Preface
Contents
Contributors
Part I: In Vivo Approaches
Chapter 1: Shedding Light on Bacterial Chromosome Structure: Exploring the Significance of 3C-Based Approaches
1 Introduction
2 A View of Bacterial Chromosome Structure Before the 3C Era
3 The Emergence of 3C Methods
4 Visualization of Bacterial Chromosome Organization Through Hi-C: The Discovery of Chromosome Interaction Domains
5 The Intricate Relationship Between Transcription and CID Boundaries
6 CID and SMC Proteins
7 The Role of NAPs in Bacterial Chromosome Structure
8 Genome Organization at a Higher Scale: The Chromosomal Domains
9 Chromosome Arrangement and Segregation Within the Cell
10 Conclusions and Perspectives
References
Chapter 2: Circuit Topology Analysis of Single-Cell HiC Data
1 Introduction
2 Theory
2.1 Circuit Topology: Concepts
2.2 Representing HiC Data with Polymer Models
2.3 Circuit Topology Matrix of a Single Chromosome
2.4 Circuit Topology Matrix from HiC Data
2.5 Circuit Topology-Based Genomic Markers
2.6 Extension of Circuit Topology Analysis to Circular Genomes
3 Methods
4 Notes
References
Chapter 3: GeF-seq: A Simple Procedure for Base-Pair Resolution ChIP-seq
1 Introduction
2 Materials
2.1 Solutions
2.2 Equipment
2.3 Enzymes, Kit, and Other Materials
2.4 Software
3 Methods (See Note 3)
3.1 DNA-Protein Crosslinking for ChAP
3.2 Optimization of DNase I Digestion Condition (Important)
3.3 DNase I Digestion of Genomic DNA for ChAP
3.4 Sonication
3.5 Purification of the DNA-Protein Complex for ChAP
3.6 Decrosslinking of the DNA-Protein Complex and Purification of the DNA Fragments
3.7 GeF-seq Data Analysis (See Note 12)
4 Notes
References
Chapter 4: ChIP-qPCR of FLAG-Tagged Proteins in Bacteria
1 Introduction
2 Materials
2.1 Cell Fixation
2.2 Chromatin Shearing and Shearing Analysis
2.3 Immunoprecipitation
2.4 De-crosslinking and DNA Purification
2.5 Genomic DNA Preparation
2.6 qPCR
3 Method
3.1 Cell Fixation
3.2 Chromatin Shearing
3.3 Chromatin Shearing Analysis
3.4 Immunoprecipitation (IP)
3.5 De-crosslinking and DNA Purification
3.6 Genomic DNA Preparation
3.7 qPCR: Experimental Design
3.8 qPCR: Data Analysis
4 Notes
References
Chapter 5: Genomic SELEX Screening of Regulatory Targets of Transcription Factors
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 6: High-Resolution Characterization of DNA/Protein Complexes in Living Bacteria
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents
3 Methods
3.1 In Vivo Cross-Linking with Formaldehyde
3.2 Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo) Analysis
3.3 Chromatin Endogenous Cleavage (ChEC) Analysis
3.4 Ligation-Mediated Polymerase Chain Reaction (LM-PCR)
3.4.1 Primer Design
3.4.2 Phi29 Extension and Linker Ligation for ChIP-Exo and ChEC Samples
3.4.3 PCR Amplification of ChIP-Exo and ChEC Samples
3.5 Southern Blot Analysis
3.5.1 Generation of Sequencing Ladders as Markers Within the Gel Blot
3.5.2 Gel Electrophoresis of ChIP-Exo and ChEC Samples and Probe Synthesis
3.5.3 DNA Transfer and Southern Blotting Protocol
3.5.4 Blot Washing and Exposure
4 Notes
References
Chapter 7: High-Throughput Mapping of Chromosomal Conformations in E. coli Under Physiological Conditions Using Massively Mult...
