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Experiments in Bacterial Genetics: A Laboratory Manual


Subject Area(s):  BacteriaGeneticsLaboratory Techniques

Edited by Lionello Bossi, Université Paris-Saclay; Andrew Camilli, Tufts University School of Medicine; Angelika Gründling, Imperial College London

Download a Free Excerpt from Experiments in Bacterial Genetics: A Laboratory Manual:

Preface
High-Throughput Mutant Screening in Vibrio cholerae via Transposon Sequencing
Index


© 2024 • 352 pages, illustrated (52 color), index
Paperback •
ISBN  978-1-621824-49-7

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  •     Description    
  •     Contents    

Description

Bacteria are powerful systems for genetic research. They are fast growing and easy to propagate and maintain, and many tools have been developed for their genomic analyses and genetic manipulation. The study of bacteria and their viruses allowed deciphering the genetic code and has shaped our understanding of fundamental biological processes such as DNA replication, transcription, and translation.

This manual provides a collection of experimental procedures for genetic research in bacteria. It describes basic methods for the isolation and propagation of bacteria and phages, purification of genomic DNA and plasmids, and introduction of DNA into bacteria. Diverse strategies for generating and confirming mutations and for creating gene deletions and gene fusions are described, using allelic exchange, random transposition, recombineering, and CRISPR–Cas technologies. Protocols for bacterial genome sequencing and resequencing, as well as for high-throughput mutant screening via transposon sequencing, are provided.

Although specific bacterial species such as Escherichia coli, Salmonella enterica, Vibrio cholerae, and Staphylococcus aureus are used for some of the protocols, the methodologies herein described can readily be adapted for use with other bacteria. This manual thus represents a valuable resource for those seeking to perform genetic experiments in bacteria.

