Springer protocols feed by Microbiology 
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.
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.Use of RNA Polymerase Molecular Beacon Assay to Measure RNA Polymerase Interactions with Model Promoter Fragments
RNA polymerase–promoter interactions that keep the transcription initiation complex together are complex and multipartite, and formation of the RNA polymerase–promoter complex proceeds through multiple intermediates. Short promoter fragments can be used as a tool to dissect RNA polymerase–promoter interactions and to pinpoint elements responsible for specific properties of the entire promoter complex. A recently developed fluorometric molecular beacon assay allows one to monitor the enzyme interactions with various DNA probes and quantitatively characterize partial RNA polymerase–promoter interactio...
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
Biochemical Analysis of Transcription Termination by RNA Polymerase III from Yeast Saccharomyces cerevisiae
Eukaryotic RNA polymerase III (pol III) transcribes short noncoding RNA genes such as those encoding tRNAs, 5S rRNA, U6 snRNA, and a few others. As compared to its pol II counterpart, Pol III has several advantages, including the relative simplicity, stability, and more direct connectivity of its transcription machinery. Only two transcription factor complexes, TFIIIB and TFIIIC, are required to faithfully initiate and direct multiple rounds of transcription by pol III. Moreover, in contrast to an intricate multipartite mechanism of pol II termination, pol III termination is extremely simple, responsive to a monopartite si...
Source: Springer protocols feed by Microbiology - February 13, 2015 Category: Microbiology Source Type: news
The Pyrosequencing Protocol for Bacterial Genomes
The pyrosequencing methodology was applied in 2005 by 454 Lifesciences to the emerging field of next generation sequencing (NGS), revolutionizing the way of DNA sequencing. In the last years the same strategy grew up and was technologically updated, reaching a high throughput in terms of amount of generated sequences (reads) per run and in terms of length of sequence up to values of 800–1,000 bases. These features of pyrosequencing perfectly fit to bacterial genome sequencing for the de novo assemblies and resequencing as well. The approaches of shotgun and paired ends sequencing allow the bacterial genome finishing ...
Source: Springer protocols feed by Microbiology - January 1, 2015 Category: Microbiology Source Type: news
The Illumina-Solexa Sequencing Protocol for Bacterial Genomes
Based on reversible dye-terminators technology, the Illumina-solexa sequencing platform enables rapid sequencing-by-synthesis (SBS) of large DNA stretches spanning entire genomes, with the latest instruments capable of producing hundreds of gigabases of data in a single sequencing run. Illumina’s NGS instruments powerfully combine the flexibility of single reads with short- and long-insert paired-end reads, and enable a wide range of DNA sequencing applications. Here, we describe the paired-end library preparation with an average insert size of 470 bp, 2 kbp, and 6 kbp, together with the DNA cluster generation and se...
Source: Springer protocols feed by Microbiology - January 1, 2015 Category: Microbiology Source Type: news
Bacterial Metabarcoding by 16S rRNA Gene Ion Torrent Amplicon Sequencing
Ion Torrent is a next generation sequencing technology based on the detection of hydrogen ions produced during DNA chain elongation; this technology allows analyzing and characterizing genomes, genes, and species. Here, we describe an Ion Torrent procedure applied to the metagenomic analysis of 16S rRNA gene amplicons to study the bacterial diversity in food and environmental samples. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - January 1, 2015 Category: Microbiology Source Type: news
Comparative Analysis of Gene Expression: Uncovering Expression Conservation and Divergence Between Salmonella enterica Serovar Typhimurium Strains LT2 and 1428S
Different strains of the same organism can share a large amount of their genetic material, the so called core pangenome. Nevertheless, these species can display different lifestyles and it is still not well known to what extent the core pangenome plays a role in the divergence of lifestyles between the two organisms. Here, we present a procedure for uncovering the conservation and divergence of gene expression by using large expression compendia. We will use data from two Salmonella enterica serovar Typhimurium strains as an example here, strain LT2 and strain 14028S, to assess if there are orthologous gene pairs with diff...
Source: Springer protocols feed by Microbiology - January 1, 2015 Category: Microbiology Source Type: news
High-Throughput Phenomics
Standard protocols are available in order to apply Phenotype MicroArray (PM) technology to characterize different groups of microorganisms. Nevertheless, there is the need to pay attention to several crucial steps in order to obtain high-quality and reproducible data from PM, such as the choice of the Dye mix, the type and concentration of the carbon source in metabolic experiments, the use of a buffered medium. A systematic research of auxotrophies in strains to be tested should be carefully evaluated before starting with PM experiments. Detailed protocols to obtain defined and reproducible phenotypic profiles for bacteri...
