12 Electroporation of Mycobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
Tanya Parish
13 Gene Switching and Essentiality Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Amanda Claire Brown
14 Oligo-Mediated Recombineering and its Use for Making SNPs,
Knockouts, Insertions, and Fusions in Mycobacterium tuberculosis. . . . . . . . . . . . . 301
Kenan C. Murphy
15 Using Proteolytic Hypomorphs to Detect Small Molecule
Mechanism of Action. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
Eachan O. Johnson and Deborah T. Hung
16 CRISPR Interference (CRISPRi) for Targeted Gene Silencing
in Mycobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Andrew I. Wong and Jeremy M. Rock
17 Exploiting Fluorescent Proteins to Understand Mycobacterium
tuberculosis Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
David Giacalone, Lu Huang, and Shumin Tan
18 Metabolic Labeling of Live Mycobacteria with Trehalose-Based Probes. . . . . . . . 385
Nicholas Banahene and Benjamin M. Swarts
19 Identification and Characterization of Mycobacterial Species
Using Whole-Genome Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
Marco A. Riojas, Andrew M. Frank, Samuel R. Greenfield,
Stephen P. King, Conor J. Meehan, Michael Strong,
Alice R. Wattam, and Manzour Hernando Hazb on
20 Whole-Genome Sequencing of Mycobacterium tuberculosis
Directly from Sputum Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
Amanda Claire Brown
21 Experimental and Computational Workflow for RNA Sequencing
in Mycobacterium tuberculosis: From Total RNA to Differentially
Expressed Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481
Shuyi Ma, Richard M. Jones Jr., Natalie S. Gleason,
Jessica Farrow-Johnson, and David R. Sherman
22 RNA Sequencing for Transcript 5
0 -End Mapping in Mycobacteria . . . . . . . . . . . . 513
M. Carla Martini, Huaming Sun, and Scarlet S. Shell
23 Methods for Proteomic Analyses of Mycobacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . 533
Carolina Mehaffy, Megan Lucas, Nicole A. Kruh-Garcia,
and Karen M. Dobos
24 Targeted Lipidomics of Mycobacterial Lipids and Glycolipids . . . . . . . . . . . . . . . . 549
Emilie Layre
25 Metabolomics of Mycobacterium tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
Kyle A. Planck and Kyu Rhee
26 Determining Minimum Inhibitory Concentrations in Liquid
Cultures or on Solid Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595
Qinglan Wang and Helena I. M. Boshoff
viii
Contents
Tanya Parish
13 Gene Switching and Essentiality Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Amanda Claire Brown
14 Oligo-Mediated Recombineering and its Use for Making SNPs,
Knockouts, Insertions, and Fusions in Mycobacterium tuberculosis. . . . . . . . . . . . . 301
Kenan C. Murphy
15 Using Proteolytic Hypomorphs to Detect Small Molecule
Mechanism of Action. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
Eachan O. Johnson and Deborah T. Hung
16 CRISPR Interference (CRISPRi) for Targeted Gene Silencing
in Mycobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Andrew I. Wong and Jeremy M. Rock
17 Exploiting Fluorescent Proteins to Understand Mycobacterium
tuberculosis Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
David Giacalone, Lu Huang, and Shumin Tan
18 Metabolic Labeling of Live Mycobacteria with Trehalose-Based Probes. . . . . . . . 385
Nicholas Banahene and Benjamin M. Swarts
19 Identification and Characterization of Mycobacterial Species
Using Whole-Genome Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
Marco A. Riojas, Andrew M. Frank, Samuel R. Greenfield,
Stephen P. King, Conor J. Meehan, Michael Strong,
Alice R. Wattam, and Manzour Hernando Hazb on
20 Whole-Genome Sequencing of Mycobacterium tuberculosis
Directly from Sputum Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
Amanda Claire Brown
21 Experimental and Computational Workflow for RNA Sequencing
in Mycobacterium tuberculosis: From Total RNA to Differentially
Expressed Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481
Shuyi Ma, Richard M. Jones Jr., Natalie S. Gleason,
Jessica Farrow-Johnson, and David R. Sherman
22 RNA Sequencing for Transcript 5
0 -End Mapping in Mycobacteria . . . . . . . . . . . . 513
M. Carla Martini, Huaming Sun, and Scarlet S. Shell
23 Methods for Proteomic Analyses of Mycobacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . 533
Carolina Mehaffy, Megan Lucas, Nicole A. Kruh-Garcia,
and Karen M. Dobos
24 Targeted Lipidomics of Mycobacterial Lipids and Glycolipids . . . . . . . . . . . . . . . . 549
Emilie Layre
25 Metabolomics of Mycobacterium tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
Kyle A. Planck and Kyu Rhee
26 Determining Minimum Inhibitory Concentrations in Liquid
Cultures or on Solid Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595
Qinglan Wang and Helena I. M. Boshoff
viii
Contents