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This book contains a comprehensive collection of experimental and computational strategies and techniques for microbial genome-scale essentiality studies, developed and presented by the leading groups in the field. In addition to wet-lab protocols, the book describes (i) statistical methods essential for planning successful large-scale essentiality screens, as well for data evaluation and analysis; (ii) in-silico prediction of gene essentiality using genome-scale reconstructed metabolic models; and (iii) data integration and comparative analysis in the context of genomic databases. All protocols follow the successful Methods in Molecular Biology series format, each offering an introduction outlining the principles behind the techniques, step-by-step instructions, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding pitfalls, and are intended for both novice and expert scientists.
Overview of Whole Genome Essentiality Analysis.- Transposon-based strategies for the identification of essential bacterial genes.- Identification and analysis of essential genes in Haemophilus influenzae.- Transposon site hybridization TraSH. in Mycobacterium tuberculosis.- Essential genes in the infection model of P.aeruginosa PCR-based signature-tagged mutagenesis.- Whole-genome detection of conditionally essential and dispensable genes in E. coli via genetic footprinting.- Generating a Collection of Insertion Mutations in the S. aureus Genome Using bursa aurealis.- Multipurpose Transposon Insertion Libraries for Large-Scale Analysis of Gene Function in Yeast.- How to: make a defined near-saturation mutant library.- Comparing insertion libraries in two Pseudomonas aeruginosa strains to assess gene essentiality.- The Construction of Systematic In- frame, Single-gene Knockout Mutant Collection in Escherichia coli K-12.- The Applications of Systematic In- frame, Single-gene Knockout Mutant Collection of Escherichia coli K-12.- A Novel, Simple, High-throughput Method for Isolation of Genome-wide Transposon Insertion Mutants of Escherichia coli K12.- High-throughput creation of a whole-genome collection of yeast knockout strains.- Analysis of Genetic Interactions on a Genome-Wide Scale in Budding Yeast: Diploid-based Synthetic Lethal Analysis by Microarray dSLAM.- Scarless engineering of the Escherichia coli genome.- Minimization of the Escherichia coli genome using Tn5-targeted Cre/loxP excision system.- Construction of long chromosomal deletion mutants of Escherichia coli and minimization of the genome.- Identification of essential genes in Staphylococcus aureus by construction and screening of conditional mutant library.- Techniques for the Isolation and Utilization of Conditionally Expressed Antisense RNA to Achieve Essential Gene Knockdowns in the Pathogen Staphylococcus aureus.- Introduction of conditional lethal amber mutations in Escherichia coli.- Statistical Methods for Building Random Transposon Mutagenesis Libraries.- Statistical evaluation of genetic footprinting data.- Modeling competitive outgrowth of mutant populations: Why do different techniques yield divergent lists of essential genes?.- Statistical Analysis of Fitness Data Determined by TAG Hybridization on Microarrays.- Profiling of E. coli Chromosome PEC. Database.- Gene essentiality analysis based on DEG, a database of essential genes.- Detection of Essential Genes in Streptococcus pneumoniae Using Bioinformatics and Allelic Replacement Mutagenesis.- Design and application of genome-scale reconstructed metabolic models.- Predicting gene essentiality using genome-scale in silico models.