1 Introduction
2 Materials
2.1 Equipment
2.2 Commercially Available Kits/Reagents
2.3 Buffers and Media
2.4 Primers, Plasmids, and Strains
3 Methods
3.1 Generation of the Donor Substrate
3.2 Generation of Barcoded Mu Lysate
3.3 Generation of a Primary Barcoded Mu Integrant Library
3.4 Induction of Single-Hop Mu Transposition to Generate the Secondary Library
3.5 Purification of Genomic DNA from the Primary Library
3.6 DNA Fragmentation and Inverse PCR-Based Footprinting
3.7 Library Quantitation and Sequencing
4 Notes
References
Chapter 8: In Vivo Genomic Supercoiling Mapping Using Psora-seq
1 Introduction
2 Materials and Equipment
2.1 Materials
2.2 Equipment
3 Methods
3.1 Cell Preparation and Psoralen Cross-Linking
3.2 CTAB DNA Isolation
3.3 DNA Shearing
3.4 Affinity Purification and Cross-Link Reversal
3.5 DNA Quantification and Sequencing
3.6 Data Analysis and Assessment
4 Notes
References
Chapter 9: Modular Assembly of Synthetic Secondary Chromosomes
1 Introduction
2 Materials
2.1 Software
2.2 Plasmids
2.3 DNA Oligonucleotides
2.4 Enzymes
2.5 Antibiotics and Supplements
2.6 Chemicals
3 Methods
3.1 Overall Design of Synthetic Chromosomes
3.2 Sequence and Oligonucleotide Design for Generation of Basic Chromosome Building Blocks
3.2.1 Primer Design for Fully Defined Basic Building Blocks
3.2.2 Design of DNA Oligonucleotides for Synthetic Sequence Libraries
3.3 Generation of Level-1 Building Blocks
3.4 Consecutive MoClo Assembly Steps
3.5 Extraction of Synthetic Secondary Chromosomes
3.6 Gel Electrophoretic Analysis of Synthetic Secondary Chromosomes
3.7 Transformation of Cells with Synthetic Secondary Chromosome
3.8 Introduction of Modifications into the Synthetic Secondary Chromosome
3.8.1 Design of DNA Oligonucleotides for Lambda Red Recombineering
3.8.2 Lambda Red Recombineering Protocol
4 Notes
References
Chapter 10: Visualization, Quantification, and Statistical Evaluation of Dynamics for DNA-Binding Proteins in Bacteria by Sing...
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 Fluorescence Microscopy
2.3 Data Curation
3 Methods
3.1 Sample Preparation
3.2 Fluorescence Microscopy
3.3 Movie Preparation with FIJI (ImageJ)
3.4 Drawing Cell Outlines with Oufti
3.5 U-Track
3.6 SMTracker
3.6.1 Import
3.6.2 Stationary Localization Analysis (SLA)
3.6.3 Mean Squared Displacement (MSD)
3.6.4 Clustering
3.6.5 Gaussian Mixture Model (GMM)
3.6.6 Squared Displacement Analysis (SQD)
3.6.7 Apparent Diffusion (APPD)
3.6.8 Spatial Distribution (SDA)
3.6.9 SMTracker Tools
Distance Measurements
Molecule Quantification
4 Notes
References
Chapter 11: Genetic Approaches to Study the Interplay Between Transcription and Nucleoid-Associated Proteins in Escherichia co...
