv proteins, thereby promoting inter-scFv interaction and the formation of “diabodies.” With careful control, this can generate scFv-based proteins able to bind two very distinct targets, “bispecific diabodies.” Further manipulation enables the assembly of higher-order complexes. (Source: Springer protocols... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2516991 Sun, 01 Mar 2009 00:00:00 +0100 2516991 http://www.medworm.com/index.php?rid=2516990&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_13 A method is described for the identification of the antigen recognised by an scFv isolated from an antibody phage display library using selection against a complex mixture of proteins (e.g. intact cells, purified cell surface membranes, and tissue sections). The method takes advantage of a yeast two-hybrid system that additionally allows for reorganization of post-translational modifications to the bait and target proteins. This technique is therefore especially useful for identifying surface-expressed antigens. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2516990 Sun, 01 Mar 2009 00:00:00 +0100 2516990 http://www.medworm.com/index.php?rid=2516989&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_12 This chapter outlines a protocol for the selection by phage display of single-chain variable antibody fragments with dual properties-specificity for tumor cells and the ability to be internalized. The protocol is based on a direct incubation of living target cells with antibody phage display libraries under conditions that allow active endocytosis of phage particles by cancer cells as well as recovery of intracellular phage particles that retain infectivity. This “functional” selection helps avoid the isolation of irrelevant phages that may be obtained when selection is performed on heterogeneous material as a source of antigen. Internalizing antibodies recovered from human antibody repertoires are promising reagents for various therapeutic applications including the delivery o... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2516989 Sun, 01 Mar 2009 00:00:00 +0100 2516989 http://www.medworm.com/index.php?rid=2516988&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_11 Many phage display techniques drive selection toward the isolation of highly specific antibodies. However, the identification of monoclonal antibodies that are cross-reactive has implications for the development of diagnostics, therapeutics, and vaccines against pathogens or cancer cells that are able to rapidly generate variants and escape mutants. To identify human monoclonal antibodies with high activity against HIV and broad-spectrum activity, we developed a technique termed sequential antigen panning. This methodology could be used to isolated recombinant antibodies against any antigen that shares epitopes with other antigens. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2516988 Sun, 01 Mar 2009 00:00:00 +0100 2516988 http://www.medworm.com/index.php?rid=2516987&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-302-2_10 We provide procedures for the panning of fully humanized Fab antibodies using guided selection. Human heavy and light chain genes are amplified. A parental light chain is cloned into a phage display vector and combined with the heavy chain library. After several rounds of panning, positive clones are identified and the heavy chain sequences that are recovered are combined with light chains for further selection by phage display. Human Fab antibodies are obtained that bind the same epitope as the parental antibody. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2516987 Sun, 01 Mar 2009 00:00:00 +0100 2516987 http://www.medworm.com/index.php?rid=2387502&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_10 Bacteriophages manipulate bacterial gene expression in order to express their own genes or influence bacterial metabolism. Gene expression can be studied using real-time PCR or microarrays. Either technique requires the prior isolation of high quality RNA uncontaminated by the presence of genomic DNA. We outline the considerations necessary when working with bacteriophage infected bacterial cells. We also give an example of a protocol for extraction and quantification of high quality RNA from infected bacterial cells, using the marine cyanobacterium WH7803 and the phage S-PM2 as a case study. This protocol can be modified to extract RNA from the host/bacteriophage of interest. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2387502 Tue, 28 Oct 2008 04:00:00 +0100 2387502 http://www.medworm.com/index.php?rid=2040770&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_9 A common approach for investigating evolutionary relationships between genes and organisms is to compare extant DNA or protein sequences and infer an evolutionary tree. This methodology is known as molecular phylogenetics and may be the most informative means for exploring phage evolution, since there are few morphological features that can be used to differentiate between these tiny biological entities. In addition, phage genomes can be mosaic, meaning different genes or genomic regions can exhibit conflicting evolutionary histories due to lateral gene transfer or homologous recombination between different phage genomes. Molecular phylogenetics can be used to identify and study such genome mosaicism. This chapter provides a general introduction to the theory and methodology used to recons... