Springer protocols feed by MicrobiologyThis is an RSS file. You can use it to subscribe to this data in your favourite RSS reader, such as GoogleReader, or to display this data on your own website or blog. Subscribe to this data using MyMedWorm.Subscribe to this data using GoogleReader.Subscribe to this data using Bloglines.Subscribe to this data using MyYahoo.

This page shows you the latest items in this publication.

Pages: 1 2

Guided Selection Methods Through Chain ShufflingEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - March 1, 2009 Category: Microbiology Source Type: info

Sequential Antigen Panning for Selection of Broadly Cross-Reactive HIV-1-Neutralizing Human Monoclonal AntibodiesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - March 1, 2009 Category: Microbiology Source Type: info

Selection of Antibodies Able to Rapidly Enter Mammalian CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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: Springer protocols feed by Microbiology - March 1, 2009 Category: Microbiology Source Type: info

Identification of the Specificity of Isolated Phage Display Single-Chain Antibodies Using Yeast Two-Hybrid ScreensEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - March 1, 2009 Category: Microbiology Source Type: info

Generation of Bispecific and Tandem DiabodiesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ab...
Source: Springer protocols feed by Microbiology - March 1, 2009 Category: Microbiology Source Type: info

Intrabody Expression in Eukaryotic CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - March 1, 2009 Category: Microbiology Source Type: info

High-Level Periplasmic Expression and Purificationof scFvsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - March 1, 2009 Category: Microbiology Source Type: info

Expression of Single-Chain Fv Fragments in E. coli CytoplasmEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - March 1, 2009 Category: Microbiology Source Type: info

High-Level Expression of a Phage Display-Derived scFv in Pichia pastorisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 (de...
Source: Springer protocols feed by Microbiology - March 1, 2009 Category: Microbiology Source Type: info

Antibody Phage Display: Overview of a Powerful Technology that Has Quickly Translated to the ClinicEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 antige...
Source: Springer protocols feed by Microbiology - March 1, 2009 Category: Microbiology Source Type: info

Design and Construction of Synthetic Phage-Displayed Fab LibrariesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 gen...
Source: Springer protocols feed by Microbiology - March 1, 2009 Category: Microbiology Source Type: info

Affinity Isolation of Antigen-Specific Circulating B Cells for Generation of Phage Display-Derived Human Monoclonal AntibodiesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ph...
Source: Springer protocols feed by Microbiology - March 1, 2009 Category: Microbiology Source Type: info

Construction of Phage Antibody Repertoires from the Blood of West Nile Virus-Infected DonorsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - March 1, 2009 Category: Microbiology Source Type: info

Anti-β2GP-I and Anti-Prothrombin Antibodies Generated by Phage DisplayEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - March 1, 2009 Category: Microbiology Source Type: info

The Generation and Selection of Single-Domain, V Region Libraries from Nurse SharksEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 clonin...
Source: Springer protocols feed by Microbiology - March 1, 2009 Category: Microbiology Source Type: info

The Isolation of scFvs Against Small Target MoleculesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - March 1, 2009 Category: Microbiology Source Type: info

Display and Selection of scFv Antibodies on HEK-293T CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - March 1, 2009 Category: Microbiology Source Type: info

Isolation of scFvs that Inhibit the NS3 Protease of Hepatitis C Virus by a Combination of Phage Display and a Bacterial Genetic ScreenEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - March 1, 2009 Category: Microbiology Source Type: info

Preparation of Bacteriophage Lysates and Pure DNAEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - October 28, 2008 Category: Microbiology Source Type: info

Preparation of RNA from Bacteria Infected with Bacteriophages: A Case Study from the Marine Unicellular Synechococcus sp. WH7803 Infected by Phage S-PM2Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ...
Source: Springer protocols feed by Microbiology - October 28, 2008 Category: Microbiology Source Type: info

Quantification of Host and Phage mRNA Expression During Infection Using Real-Time PCREmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ac...
Source: Springer protocols feed by Microbiology - October 28, 2008 Category: Microbiology Source Type: info

Oligonucleotide Microarrays for Bacteriophage Expression StudiesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - October 28, 2008 Category: Microbiology Source Type: info

Purification of Bacteriophages and SDS-PAGE Analysis of Phage Structural Proteins from Ghost ParticlesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - October 28, 2008 Category: Microbiology Source Type: info

Phage Proteomics: Applications of Mass SpectrometryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - October 28, 2008 Category: Microbiology Source Type: info

Isolation Independent Methods of Characterizing Phage Communities 1: Strain Typing Using Fingerprinting MethodsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 p...
Source: Springer protocols feed by Microbiology - October 28, 2008 Category: Microbiology Source Type: info

Isolation Independent Methods of Characterizing Phage Communities 2: Characterizing a MetagenomeEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 availabilit...
Source: Springer protocols feed by Microbiology - October 28, 2008 Category: Microbiology Source Type: info

Phage TypingEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - October 28, 2008 Category: Microbiology Source Type: info

A Genetic Screen to Identify Bacteriophage LysinsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 t...
Source: Springer protocols feed by Microbiology - October 28, 2008 Category: Microbiology Source Type: info

General M13 Phage Display: M13 Phage Display in Identification and Characterization of Protein–Protein InteractionsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - October 28, 2008 Category: Microbiology Source Type: info

Approaches to the Compositional Analysis of DNAEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - October 28, 2008 Category: Microbiology Source Type: info

Isolation of Monoclonal Antibody Fragments from Phage Display LibrariesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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...
Source: Springer protocols feed by Microbiology - October 28, 2008 Category: Microbiology Source Type: info

Internet Resources of Interest to Bacteriophage WorkersEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - October 28, 2008 Category: Microbiology Source Type: info