Contents

Preface
Overview
Cold Spring Harbor Laboratory Courses in Phage and Bacterial Genetics: A Catalyst for Innovation in Molecular Biology, Genetics, and Microbiology
Stanley Maloy
SECTION 1. Fundamental Procedures in Bacterial Molecular Genetics
INTRODUCTION
Working with Bacteria, Phage, and Plasmids
Nara Figueroa-Bossi, Roberto Balbontín, and Lionello Bossi
PROTOCOLS
1 Basic Bacteriological Routines
Nara Figueroa-Bossi, Roberto Balbontín, and Lionello Bossi
2 Working with Phage P22
Nara Figueroa-Bossi, Roberto Balbontín, and Lionello Bossi
3 Preparing Plasmid DNA from Bacteria
Nara Figueroa-Bossi, Roberto Balbontín, and Lionello Bossi
4 Preparing Bacterial Genomic DNA
Nara Figueroa-Bossi, Roberto Balbontín, and Lionello Bossi
5 Quick Transformation with Plasmid DNA
Nara Figueroa-Bossi, Roberto Balbontín, and Lionello Bossi
SECTION 2. Isolation and Sequencing of Novel Bacterial Species
INTRODUCTION
De Novo Genome Sequencing, Annotation, and Taxonomy ofUnknown Bacteria
Andrew Camilli
PROTOCOLS
1 Isolation and Sequencing of Novel Vibrio Species
Andrew Camilli
SECTION 3. Selection of Antibiotic-Resistant Bacterial Strains and Identification of Mutations by Whole-Genome Sequencing
INTRODUCTION
Selection of Antibiotic-Resistant Bacterial Strains and Identification of Genomic Alterations by Whole-Genome Sequencing: Using Staphylococcus aureus and Oxacillin Resistance as an Example
Merve S. Zeden and Angelika Gründling
PROTOCOLS
1 Agar Plate–Based Method for the Selection of Antibiotic-Resistant Bacterial Strains
Merve S. Zeden and Angelika Gründling
2 Preparation of Staphylococcus aureus Genomic DNA Using Promega Nuclei Lysis and Protein Precipitation Solutions, Followed by Additional Cleanup and Quantification Steps
Merve S. Zeden and Angelika Gründling
3 Preparation of Staphylococcus aureus Genomic DNA Using a Chloroform Extraction and Ethanol Precipitation Method, Followed by Additional Cleanup and Quantification Steps
Merve S. Zeden and Angelika Gründling
4 Small-Scale Illumina Library Preparation Using the Illumina Nextera XT DNA Library Preparation Kit
Merve S. Zeden and Angelika Gründling
5 Bacterial Whole-Genome-Resequencing Analysis: Basic Steps Using the CLC Genomics Workbench Software
Merve S. Zeden and Angelika Gründling
SECTION 4. Transposon Sequencing Genetic Screening in Bacteria
INTRODUCTION
High-Throughput Mutant Screening via Transposon Sequencing
Jacob Bourgeois and Andrew Camilli
PROTOCOLS
1 High-Throughput Mutant Screening in Vibrio cholerae via Transposon Sequencing
Jacob Bourgeois and Andrew Camilli
SECTION 5. Making Genome-Wide Reporter Gene Fusions by Random Transposition
INTRODUCTION
Use of Transposable Reporters in the Analysis of Bacterial Regulatory Networks
Nara Figueroa-Bossi, Roberto Balbontín, and Lionello Bossi
PROTOCOLS
1 Generating Libraries of Random lacZY or GFP Gene Fusions in the Bacterial Chromosome: Screening for Differentially Expressed Fusions
Nara Figueroa-Bossi, Roberto Balbontín, and Lionello Bossi
2 Mapping Transposon Insertion Sites by Inverse Polymerase Chain Reaction and Sanger Sequencing
Nara Figueroa-Bossi, Roberto Balbontín, and Lionello Bossi
SECTION 6. Generating Gene Deletions by Allelic Exchange
INTRODUCTION
Allelic Exchange: Construction of an Unmarked In-Frame Deletion in Staphylococcus aureus
Merve S. Zeden, Christopher F. Schuster, and Angelika Gründling
PROTOCOLS
1 Construction of a Staphylococcus aureus Gene-Deletion Allelic-Exchange Plasmid by Gibson Assembly and Recovery in Escherichia coli
Merve S. Zeden, Christopher F. Schuster, and Angelika Gründling
2 Preparation of Electrocompetent Staphylococcus aureus Cells and Plasmid Transformation
Merve S. Zeden, Christopher F. Schuster, and Angelika Gründling
3 Allelic-Exchange Procedure in Staphylococcus aureus
Merve S. Zeden, Christopher F. Schuster, and Angelika Gründling
4 Staphylococcus aureus Colony Polymerase Chain Reaction
Merve S. Zeden, Christopher F. Schuster, and Angelika Gründling
SECTION 7. Genome Editing by l Red Recombination
INTRODUCTION
DNA Recombineering Applications
Nara Figueroa-Bossi, Roberto Balbontín, and Lionello Bossi
PROTOCOLS
1 Recombineering 101: Making an In-Frame Deletion Mutant
Nara Figueroa-Bossi, Roberto Balbontín, and Lionello Bossi
2 Scarless DNA Recombineering
Nara Figueroa-Bossi, Roberto Balbontín, and Lionello Bossi
3 Construction of Single-Copy Fluorescent Protein Fusions by One-Step Recombineering
Roberto Balbontín, Nara Figueroa-Bossi, and Lionello Bossi
4 Converting an FRT-Tagged Gene into a Fluorescent Protein Gene Fusion by Flp-Mediated Site-Specific Recombinatio
Roberto Balbontín, Mathilde Ratel, Nara Figueroa-Bossi, and Lionello Bossi
SECTION 8. Genome Editing in Staphylococcus aureus
Using CRISPR–Cas9 Technologies
INTRODUCTION
Using CRISPR–Cas9-Based Methods for Genome Editing in Staphylococcus aureus
Angelika Gründling, Quanjiang Ji, and Stephen J. Salipant
PROTOCOLS
1 Oligonucleotide Design and Construction of a Gene-Targeting CRISPR–Cas9 Plasmid in Escherichia coli for Generating a Gene-Deletion Strain in Staphylococcus aureus
Angelika Gründling and Stephen J. Salipante
2 Introduction of a Recombineering Oligonucleotide and a CRISPR–Cas9 Gene-Targeting Plasmid into Staphylococcus aureus for Generating a Gene-Deletion Strain
Angelika Gründling and Stephen J. Salipante
3 Identification of Editable Sites, Spacer Oligonucleotide Design, and Generation of the Gene-Targeting CRISPR–nCas9 Plasmid for Gene Disruption in Staphylococcus aureus Using the CRISPR–nCas9 and Cytidine Deaminase System
Angelika Gründling and Quanjiang Ji
4 Introduction of a CRISPR–nCas9 Gene-Targeting Plasmid into Staphylococcus aureus for Gene Disruption
Angelika Gründling and Quanjiang Ji
SECTION 9. Genome Editing of Bacteriophages Using CRISPR–Cas
INTRODUCTION
CRISPR Gene Editing of a Virulent Bacteriophage
Andrew Camilli
PROTOCOLS
1 CRISPR Gene Editing of Virulent Bacteriophage ICP1
Andrew Camilli
Appendix: General Safety and Hazardous Material Information
Index