Table of Contents INTRODUCTION 1. Karen Joy Shaw. Overview of Whole Genome Essentiality Analysis Part I. EXPERIMENTAL PROTOCOLS I.1 Populational genome-wide essentiality screens 2. Bill Reznikoff and Kelly Winterberg. Transposon-based strategies for the identification of essential bacterial genes. 3. Sandy M.S. Wong and Brian J. Akerley: Identification and analysis of essential genes in Haemophilus influenzae. 4. Jeffrey P. Murry, Christopher M. Sassetti, James M. Lane, Zhifang Xie, and Eric J. Rubin 'Transposon site hybridization TraSH. in Mycobacterium tuberculosis' 5. François Sanschagrin, Irena Kukavica-Ibrulj, and Roger C. Levesque Essential genes in the infection model of P.aeruginosa PCR-based signature-tagged mutagenesis 6. Michael Scholle, Svetlana Gerdes 'Whole-genome detection of conditionally essential and dispensable genes in E. coli via genetic footprinting' 7. Taeok Bae, Elizabeth Marland Glass, Olaf Schneewind, and Dominique Missiakas Generating a Collection of Insertion Mutations in the S. aureus Genome Using bursa aurealis 8. Anuj Kumar Multipurpose Transposon Insertion Libraries for Large-Scale Analysis of Gene Function in Yeast I.2. Systematic collections of knockout mutants 9. Michael Jacobs 'How to: make a defined near-saturation mutant library. Case one: Pseudomonas aeruginosa PAO1' 10. Nicole T. Liberati, Jonathan M. Urbach, Tara K. Thurber, Gang Wu, and Frederick M. Ausubel Comparing insertion libraries in two Pseudomonas aeruginosa strains to assess gene essentiality. 11. Tomoya Baba and Hirotada Mori 'The Construction of Systematic In- frame, Single-gene Knockout MutantCollection in Escherichia coli K-12' 12. Tomoya Baba, Hsuan-Cheng Huan, Kirill Datsenko, Barry L. Wanner, and Hirotada Mori: 'The Applications of Systematic In- frame, Single-gene Knockout Mutant Collection of Escherichia coli K-12' 13. Takeyoshi Miki, Yoshihiro Yamamoto, and Hideo Matsuda. A Novel, Simple, High-throughput Method for Isolation of Genome-wide Transposon Insertion Mutants of Escherichia coli K12 14. Angela M. Chu and Ronald W. Davis 'High-throughput creation of a whole-genome collection of yeast knockout strains' 15. Pamela B. Meluh, Xuewen Pan, Daniel S. Yuan, Carol Tiffany, Ou Chen, Sharon Sookhai-Mahadeo, Xaioling Wang, Brian Peyser, Rafael Irizarry, Forrest Spencer, and Jef D. Boeke Analysis of Genetic Interactions on a Genome-Wide Scale in Budding Yeast: Diploid-based Synthetic Lethal Analysis by Microarray dSLAM. I.3 Genome minimization 16. Tamás Fehér, Ildikó Karcagi, Zsuzsa Gyorfy, Kinga Umenhoffer, Bálint Csörgo and György Pósfai 'Scarless engineering of the Escherichia coli genome' 17. Byung Jo Yu, and Sun Chang Kim: Minimization of the Escherichia coli genome using Tn5-targeted Cre/loxP excision system 18. Jun-ichi Kato and Masayuki Hashimoto Construction of long chromosomal deletion mutants of Escherichia coli and minimization of the genome I.4 Conditional knockouts 19. Dezhong Yin and Yinduo Ji. 'Identification of essential genes in Staphylococcus aureus by construction and screening of conditional mutant library' 20. Allyn Forsyth and Liangsu Wang Techniques for the Isolation and Utilization of Conditionally Expressed Antisense RNA to Achieve Essential Gene Knockdowns in the Pathogen Staphylococcus aureus 21. Christopher D. Herring
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. It contains detailed description of the procedures, discussion of potential difficulties and failures. All protocols follow the successful Methods in Molecular Biology(TM) series format.
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(TM) 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.