1 Introduction
2 Materials
2.1 Antibiotics and Media
2.2 Plasmids and Strains
3 Methods
3.1 Chromosomal and Plasmidic Promoter-lacZ Fusions
3.1.1 Integration at the Chromosomal Phage Lambda Attachment Site, attB
3.1.2 Low-Copy Plasmids for Transcriptional and Translational Promoter-lacZ Fusions
3.2 Integration of Promoter Cassettes into the Genome
3.3 Construction of Isogenic Mutants (hns and Others) by Transduction
4 Notes
References
Chapter 12: Genetic Engineering of Bacillus subtilis Using Competence-Induced Homologous Recombination Techniques
1 Introduction
2 Materials
2.1 Growth Media and Antibiotics
2.2 Media for Competent Cells
2.3 Media for Ξ²-Galactosidase Assay
2.4 Materials for Flow Cytometry
2.5 Plasmids, Strains, and Oligonucleotides
3 Methods
3.1 B. subtilis Competent Cells
3.2 Chromosomally Located Fusions to Reporter Gene lacZ or gfp
3.2.1 Detection of Reporter Gene lacZ: Ξ²-Galactosidase Assay
3.2.2 Detection of Reporter Gene gfp. Flow Cytometry
3.3 Ectopic Expression of Genes from Inducible Promoters
3.3.1 amyE Integration Vectors pDR110 and pDR111
3.3.2 lacA Integration Vector pAX01
3.4 Construction of B. subtilis Strains Containing Deletions or Insertions
3.4.1 Disruption of a Gene by Replacement with a Selectable Marker
3.4.2 cre-lox Recombination System
3.4.3 pMinimad2 Vector
4 Notes
References
Part II: In Vitro Approaches
Chapter 13: Atomic Force Microscopy Imaging and Analysis of Prokaryotic Genome Organization
1 Introduction
2 Materials
2.1 Culture Medium
2.2 Buffers
2.3 Chemicals and Enzymes
2.4 Equipment and Other Materials
3 Methods
3.1 Dissection of Bacterial Cells and Structural Analysis of the Nucleoid by AFM
3.2 Further Enzymatic Treatment of Dissected Nucleoid Fibers
3.3 Dissection of Archaea Cells and Structural Analysis of the Nucleoid by AFM
3.4 AFM Analysis of Distinct Chromosome Structures Separated Through Sucrose Gradient Sedimentation
3.5 Reconstitution of Nucleoid Fibers
3.6 Dissection of Chloroplasts to Analyze Nucleoid Structure by AFM
3.7 Dissection of Mitochondria to Analyze Nucleoid Structure by AFM
4 Notes
References
Chapter 14: Atomic Force Microscopy Characterization of Reconstituted Protein-DNA Complexes
1 Introduction
2 Materials
3 Methods
3.1 In Vitro Chromatin Reconstitution
3.2 AFM Imaging
3.3 AFM Image Analysis
4 Notes
References
Chapter 15: Approaches for Determining DNA Persistence Length Using Atomic Force Microscopy
1 Introduction
2 Materials
2.1 Equipment
2.2 PCR Reagents
2.3 AFM Reagents
3 Methods
3.1 Sample Preparation
3.2 AFM Imaging
3.3 Image Processing and Data Analysis
3.4 WLC Fitting and Persistence Length Determination
4 Notes
References
Chapter 16: Dynamic Light Scattering of DNA-Ligand Complexes
1 Introduction
1.1 Particle Size
1.2 Electrophoretic Mobility and Zeta Potential of Particle
1.3 DNA Condensation and Electrophoretic Mobility of DNA-Ligand Complex
2 Materials and Experimental Conditions
3 Methods
3.1 Particle Size Measurement
3.2 Zeta Potential and Electrophoretic Mobility Measurement
3.3 Conclusions
4 Notes
References
Chapter 17: Microscale Thermophoresis Analysis of Chromatin Interactions
1 Introduction
1.1 Microscale Thermophoresis (MST)
