Archaea: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Archaea: A Goldmine for Molecular Biologists and Evolutionists
1 Breaking the Eukaryote-Prokaryote Dichotomy
2 A Single Molecule Can Tell the Truth
3 A Third Type of Molecular Biology, with Eukaryotic Flavor
4 The Impact of Methodologies
5 Mining Archaeal Genomes
6 The Expanding Archaeal Diversity
7 Reconstruct the Past from Comparative Molecular Biology, the Pivotal Role of Archaea
8 The Expanding Archaeal Eukaryome
References
Part I: Genetic Manipulation of Archaea
Chapter 2: Progress and Challenges in Archaeal Genetic Manipulation
1 Halophiles
2 Methanogens
3 Thermococcales
4 Sulfolobales
5 Future
References
Chapter 3: Genetic Manipulation of Haloferax Species
Abbreviations
1 Introduction
2 Materials
2.1 Plasmids and Strains
2.2 Hv-YPC Rich Media
2.3 Hv-Ca Semiselective Media
2.4 Hv-min Minimal Selective Media
2.5 Transformation
2.6 Colony Hybridization and Southern Blotting
3 Methods
3.1 Preparation of Media
3.2 Construction of Gene Deletion/Insertion Vector
3.3 Generating ``Pop-In´´ Strains-Transformation of Haloferax with Plasmid DNA
3.4 Generating ``Pop-Out´´ Strains
3.5 Testing for a Gene Deletion Event
3.6 Gene Restoration in Deletion Strains
3.7 Deleting Essential Genes
3.8 Generating a Tryptophan-Inducible Allele of an Essential Gene
4 Notes
References
Chapter 4: CRISPR Interference as a Tool to Repress Gene Expression in Haloferax volcanii
1 Introduction
2 Materials
2.1 Cloning
2.1.1 Enzymes
2.1.2 Vectors, Oligonucleotides, and Technical Equipment
2.2 Microbiological Culture
2.2.1 Escherichia coli Strains
2.2.2 E. coli Medium
2.2.3 H. volcanii Strains
2.2.4 H. volcanii Medium (See Note 3)
2.3 Transformation of H. volcanii
2.4 Plasmid Screening, Strain Control, Cultivation, and Harvesting
2.5 RNA Preparation
3 Methods
3.1 Preparation of Solutions
3.1.1 Preparation of H. volcanii Medium Components (See Notes 4 and 5)
General Medium Solutions
Solutions for Hv-Min and Hv-Ca Agar Plates
Solutions for Liquid Medium (MinTE, Cab, YPCab)
3.1.2 Preparation of Solid Medium/Agar Plates (Hv-Min + Ura + Trp/Hv-Ca)
3.1.3 Preparation of Liquid Media
3.1.4 Preparation of Transformation Solutions
3.2 Selection of Spacer Sequences
3.3 Design and Cloning of crRNA Expression Constructs
3.3.1 Method A: crRNAs Matured Independently of Cas6b by RNase P and Z
3.3.2 Method B: crRNAs Expressed from a Synthetic CRISPR-Locus (CRISPRS Plasmids)
3.4 Transformation and Selection of H. volcanii
3.4.1 Transformation of H. volcanii
3.4.2 Selection of Positive Transformants
3.5 Further Experiments to Characterize the CRISPRi Effect
3.5.1 Growth Experiment
3.5.2 RNA Analysis
4 Notes
References
Chapter 5: Transformation Techniques for the Anaerobic Hyperthermophile Thermococcus kodakarensis
1 Introduction
2 Materials
2.1 Microbial Culture
2.1.1 Escherichia coli Media
2.1.2 Thermococcus kodakarensis Media
2.1.3 Equipment
3 Methods
3.1 Microbial Methods
3.1.1 E. Coli Media and Cultivation
3.1.2 Thermococcus kodakarensis Media and Cultivation
3.2 Design and Construction of Plasmid DNAs Used to Transform Thermococcus kodakarensis
3.2.1 Generating Linearized pTS700 to Accept Amplicons for Genomic Modifications. Construction of ``A´´-Plasmids
3.2.2 Generating ``B´´- and ``C´´-Plasmids from ``A´´-Plasmids
