实验基础

A method is described for determination of the concentration of infectious phage particles by the direct plating plaque assay, which is simpler and faster than the double agar overlay plaque procedure outlined in the previous chapter.

The determination of the concentration of infectious phage particles is fundamental to many protocols in phage biology, genetics, and molecular biology. In this chapter the classical overlay protocol is described.

Recent studies have established that the most abundant life form, that of phages, has had major influence on the biosphere, bacterial evolution, bacterial genome, and lateral gene transmission. Importantly the phages have served and continue to serve as valuable model systems. Such stud ...

The determination of the concentration of infectious phage particles is fundamental to many protocols in phage biology, genetics, and molecular biology. Described here is a drop plaque assay, which, being simpler, faster and more efficient than either the classical overlay or direct pla ...

The fate of lysogens following prophage induction has assumed added significance with the finding that in many pathogens virulence genes are carried on prophages and, in some, the production and/or release of the virulence factor is under control of the phage lytic regulatory program. We out ...

Transduction is the process in which bacterial DNA is transferred from one bacterial cell to another by means of a phage particle. There are two types of transduction, generalized transduction and specialized transduction. In this chapter two of the best-studied systems – Escherichia col ...

As interest in lytic phages as antimicrobial therapies or as treatments to reduce environmental contamination with pathogenic bacteria has increased, so has the need to determine if the use of lytic phages may lead to dissemination of virulence factors through generalized transduct ...

This chapter describes a method for the generation of polyclonal antibodies against bacteriophages and how these may be assayed immunochemically and biologically.

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 ly ...

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 ...

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. Spec ...

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 ...

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 ...

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 bacterio ...

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 c ...

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 (- or -single-strand extensions), circ ...

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 ev ...

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 p ...

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 expres ...

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 concentrat ...