1.2 MST of Chromatin Systems
2 Materials
2.1 Fluorescent Labeling
2.2 MST Materials, Equipment, and Software
2.3 Stock Solutions
3 Methods
3.1 Design of MST Experiment: Choice of Fluorescent Labeling
3.1.1 Protein Labeling
3.1.2 DNA Preparation
3.2 Optimization of Experimental Conditions
3.3 Preparation of Dilution Series
3.4 MST Measurement
3.5 MST Data Analysis
3.6 Description of Examples
3.6.1 Different DNA Compaction Modes of HMfB
3.6.2 Histone Binding by Histone Chaperone APLF
3.6.3 Binding of a Nucleosome-Mimicking Peptide to a Reader Protein
3.6.4 Analysis of a Nucleosome-Peptide Interaction
4 Notes
References
Chapter 18: In Vitro Transcription Assay to Quantify Effects of H-NS Filaments on RNA Chain Elongation by RNA Polymerase
1 Introduction
2 Materials
2.1 DNA Preparation
2.2 In Vitro Transcription and RNA Purification
2.2.1 In Vitro Transcription Reaction
2.2.2 RNA Purification
2.3 Electrophoretic Mobility Shift Assay
2.3.1 Gel Setup
2.3.2 Filament Formation on Non-EC DNA Controls
2.4 Urea-PAGE
2.5 Data Analysis
3 Methods
3.1 DNA Preparation
3.1.1 PCR Amplify and Concentrate DNA
3.1.2 Gel Purify DNA
3.1.3 DNA Electroelution
3.1.4 DNA Cleanup and Concentration
3.2 In Vitro Transcription
3.2.1 In Vitro Transcription Setup
3.2.2 Performing In Vitro Transcription Reaction
3.2.3 RNA Cleanup
3.3 Electrophoretic Mobility Shift Assay
3.3.1 Pour and Set Up 3% Native Polyacrylamide Gel
3.3.2 Form Filaments on DNA Not Containing ECs
3.3.3 Perform Native PAGE to Confirm Protein-DNA Interactions
3.4 Urea-PAGE
3.5 Data Analysis
3.5.1 Conversion of Pixels to Nucleotides
3.5.2 Making Pseudo-Densitometry Plots
3.5.3 Calculating Average Transcript Lengths (ATLs)
4 Notes
References
Chapter 19: Methods to Quantitatively Measure Topological Changes Induced by DNA-Binding Proteins In Vivo and In Vitro
1 Introduction
2 Materials
2.1 Capturing Changes in DNA Supercoiling In Vivo
2.2 Capturing Changes in DNA Supercoiling In Vitro
2.3 Preparation for Chloroquine Gel Electrophoresis
2.4 Loading, Running, and Imaging of 1D Chloroquine Gels
2.5 Interpretation and Quantitation of Plasmid Topoisomers
3 Methods
3.1 Capturing Changes in DNA Supercoiling In Vivo
3.2 Capturing Changes in DNA Supercoiling In Vitro Using the Topoisomerase I-Mediated Relaxation Assay (TMRA) or the Ligase-Me...
3.2.1 DNA-Protein Binding Reaction
3.2.2 Validating DNA-Protein Interactions by EMSA
3.2.3 Fixing the Topological Effect Enzymatically
3.2.4 Purification of DNA for Analysis
3.3 Preparation for Chloroquine Gel Electrophoresis
3.4 Loading, Running, and Imaging of 1D Chloroquine Gels
3.5 Interpretation and Quantitation of Plasmid Topoisomers
3.5.1 Quantitation of Images of Gels Showing Samples Collected In Vivo
3.5.2 Quantitation of Images of Gels Showing Samples Generated In Vitro
4 Notes
References
Chapter 20: Quantitative Determination of DNA Bridging Efficiency of Chromatin Proteins
1 Introduction
2 Materials
2.1 Stock Solutions
2.2 Generation of DNA Substrates Using PCR
2.3 Bridging Assay Equipment
2.4 Quantifying DNA Bridging Through Radioactivity
3 Methods
3.1 Generation of DNA Substrates Using PCR
3.2 Radiolabeling DNA