3.2.3 Generating pLC70-Based Expression Plasmids
3.2.4 Thermococcus kodakarensis Strain Construction
4 Notes
References
Chapter 6: Genetic Methods and Construction of Chromosomal Mutations in Methanogenic Archaea
1 Introduction
2 Materials
2.1 Microbiological Culture
2.1.1 Media and Cultivation
2.1.2 Strains
2.2 Liposome-Mediated Transformation
2.3 Selection and Singulation of Successfully Transformed M. mazei Cells
2.4 Selection and Singulation of Successfully Generated M. mazei Δhpt Strain
2.5 Generation of a M. mazei Mutant Within the Δhpt Strain
3 Methods
3.1 Construct Plasmids
3.2 Growth of M. mazei
3.3 Liposome-Mediated Transformation of M. mazei
3.4 Selection and Singulation of Successfully Transformed M. mazei Cells
3.5 Selection and Singulation of Successfully Generated M. mazei Δhpt Strain
3.6 Generation of a M. mazei Mutant Within the Δhpt Strain
4 Notes
References
Chapter 7: An Interdomain Conjugation Protocol for Plasmid-DNA Transfer into Methanothermobacter thermautotrophicus ΔH
1 Introduction
2 Materials
2.1 Microbial Cultivation
2.1.1 Mineral Salt (MS) Medium for Methanothermobacter thermautotrophicus
2.1.2 LB Medium for Escherichia coli
2.1.3 MS/LB Medium for Conjugation Experiments
2.1.4 Antibiotics
2.1.5 Solid Media Plates
2.1.6 Gases (See Note 3)
2.1.7 Microbial Strains
2.1.8 Anaerobic Cultivation Container (Anaerobic Jars)
2.2 Molecular Methods
2.2.1 Plasmids
2.2.2 Preparation of Recombinant Pseudomurein Endoisopeptidase
2.2.3 Plasmid Extraction from M. thermautotrophicus
2.2.4 ``Colony´´ PCR
3 Methods
3.1 Cultivation Procedures
3.1.1 M. thermautotrophicus Media
3.1.2 M. thermautotrophicus Cultivation
3.1.3 E. coli Cultivation
3.2 Interdomain Conjugation (See Fig. 4)
3.2.1 Preparation of Precultures
3.2.2 Conjugation Procedure
3.3 Molecular Methods
3.3.1 ``Colony´´ PCR
3.3.2 Purification of Recombinant PeiP
3.3.3 Plasmid-DNA Extraction from M. thermautotrophicus
3.3.4 Retransformation of E. coli
4 Notes
References
Chapter 8: Methods for Markerless Gene Deletion and Plasmid-Based Expression in Sulfolobus acidocaldarius
1 Introduction
2 Materials
2.1 Brock Medium
2.2 Brock Plates
2.3 Growth of Sulfolobus
2.4 Glycerol Stocks
2.5 Sulfolobus Competent Cells and Transformation
2.6 First Selection, Second Selection and Mutant Screening
3 Methods
3.1 Preparing Brock Medium
3.2 Preparing Selection Plates
3.3 Growth of Sulfolobus Cells
3.4 Glycerol Stocks
3.5 Preparing Competent Cells
3.6 Transformation of DNA to S. acidocaldarius
3.7 First Selection
3.8 Complementation or Overexpression
3.9 Second Selection
3.10 Mutant Screening
4 Notes
References
Chapter 9: A Rapid Targeted Gene Inactivation Approach in Sulfolobus islandicus
1 Introduction
2 Materials
2.1 Strains and Plasmids
2.2 Media and Solutions
2.3 Cultivation of S. Islandicus Strains (See Note 2)
2.4 Preparation of Highly Concentrated Gene Deletion Cassettes
2.5 S. islandicus Competent Cells Preparation and Electroporation-Mediated Transformation
2.6 Mutant Screening Via Colony PCR
2.7 DNA Gel Electrophoresis
3 Methods
3.1 Generation of the Gene Deletion Cassette for Electroporation-Mediated Transformation
3.1.1 Preparation of Plasmid pSeSd-StoargD
3.1.2 Linearization of Plasmid pSeSd-StoargD (See Note 8)
3.1.3 Preparation of Highly Concentrated Gene Deletion Cassettes
