This collection of experimental and computational strategies and techniques for microbial genome-scale essentiality studies was developed and written by the leading research groups in the field. In addition to wet-lab protocols, the book describes statistical methods essential for planning and evaluating successful large-scale essentiality screens; in-silico prediction of gene essentiality using genome-scale reconstructed metabolic models; and data integration and comparative analysis of genomic databases. Each protocol follows the successful Methods in Molecular Biology™ series format, offering an introduction outlining the underlying principles, step-by-step instructions, a list of necessary equipment and reagents, and tips on troubleshooting and avoiding pitfalls.

Offers a comprehensive, up-to-date collection of all the main strategies of experimental genome-scale gene essentiality analysis Contains a detailed description of the procedures as well as a discussion of potential difficulties and failures Includes chapters on statistical analysis of the generated datasets and their integration into existing genomic databases Includes supplementary material: sn.pub/extras

Inhalt
Overview of Whole-Genome Essentiality Analysis.- Experimental Protocols.- Transposon-Based Strategies for the Identification of Essential Bacterial Genes.- Identification and Analysis of Essential Genes in Haemophilus influenzae.- Transposon Site Hybridization in Mycobacterium tuberculosis.- Essential Genes in the Infection Model of Pseudomonas aeruginosa PCR-Based Signature-Tagged Mutagenesis.- Whole-Genome Detection of Conditionally Essential and Dispensable Genes in Escherichia coli via Genetic Footprinting.- Generating a Collection of Insertion Mutations in the Staphylococcus 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. Case 1: Pseudomonas aeruginosa PAO1.- 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 K-12.- 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 Lethality Analysis by Microarray.- Scarless Engineering of the Escherichia coli Genome.- Minimization of the Escherichia coli Genome Using the 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 Use of Conditionally Expressed Antisense RNA to Achieve Essential Gene Knockdowns in Staphylococcus aureus.- of Conditional Lethal Amber Mutations in Escherichia coli.- Bioinformatics.- Statistical Methods for Building Random Transposon Mutagenesis Libraries.- Statistical Evaluation of Genetic Footprinting Data.- Modeling Competitive Outgrowth of Mutant Populations: Why Do Essentiality Screens Yield Divergent Results?.- Statistical Analysis of Fitness Data Determined by TAG Hybridization on Microarrays.- Profiling of Escherichia coli Chromosome 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.- Comparative Approach to Analysis of Gene Essentiality.