3.3 Bridging Assay
4 Results
4.1 DNA Bridging Efficiency as a Function of Protein Concentration
4.2 DNA Bridging Efficiency of Proteins as a Function of Physicochemical Conditions and Protein-Protein Interactions
5 Notes
References
Chapter 21: Measuring Partition Coefficients of In Vitro Biomolecular Condensates Using Fluorescence Correlation Spectroscopy
Abbreviations
1 Introduction
2 Materials
3 Methods
3.1 Coverslip Passivation
3.2 Condensate Production and Sealing
3.3 Optical Alignment and Calibration for FCS
3.4 FCS Data Acquisition
3.5 Basic FCS Data Analysis for Partition Coefficient Determination
3.6 Background Correction
3.7 Bleaching Correction
3.8 Afterpulsing Correction
4 Notes
References
Chapter 22: Tethered Particle Motion Analysis of DNA-Binding Properties of Architectural Proteins
1 Introduction
1.1 Tethered Particle Motion
1.2 Tethered Particle Motion Studies of Architectural Proteins
2 Materials
2.1 Stock Solutions
2.2 Generation of DNA Substrates Using PCR
2.3 Preparation and Assembly of Incubation Chamber
2.4 Microscopy Equipment
2.5 Particle Tracking and Analysis
3 Methods
3.1 Generation of DNA Substrates Using PCR
3.2 Making Flow Cells
3.3 Preparing the Bead Solution
3.4 Preparing Flow Cells
3.5 Microscopy (for the Instrument See Fig. 5)
3.6 Measurements
3.7 Data Analysis
3.8 Analysis of Protein-DNA Complexes Using TPM
3.8.1 DNA-Bending Proteins
3.8.2 DNA-Wrapping Proteins
3.8.3 DNA-Binding Proteins Capable of Multimerizing Along the DNA
3.8.4 Highly Dynamic DNA Binding
3.8.5 Further Analysis of DNA Binding
4 Notes
References
Chapter 23: Quantitation of DNA Binding Affinity Using Tethered Particle Motion
1 Introduction
2 Materials
3 Methods
3.1 Parafilm Semicircles
3.2 Sample Chambers
3.3 Bead Suspension
3.4 DNA Substrate
3.5 Anti-digoxygenin Solution
3.6 DNA-Tethered Bead in Sample Chamber
3.7 Dilution Series
3.8 Imaging
3.9 Data Analysis
3.10 Analysis of Specific Protein-DNA Binding
3.11 Example of KD Estimation for DNA-Binding Protein IHF
3.12 Analysis of Structural Features Using End-to-End Distance
3.13 Example of Tetrameric DNA Binding by Archaeal Histone HMfA
3.14 Data Representation
4 Notes
References
Chapter 24: Unravelling the Biophysical Properties of Chromatin Proteins and DNA Using Acoustic Force Spectroscopy
1 Introduction
2 Materials
2.1 Stock Solutions and Beads
2.1.1 Buffer for Analysis by Agarose Gel Electrophoresis (See Subheading 3.1)
2.1.2 Solutions for Reference Bead Preparation (See Subheading 3.2)
2.1.3 Solutions for Cleaning Protocol (See Subheading 3.4)
2.1.4 Solutions for Passivation of the Flow Cell (See Subheading 3.6)
2.1.5 Solutions for Passivation of the Flow Cell (See Subheadings 3.2 and 3.6)
2.2 DNA Substrates
2.3 AFS Instrument
2.4 Particle Tracking, Control, and Analysis
3 Methods
3.1 Generation of DNA Substrate Using PCR
3.2 Preparation of Reference Beads
3.3 Preparation of Tether Beads
3.4 Flow Cell and Tubing Cleaning
3.5 Preparation of Flow Cell and Bead Tethers
3.6 Measurements
3.7 Data Analysis
3.7.1 Generate FD Curves
3.7.2 Extensible Wormlike Chain (eWLC) Model Fitting
4 Analysis of Protein-DNA Complexes Using AFS
4.1 Force-Extension Curves of HU-DNA Complex