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040770 Tue, 28 Oct 2008 04:00:00 +0100 2040770 http://www.medworm.com/index.php?rid=2040769&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_8 Knowledge of the regulatory elements contained within bacteriophage genomes forms the basis for understanding genomic expression and organization. The in silico prediction of promoter and terminator sequences in phage genomes is a first step towards this understanding. In this chapter, a number of programs and resources to identify regulatory elements are listed and discussed. Combining the available web-resources and literature data optimizes these predictions and can thus aid in a more directed experimental identification of these regulatory elements. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040769 Tue, 28 Oct 2008 04:00:00 +0100 2040769 http://www.medworm.com/index.php?rid=2040768&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_7 Tailed-bacteriophage virions contain a single linear dsDNA chromosome which can range in size from about 18 to 500 kbp across the known tailed-phage types. These linear chromosomes can have one of several known types of termini as follows: cohesive ends ( $5^{\prime}$ - or $3^{\prime}$ -single-strand extensions), circularly permuted direct terminal repeats, short or long exact direct terminal repeats, terminal host DNA sequences, or covalently bound terminal proteins. These different types of ends reflect differing DNA replication strategies and especially differing terminase actions during DNA packaging. In general, complete genome sequence determination does not by itself elucidate the nature of these ends, so directed experimental analysis is usually requir... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040768 Tue, 28 Oct 2008 04:00:00 +0100 2040768 http://www.medworm.com/index.php?rid=2040767&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_6 One of the most satisfying aspects of a genome sequencing project is the identification of the genes contained within it.These are of two types: those which encode tRNAs and those which produce proteins. After a general introduction on the properties of protein-encoding genes and the utility of the Basic Local Alignment Search Tool (BLASTX) to identify genes through homologs, a variety of tools are discussed by their creators. These include for genome annotation: GeneMark, Artemis, and BASys; and, for genome comparisons: Artemis Comparison Tool (ACT), Mauve, CoreGenes, and GeneOrder. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040767 Tue, 28 Oct 2008 04:00:00 +0100 2040767 http://www.medworm.com/index.php?rid=2040766&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_5 PCR is a quick and effective way of identifying the presence and ‘affiliation’ of bacteriophages, or phage-encoded genes from environmental samples, bacterial cells or purified viruses. The limitations are that you have to know what you are looking for in order to find it. Although the bacteriophage world does not have the advantage of a conserved gene, present in all members, there are many phage genes that do show nucleotide conservation even between phages which infect fairly divergent taxa. As more sequence data become available through both metagenomic approaches and the sequencing of complete bacteriophage genomes, PCR primers can be further refined and thus it should be an increasingly useful tool for bacteriophage biology. (Source: Springer protocols feed by Microbiolog... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040766 Tue, 28 Oct 2008 04:00:00 +0100 2040766 http://www.medworm.com/index.php?rid=2040765&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_4 The most efficient method to determine the genomic sequence of a dsDNA phage is to use a whole genome shotgun approach (WGSA). Preparation of a library where each genomic fragment has an equal chance of being represented is critical to the success of the WGSA. For many phages, there are regions of the genome likely to be under-represented in the shotgun library, which results in more gaps in the shotgun assembly than predicted by the Poisson distribution. However, as phage genomes are relatively small, this increased number of gaps does not present an insurmountable impediment to using the WGSA. This chapter will focus on construction of a high-quality random library and sequence analysis of this library in a 96-well format. Techniques are described for the mechanical fragmentation of geno... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040765 Tue, 28 Oct 2008 04:00:00 +0100 2040765 http://www.medworm.com/index.php?rid=2040764&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_3 Standard agarose gel electrophoresis is extensively used to resolve DNA fragments from 0.2 to 40–50 kb. Larger fragments of genomic DNA or whole viral genomes can only effectively be resolved by pulsed-field gel electrophoresis (PFGE), which extends the range of molecular separation from 200 bp to 12 Mb. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040764 Tue, 28 Oct 2008 04:00:00 +0100 2040764 http://www.medworm.com/index.php?rid=2040763&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_21 The Internet provides a myriad of useful tools for the phage worker including access to culture collections, specific databases, tools for gene identification, and whole genome comparisons, lecture notes, information on upcoming scientific meetings, books, etc. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040763 Tue, 28 Oct 2008 04:00:00 +0100 2040763 http://www.medworm.com/index.php?rid=2040762&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_20 Techniques developed over the past 20 years for the display of foreign peptides and proteins on the surfaces of filamentous bacteriophages have been a major driving force in the rapid development of recombinant antibody technology in recent years. With phage display of antibodies as one of its key components, recombinant antibody technology has led to the development of an increasing number of therapeutic monoclonal antibodies. Antibody gene libraries are fused to a gene encoding a phage coat protein. Recombinant phage expressing the resulting antibody libraries in fusion with the coat protein are propagated in Escherichia coli. Phage displaying monoclonal antibodies with specificities for target antigens are isolated from the libraries by a process called panning. The genes encoding the d... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040762 Tue, 28 Oct 2008 04:00:00 +0100 2040762 http://www.medworm.com/index.php?rid=2040761&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_2 DNA base compositional analysis is something which is rarely undertaken today, but it is still a useful criterion for phage taxonomy. A variety of techniques are described including hydrolysis of the DNA to the level of bases or nucleosides and separation by paper chromatography or HPLC. Spectroscopic and spectrofluorometric procedures are also outlined. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040761 Tue, 28 Oct 2008 04:00:00 +0100 2040761 http://www.medworm.com/index.php?rid=2040760&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_19 We describe the construction of fragmented whole genome and gene fragment phage display libraries and interaction selection by panning. This strategy allows the identification and characterization of interacting proteins on a genomic scale by screening the fragmented “proteome” against protein baits. Gene fragment libraries allow a more in depth characterization of the protein–protein interaction site by identification of the protein region involved in the interaction. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040760 Tue, 28 Oct 2008 04:00:00 +0100 2040760 http://www.medworm.com/index.php?rid=2040759&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_18 Lysins are phage-encoded, peptidoglycan (cell wall) hydrolases that accumulate in the bacterial cytoplasm during a lytic infection cycle. Late during infection, the lysins undergo holin-mediated translocation across the inner membrane into the peptidoglycan matrix where they cleave cell wall covalent bonds required for wall stability and allow bacterial lysis and progeny phage release. This potent hydrolytic activity is now the foundation of a powerful genetic-based screening process for the identification and analysis of phage lysin proteins. Here, we describe a method for identifying a lysin, PlyG, from a bacteriophage that specifically infects the Gram-positive organism Bacillus anthracis; however, the techniques described can be adapted to clone, express, and analyze lysins from any ph... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040759 Tue, 28 Oct 2008 04:00:00 +0100 2040759 http://www.medworm.com/index.php?rid=2040758&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_17 Phage typing is a rapid, economical, reliable, and reproducible technique, requiring no specialized equipment, for fingerprinting disease-causing agents for epidemiological investigation and surveillance. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040758 Tue, 28 Oct 2008 04:00:00 +0100 2040758 http://www.medworm.com/index.php?rid=2040757&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_16 Current appreciation of the vast expanse of prokaryotic diversity has largely come through molecular phylogenetic exploration of sequence diversity within the universally conserved gene for small subunit ribosomal RNA (16S rDNA). A plethora of methodologies for characterizing the diversity and composition of bacterial communities is based on sequence polymorphisms within this single gene. By comparison, no gene is universally shared among viruses or bacteriophages, which has prevented broad scale characterization of viral diversity within microbial ecosystems. With the reduction in DNA sequencing costs and wide availability of bioinformatics software, the tools of whole genome shotgun sequencing are now beginning to be applied to the characterization of genetic diversity within whole micro... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040757 Tue, 28 Oct 2008 04:00:00 +0100 2040757 http://www.medworm.com/index.php?rid=2040756&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_15 Since most of the phage genomes isolated from natural samples are previously unknown sequences, an isolation-independent approach is necessary to quantify the diversity of natural viral communities. Currently, two different methodological approaches are widely used to obtain genetic fingerprints of natural phage communities. While the separation of different viral genomes with pulsed field gel electrophoresis (PFGE) is based on the size of the genome, denaturing gradient gel electrophoresis (DGGE) uses minor differences in gene base composition to separate fragments of amplified DNA from natural viral communities. Finger printing techniques are a relatively fast and cheap tool to assess the diversity of environmental viruses. Together, PFGE and DGGE provide useful tools to study viral ecol... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040756 Tue, 28 Oct 2008 04:00:00 +0100 2040756 http://www.medworm.com/index.php?rid=2040755&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_14 Current techniques in mass spectrometry (MS) allow sensitive and accurate identification of proteins thanks to the in silico availability of these protein sequences within databases. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040755 Tue, 28 Oct 2008 04:00:00 +0100 2040755 http://www.medworm.com/index.php?rid=2040754&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_13 Concentration and purification of infectious particles are prerequisites for structural and functional characterization of bacteriophages. The methods detailed in the first part of this chapter outline the protocols commonly used to obtain purified phages: the concentration of phage particles by precipitation with polyethylene glycol and their purification by centrifugation in CsCl step gradients and subsequently by equilibrium centrifugation. This sequence of procedures, if carried out as a whole, ensures a purification of high quality, which is well suited for most analytical techniques used to characterize bacteriophage particles. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040754 Tue, 28 Oct 2008 04:00:00 +0100 2040754 http://www.medworm.com/index.php?rid=2040753&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_12 Gene expression microarrays offer the ability to monitor the expression of all phage genes over an infection cycle. However, there are relatively few reports to date of microarrays being used to investigate phage biology. This chapter aims to provide an overview of how to design and implement a microarray experiment to investigate phage biology. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040753 Tue, 28 Oct 2008 04:00:00 +0100 2040753 http://www.medworm.com/index.php?rid=2040752&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_11 Real-time, or quantitative PCR, is a valuable technique useful in bacteriophage research to quantify the abundance of phage or host gene transcripts. It can be used during the infection cycle both to monitor the expression of individual viral transcripts and to compare relative gene expression levels throughout the infection cycle. It is fairly economical to conduct and is useful in bacteria–phage systems where obtaining high yields of RNA is problematic. To perform real-time PCR, it is simply necessary to know the DNA sequence of the genes to be monitored, to have accurately quantified mRNA good quality cDNA, and access to a light-cycler. Although this chapter briefly reviews the basic principles of real-time PCR, the emphasis is on aspects of technique that are specific to the stud... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040752 Tue, 28 Oct 2008 04:00:00 +0100 2040752 http://www.medworm.com/index.php?rid=2040751&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_10 Bacteriophages manipulate bacterial gene expression in order to express their own genes or influence bacterial metabolism. Gene expression can be studied using real-time PCR or microarrays. Either technique requires the prior isolation of high quality RNA uncontaminated by the presence of genomic DNA. We outline the considerations necessary when working with bacteriophage infected bacterial cells. We also give an example of a protocol for extraction and quantification of high quality RNA from infected bacterial cells, using the marine cyanobacterium WH7803 and the phage S-PM2 as a case study. This protocol can be modified to extract RNA from the host/bacteriophage of interest. (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040751 Tue, 28 Oct 2008 04:00:00 +0100 2040751 http://www.medworm.com/index.php?rid=2040750&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-565-1_1 Preparation of pure bacteriophage DNA used to rely on using CsCl gradients to give high purity or methods that yielded DNA that was either of low recovery or subject to significant genomic contamination. Recently though, new methods have come along that allow the purification of DNA from plate lysates that are not only capable of high yield but also, for all intents and purposes, free of genomic contamination (i.e. no visible genomic contamination on restriction analysis or when used for bacteriophage sequencing). (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2040750 Tue, 28 Oct 2008 04:00:00 +0100 2040750 http://www.medworm.com/index.php?rid=2505909&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-254-4_12 (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2505909 Tue, 30 Sep 2008 23:00:00 +0100 2505909 http://www.medworm.com/index.php?rid=2505908&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-254-4_11 (Source: Springer protocols feed by Microbiology) Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2505908 Tue, 30 Sep 2008 23:00:00 +0100 2505908 http://www.medworm.com/index.php?rid=2505907&cid=s_37126_77_f&fid=37126&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-254-4_10 This chapter deals with control charts to monitor the performance of Indirect ELISAs. An Indirect ELISA kit for the detection of antibodies against Brucella is used to demonstrate the methods. Many of the features explained in Chapter 9 are relevant to this chapter; some repetition is intended, as this chapter may be read independently. Figure 1 gives an overview of the indirect ELISA scheme used. The details of the procedure, which involves plotting the data graphically (charting methods), are explained. As a reminder, the objectives of charting data are as follows: 1.To keep a constant record of all data.2.To monitor the assay from plate to plate in any one day's testing.3.To monitor the tests made from day to day, week to week, year to year.4.To allow rapid identification of unacceptabl... Springer protocols feed by Microbiology news http://www.medworm.com/rss/comments.php?id=2505907 Tue, 30 Sep 2008 23:00:00 +0100 2505907