Springer protocols feed by Microbiology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Pulse Field Gel Electrophoresis
(PFGE) is a powerful genotyping technique used for the separation of large DNA molecules (entire genomic DNA) after digesting it with unique restriction enzymes and applying to a gel matrix under the electric field that periodically changes direction. PFGE is a variation of agarose gel electrophoresis that permits analysis of bacterial DNA fragments over an order of magnitude larger than that with conventional restriction enzyme analysis. It provides a good representation of the entire bacterial chromosome in a single gel with a highly reproducible restriction profile, providing clearly distinct and well-resolved DNA frag...
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
Chemical and UV Mutagenesis
The ability to create mutations is an important step towards understanding bacterial physiology and virulence. While targeted approaches are invaluable, the ability to produce genome-wide random mutations can lead to crucial discoveries. Transposon mutagenesis is a useful approach, but many interesting mutations can be missed by these insertions that interrupt coding and noncoding sequences due to the integration of an entire transposon. Chemical mutagenesis and UV-based random mutagenesis are alternate approaches to isolate mutations of interest with the potential of only single nucleotide changes. Once a standard method,...
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
Generation of a Transposon Mutant Library in Staphylococcus aureus and Staphylococcus epidermidis Using bursa aurealis
Transposon mutagenesis is a genetic process that involves the random insertion of transposons into a genome resulting in the disruption of function of the genes in which they insert. Identification of the insertion sites through DNA sequencing allows for the identification of the genes disrupted and the creation of “libraries” containing a collection of mutants in which a large number of the nonessential genes have been disrupted. These mutant libraries have been a great resource for investigators to understand the various biological functions of individual genes, including those involved in metabolism, antibio...
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
Creation of Staphylococcal Mutant Libraries Using Transposon Tn917
Non-directed mutagenesis of the staphylococcal genome is a global approach that can be used to identify the genetic basis for phenotypes of interest as well as for identifying regulators of gene expression. One such approach that has been widely used in the study of S. aureus and S. epidermidis is transposon Tn917 mutagenesis to generate random libraries of mutants. This chapter describes the use of plasmid pLTV1 (containing Tn917-lac) to generate Tn917 transposon mutants. Through the use of temperature manipulation and antibiotic selection, staphylococcal strains harboring this plasmid can be effectively mutagenized to cr...
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
Allelic Exchange
Methods used to understand the function of a gene/protein are one of the hallmarks of modern molecular genetics. The ability to genetically manipulate bacteria has become a fundamental tool in studying these organisms and while basic cloning has become a routine task in molecular biology laboratories, generating directed mutations can be a daunting task. This chapter describes the method of allelic exchange in Staphylococcus aureus using temperature-sensitive plasmids that have successfully produced a variety of chromosomal mutations, including in-frame deletions, insertion of antibiotic-resistance cassettes, and even sing...
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
Bacteriophage Transduction in Staphylococcus aureus
The genetic manipulation of Staphylococcus aureus for molecular experimentation is a valuable tool for assessing gene function and virulence. Genetic variability between strains coupled with difficult laboratory techniques for strain construction is a frequent roadblock in S. aureus research. Bacteriophage transduction greatly increases the speed and ease of S. aureus studies by allowing movement of chromosomal markers and plasmids between strains. This technique enables the S. aureus research community to focus investigations on clinically relevant isolates. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
Bacteriophage Transduction in Staphylococcus aureus: Broth-Based Method
The ability to move DNA between Staphylococcus strains is essential for the genetic manipulation of this bacterium. Often in the Staphylococci, this is accomplished through transduction using generalized transducing phage and can be performed in different ways and therefore the presence of two transduction procedures in this book. The following protocol is a relatively easy-to-perform, broth-based procedure that we have used extensively to move both plasmids and chromosomal fragments between strains of Staphylococcus aureus. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
Rapid Isolation of DNA from Staphylococcus