Determination of Bacteriophage Genome Size by Pulsed-Field Gel ElectrophoresisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - October 28, 2008 Category: Microbiology Source Type: info

Preparation of a Phage DNA Fragment Library for Whole Genome Shotgun SequencingEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ...
Source: Springer protocols feed by Microbiology - October 28, 2008 Category: Microbiology Source Type: info

PCR and Partial Sequencing of Bacteriophage GenomesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 bacteriophag...
Source: Springer protocols feed by Microbiology - October 28, 2008 Category: Microbiology Source Type: info

In Sffamily Identification of Genes in Bacteriophage DNAEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - October 28, 2008 Category: Microbiology Source Type: info

Determining DNA Packaging Strategy by Analysis of the Termini of the Chromosomes in Tailed-Bacteriophage VirionsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 DN...
Source: Springer protocols feed by Microbiology - October 28, 2008 Category: Microbiology Source Type: info

In silico Characterization of DNA Motifs with Particular Reference to Promoters and TerminatorsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 - October 28, 2008 Category: Microbiology Source Type: info

Molecular Phylogenetics: Testing Evolutionary HypothesesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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. ...
Source: Springer protocols feed by Microbiology - October 28, 2008 Category: Microbiology Source Type: info

Preparation of RNA from Bacteria Infected with Bacteriophages: A Case Study from the Marine Unicellular Synechococcus sp. WH783 Infected by Phage S-PM2Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 ...
Source: Springer protocols feed by Microbiology - October 28, 2008 Category: Microbiology Source Type: info

Overview of ELISA in Relation to Other DisciplinesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Source: Springer protocols feed by Microbiology - October 1, 2008 Category: Microbiology Source Type: info

Charting Methods for Internal Quality Control of Indirect ELISAEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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...
Source: Springer protocols feed by Microbiology - October 1, 2008 Category: Microbiology Source Type: info

Ruggedness and Robustness of Tests: Aspects of Kit Use and ValidationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Source: Springer protocols feed by Microbiology - October 1, 2008 Category: Microbiology Source Type: info

More Advanced Statistical Methods for Quality Assurance, Test Validation, and InterpretationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Source: Springer protocols feed by Microbiology - October 1, 2008 Category: Microbiology Source Type: info

Methods for the Isolation of Viruses from Environmental SamplesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Viruses are omnipresent and extraordinarily abundant in the microbial ecosystems of water, soil, and sediment. In nearly every reported case for aquatic and porous media environments (soils and sediments) viral abundance exceeds that of co-occurring host populations by 10–100-fold. If current estimates based on metagenome DNA sequence data are correct, then viruses represent the largest reservoir of unknown genetic diversity on Earth. Microscopy and molecular genetic tools have been critical in demonstrating that viruses are a dynamic component of microbial ecosystems capable of significantly influencing the producti...
Source: Springer protocols feed by Microbiology - July 1, 2008 Category: Microbiology Source Type: info

Determination of Virus Abundance by Epifluorescence MicroscopyEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Determination of virus abundance using epifluorescence microscopy is a rapid and accurate method. The protocol requires the concentration of virus particles by collection on a filter. The nucleic acid in the virus particles is then stained with a fluorescent stain and the sample viewed with an epifluorescence microscope. The method was originally developed to determine the abundance of virus particles in water samples, however the protocol has been adapted for cultures and sediment samples. Although the method provides total counts of all virus-sized particles, regardless of infectivity, the method can be used for rapidly ...
Source: Springer protocols feed by Microbiology - July 1, 2008 Category: Microbiology Source Type: info

Enumeration of Bacteriophages Using Flow CytometryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Rapid identification and enumeration of the numerically important bacteriophages has been till recently a major limitation for studies of virus ecology. The development of sensitive nucleic acid stains, in combination with flow cytometric techniques, has changed this. The flow cytometric method allows the detection and discrimination of a wide variety of viruses of different morphology, genome type, and size. The present paper describes an optimized protocol for the enumeration of bacteriophages using a standard benchtop flow cytometer.
Source: Springer protocols feed by Microbiology - July 1, 2008 Category: Microbiology Source Type: info

Basic Phage Electron MicroscopyEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Negative staining of purified viruses is the most important electron microscopical technique in virology. The principal stains are phosphotungstate and uranyl acetate, both of which have problems and advantages. Particular problems are encountered in photography, calibration of magnification, measurements, and interpretation of artifacts.
Source: Springer protocols feed by Microbiology - July 1, 2008 Category: Microbiology Source Type: info

Phage Classification and CharacterizationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Prokaryote viruses include 14 officially accepted families and at least five other potential families awaiting classification. Approximately 5,500 prokaryote viruses have been examined in the electron microscope. Classification has a predictive value and is invaluable to control experimental techniques and results. In describing viruses, the choice of methods depends on structure and taxonomical position of viruses. The study of isometric, filamentous, and pleomorphic viruses requires more detailed investigations than that of tailed species.
Source: Springer protocols feed by Microbiology - July 1, 2008 Category: Microbiology Source Type: info

Phage Host Range and Efficiency of PlatingEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The host range of a bacteriophage is defined by what bacterial genera, species and strains it can lyse; it is one of the defining biological characteristics of a particular bacterial virus. Because of host factors such as masking by O antigens that affects injection and the presence of restriction endonucleases, the relative efficiency of plating (EOP), that is, the titer of the phage on a given bacterial cell line compared to the maximum titer observed, may vary considerably. This chapter describes rapid procedures for determining the host range and relative EOP on each host of any phage.
Source: Springer protocols feed by Microbiology - July 1, 2008 Category: Microbiology Source Type: info