Source: Springer protocols feed by Microbiology - January 1, 2015 Category: Microbiology Source Type: news
Raw Sequence Data and Quality Control
Next-generation sequencing technologies are extensively used in many fields of biology. One of the problems, related to the utilization of this kind of data, is the analysis of raw sequence quality and removal (trimming) of low-quality segments while retaining sufficient information for subsequent analyses. Here, we present a series of methods useful for converting and for refinishing one or more sequence files. One of the methods proposed, based on dynamic trimming, as implemented in the software StreamingTrim allows a fast and accurate trimming of sequence files, with low memory requirement. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - January 1, 2015 Category: Microbiology Source Type: news
Separation of Basic Proteins from Leishmania Using a Combination of Free Flow Electrophoresis (FFE) and 2D Electrophoresis (2-DE) Under Basic Conditions
Basic proteins, an important class of proteins in intracellular organisms such as Leishmania, are usually underrepresented on 2D gels. This chapter describes a method combining basic proteins fractionation using Free flow electrophoresis in isoelectric focusing mode (IEF-FFE) followed by protein separation using two-dimensional gel electrophoresis (2-DE) in basic conditions. The combination of these two techniques represents a great improvement for the visualization of Leishmania proteins with basic pI using 2D gels. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - November 13, 2014 Category: Microbiology Source Type: news
A Transposon-Based Tool for Transformation and Mutagenesis in Trypanosomatid Protozoa
The ability of transposable elements to mobilize across genomes and affect the expression of genes makes them exceptional tools for genetic manipulation methodologies. Several transposon-based systems have been modified and incorporated into shuttle mutagenesis approaches in a variety of organisms. We have found that the Mos1 element, a DNA transposon from Drosophila mauritiana, is suitable and readily adaptable to a variety of strategies to the study of trypanosomatid parasitic protozoa. Trypanosomatids are the causative agents of a wide range of neglected diseases in underdeveloped regions of the globe. In this chapter w...
Source: Springer protocols feed by Microbiology - November 13, 2014 Category: Microbiology Source Type: news
Protein Microarrays for Parasite Antigen Discovery
The host serological profile to a parasitic infection, such as schistosomiasis, can be used to define potential vaccine and diagnostic targets. Determining the host antibody response using traditional approaches is hindered by the large number of putative antigens in any parasite proteome. Parasite protein microarrays offer the potential for a high-throughput host antibody screen to simplify this task. In order to construct the array, parasite proteins are selected from available genomic sequence and protein databases using bioinformatic tools. Selected open reading frames are PCR amplified, incorporated into a vector for ...
Source: Springer protocols feed by Microbiology - November 13, 2014 Category: Microbiology Source Type: news
RNA-Seq Approaches for Determining mRNA Abundance in Leishmania
High-throughput sequencing of cDNA copies of mRNA (RNA-seq) provides a digital read-out of mRNA levels over several orders of magnitude, as well as mapping the transcripts to the nucleotide level. Here we describe an RNA-seq approach that exploits the 39-nucleotide mini-exon or spliced leader (SL) sequence found at the 5′ end of all Leishmania (and other trypanosomatid) mRNAs. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - November 13, 2014 Category: Microbiology Source Type: news
The Genome-Wide Identification of Promoter Regions in Toxoplasma gondii
Parasites change their transcriptional systems in different developmental stages and in response to environmental changes. To investigate the molecular mechanisms that underlie transcriptional regulation, it is essential to identify the exact positions of the transcriptional start sites (TSSs) and characterize the upstream promoter regions. However, it has been essentially impossible to obtain comprehensive information using conventional methods. Here, we introduce our TSS-seq method, which combines full-length technology, oligo-capping, and rapidly developing next-generation sequencing technology. TSS-seq has enabled iden...
Source: Springer protocols feed by Microbiology - November 13, 2014 Category: Microbiology Source Type: news
Techniques to Study Epigenetic Control and the Epigenome in Parasites
Epigenetics is the study of heritable changes in gene expression that occur independent of the DNA sequence. Due to their intimacy with DNA, histones have a central role in chromatin structure and epigenetic regulation. Their tails are subject to posttranslational modifications (PTMs) that together with chromatin-remodeling proteins control the access of different proteins to DNA and allow a precise response to different environmental conditions. The first part of this chapter is dedicated to histone enrichment methods that allow the study of histones using techniques such as immunoblot or mass spectrometry for the mapping...
Source: Springer protocols feed by Microbiology - November 13, 2014 Category: Microbiology Source Type: news
Detection of Carbapenemases Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) Meropenem Hydrolysis Assay
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been recently introduced to many diagnostic microbiological laboratories. Besides the identification of bacteria and fungi, that technique provides a potentially useful tool for the detection of antimicrobial resistance, especially of that conferred by β-lactamases. Here, we describe an assay allowing a detection of meropenem hydrolysis in clinical isolates of Enterobacteriaceae, Pseudomonas spp., and Acinetobacter baumannii using MALDI-TOF MS. This method is able to confirm carbapenemases within 3 h. The results are import...
Source: Springer protocols feed by Microbiology - October 17, 2014 Category: Microbiology Source Type: news