Many methods exist to extract DNA from bacteria. Indeed, there is no shortage of kits available from manufacturers that allow for isolation of highly purified DNA. However, for many applications samples do not need to be extremely pure (i.e., free of contaminating proteins or RNA). Furthermore, for quick genetic screening, it is often useful to have a rapid and inexpensive option for DNA isolation from small samples. For these occasions, the method found in this chapter provides a cost-efficient, yet rapid, isolation of DNA. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
Method for Preparation and Electroporation of S. aureus and S. epidermidis
For bacterial species that are not known to be naturally competent, such as Staphylococcus aureus and Staphylococcus epidermidis, electroporation is an efficient method for introducing genetic material into the cell. The technique utilizes electrical pulses to transiently permeabilize bacterial cell membranes, which allows for the passage of plasmid DNA across the membranes. Here, we describe methods for preparing electrocompetent S. aureus and S. epidermidis cells and outline a procedure for electroporation of the prepared competent cells. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
Splicing by Overlap Extension PCR to Obtain Hybrid DNA Products
Genetic manipulation of bacteria often requires the joining together of more than one DNA segment to form a hybrid DNA molecule. This can be accomplished by PCR followed by restriction endonuclease digestions and ligations. However, this approach can often become laborious and expensive. Here is described a well-established method for using primer design and PCR to obtain hybrid products for use in cloning vectors, mutagenesis protocols, and other applications. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
Complementation Plasmids, Inducible Gene-Expression Systems, and Reporters for Staphylococci
A cornucopia of methods and molecular tools is available for genetic modification of staphylococci, as shown for at least ten different species to date (Prax et al. Microbiology 159:421–435, 2013). This chapter reviews a number of frequently used vectors for complementation purposes that usually replicate in E. coli and staphylococci and differ in parameters including copy number, mode of replication, and sequence length. Systems for the artificial control of gene expression are described that are modulated by low-molecular-weight effectors such as metal cations, carbohydrates, and antibiotics. Finally, the usefulnes...
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
Restriction–Modification Systems as a Barrier for Genetic Manipulation of Staphylococcus aureus
Genetic manipulation is a powerful approach to study fundamental aspects of bacterial physiology, metabolism, and pathogenesis. Most Staphylococcus aureus strains are remarkably difficult to genetically manipulate as they possess strong host defense mechanisms that protect bacteria from cellular invasion by foreign DNA. In S. aureus these bacterial “immunity” mechanisms against invading genomes are mainly associated with restriction–modification systems. To date, prokaryotic restriction–modification systems are classified into four different types (Type I–IV), all of which have been found in t...
Source: Springer protocols feed by Microbiology - October 22, 2015 Category: Microbiology Source Type: news
Genome-Wide Probing of RNA Structures In Vitro Using Nucleases and Deep Sequencing
RNA structure probing is an important technique that studies the secondary and tertiary conformations of an RNA. While it was traditionally performed on one RNA at a time, recent advances in deep sequencing has enabled the secondary structure mapping of thousands of RNAs simultaneously. Here, we describe the method Parallel Analysis for RNA Structures (PARS), which couples double and single strand specific nuclease probing to high throughput sequencing. Upon cloning of the cleavage sites into a cDNA library, deep sequencing and mapping of reads to the transcriptome, the position of paired and unpaired bases along cellular ...
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
Biotin-Genomic Run-On (Bio-GRO): A High-Resolution Method for the Analysis of Nascent Transcription in Yeast
Transcription is a highly complex biological process, with extensive layers of regulation, some of which remain to be fully unveiled and understood. To be able to discern the particular contributions of the several transcription steps it is crucial to understand RNA polymerase dynamics and regulation throughout the transcription cycle. Here we describe a new nonradioactive run-on based method that maps elongating RNA polymerases along the genome. In contrast with alternative methodologies for the measurement of nascent transcription, the BioGRO method is designed to minimize technical noise that arises from two of the most...
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
Translation Analysis at the Genome Scale by Ribosome Profiling
Ribosome profiling is an emerging approach using deep sequencing of the mRNA part protected by the ribosome to study protein synthesis at the genome scale. This approach provides new insights into gene regulation at the translational level. In this review we describe the protocol to prepare polysomes and extract ribosome protected fragments before to deep sequence them. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