4.2 Pulling of DNA and DNA-H-NS Complex
5 Notes
References
Chapter 25: Unravelling DNA Organization with Single-Molecule Force Spectroscopy Using Magnetic Tweezers
1 Introduction
1.1 Chromatin Organization by DNA-Binding Proteins
1.2 Magnetic Tweezers
1.3 Measurement Schemes
1.4 Data Analysis
2 Materials
2.1 Stock Solutions
2.2 Isolation of DNA Plasmids
2.3 Digestion and Labeling of DNA
2.4 Flow Cell Assembly
2.5 Bead-Tether Assembly
2.6 Microscope
3 Methods
3.1 Isolation of DNA Plasmids
3.2 Production of Torsionally Free DNA
3.3 Production of Torsionally Constrained DNA
3.4 Flow Cell Assembly
3.5 Bead-Tether Assembly
3.6 Initial Bead Selection and Height Calibration
3.7 Force Calibration
3.8 Force Spectroscopy Experiments
3.9 Rotational Spectroscopy Experiments
3.10 Data Analysis of Force Spectroscopy Experiments
3.11 Data Analysis of Rotational Spectroscopy Experiments
4 Notes
References
Chapter 26: Approaches to the Full and Partial Chemical Synthesis of Proteins
1 Introduction
2 Materials
2.1 N-Terminal Protein Fragment
2.2 C-Terminal Protein Fragment
2.3 Reagents for NCL/EPL
2.4 Reagents for Desulfurization
3 Methods
3.1 NCL/EPL Reaction
3.2 Purification of the NCL Reaction
3.3 Purification of EPL Reaction
3.4 Desulfurization of Cys to Ala
3.5 Purification of Desulfurized Product
4 Notes
References
Part III: In Silico Approaches
Chapter 27: Deciphering Sequence-Specific DNA Binding by H-NS Using Molecular Simulation
1 Structure and Function of H-NS
1.1 Function of H-NS
1.2 H-NS Structure
1.3 Binding Specificity
1.4 Molecular Simulations Provide Highly Detailed Insights
2 Methodology
2.1 Molecular Dynamics
2.2 Steered Molecular Dynamics Settings
2.3 Analysis
3 H-NS Binds Differently to AT-Rich DNA and GC-Rich DNA
3.1 Contacts Between H-NS and DNA
3.2 Conformational Differences
3.3 Ion DNA Densities
3.4 Ion-Mediated Contacts
3.5 Quantifying the Binding Specificity of H-NS
4 Concluding Remarks
4.1 Recognition Mechanism
4.2 Summary
References
Chapter 28: Molecular Dynamics Simulation of a Feather-Boa Model of a Bacterial Chromosome
1 Introduction
2 Materials
2.1 Model Specification
2.2 Implementation
3 Methods
3.1 Molecular Dynamics with Langevin Heat Bath
3.2 Program Implementation Using ESPResSo
3.3 Model Verification: Emergent Stiffness
4 Notes
References
Chapter 29: Replicating Chromosomes in Whole-Cell Models of Bacteria
1 Introduction
1.1 Chapter Overview
1.2 Background
2 Preparing Cell Architecture and Generating Initial Chromosome Configurations
3 Chromosome Model and Brownian Dynamics Simulations
4 Simulating SMC Complexes and Type-II Topoisomerases
5 Replicating Circular Chromosomes
6 Calculating Contact Maps of Replicating Chromosomes
7 Software Guide
7.1 Simulation Input/Output
7.1.1 Replication State
Replication State File (I/O)
Replication State Transforms File (I)
Topology File (O)
Fork Partition File (O)
Coarse-Graining File (O)
7.1.2 System State
Coordinate and Orientation Binary Files (I/O)
Monomer Coordinate XYZ File (O)
LAMMPS Data File (O)
7.1.3 Trajectory
LAMMPS Trajectory File (O)
7.2 Example Simulations
7.3 Visualizing Simulations of Replicating Chromosomes
References
Index