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

  “This is an outstanding book that will find a wide range of uses in various research and instructional settings. Including a full range of experimental protocols from basic microbial manipulation and storage to CRISPR-Cas9 methods for genome editing, this book is a must-have addition to any library or research enterprise using bacteria or bacteriophage as experimental organisms. 5 stars.”
      —Doody's Review Service