3.2 S. islandicus Transformation with PCR-Amplified Gene Deletion Cassettes
3.2.1 Preparation of S. islandicus Competent Cells
3.2.2 Electroporation-Mediated S. islandicus Transformation
3.3 Mutant Screening Via Colony PCR
3.4 Cultivation, Purification, and Storage of S. islandicus Mutant Strains
3.4.1 Cultivation of Mutant Strains
3.4.2 Purification of Mutant Strains Via an Overlay Plating Approach (See Note 18)
3.4.3 Storage of Mutant Strains
4 Notes
References
Chapter 10: Transposon Insertion Mutagenesis in Hyperthermophilic Crenarchaeon Sulfolobus islandicus
1 Introduction
2 Materials
2.1 Sulfolobus Growth Media and Solution
2.2 Strains and Plasmids
2.3 Oligonucleotides (5′-3′; Synthesized by Integrated DNA Technologies)
2.4 Molecular Biology Reagents
2.5 Reagents and Equipment for E. coli Transformation
2.6 Reagents and Equipment for S. islandicus Transformation
3 Methods
3.1 Construction of Plasmid pT-SsoargD Containing a Sulfolobus Selectable Marker Cassette
3.2 Generation of a Customized Mini-Transposon Tn5
3.3 Generation of Tn5 Transposome
3.4 Electroporation of S. islandicus Cells with Tn5 Transposome
3.5 Initial Screening of Tn5 Integration in S. islandicus Chromosome
3.6 Creation of Transposon Insertion Mutant Library
3.7 Storage of Transposon Insertion Mutant Library
4 Notes
References
Chapter 11: Reprogramming CRISPR-Mediated RNA Interference for Silencing of Essential Genes in Sulfolobales
1 Introduction
2 Materials
2.1 Protospacer and Overlap Extension Primer Design
2.2 MiniCR Vector Construction (See Note 1)
2.2.1 Amplification of miniCR from S. solfataricus P2 CRISPR Locus D
2.2.2 TA Cloning and Propagation of pENTRY-miniCR in E. coli TOP10
2.2.3 Three-Step Overlap Extension PCR (OE-PCR)
2.3 S. solfataricus and S. acidocaldarius Medium
2.3.1 Brock Medium and Plates
2.3.2 Strains
2.4 S. solfataricus and S. acidocaldarius Transformation
3 Methods
3.1 Spacer and Overlap Extension (OE) Primer Design
3.2 Construction of pENTRY-miniCR Vector (See Note 1)
3.2.1 Amplification of miniCR from S. solfataricus P2 CRISPR Locus D
3.2.2 TA Cloning and Propagation of pENTRY-miniCR in E. coli TOP10
3.2.3 Three-Step Overlap Extension PCR (OE-PCR)
Construction PCR 1-Spacer Flanks
Construction PCR 2-Silencing miniCR Fragment
Construction PCR 3-pENTRY-Silencing miniCR Vector
Gateway Recombination Cloning in pIZ Destination Vector (See Note 11)
3.3 S. solfataricus and S. acidocaldarius Medium and Cultivation
3.3.1 Brock Medium
3.3.2 NZ/S Gellan Gum Plates
3.4 Transformation of S. solfataricus and S. acidocaldarius
3.4.1 Electroporation
3.4.2 Overlay Plating on NZ/S Gellan Gum Plates
4 Notes
References
Part II: Molecular Biology of Archaea
Chapter 12: Progress and Challenges in Archaeal Molecular Biology
References
Chapter 13: ChIP-Seq Occupancy Mapping of the Archaeal Transcription Machinery
1 Introduction
2 Materials
2.1 Antibody Purification
2.2 Cell Cross-Linking
2.3 DNA Shearing
2.4 Chromatin Immunoprecipitation
3 Methods
3.1 Antibody Purification
3.2 Cross-Linking of Cells
3.3 DNA Shearing by Sonication
3.4 Immunoprecipitation
3.5 qPCR Analysis and Protocol Optimization
3.6 Recommendations for Deep Sequencing and Data Analysis
4 Notes
References
Chapter 14: Conversion of Ribosome-Protected mRNA to DNA and Deep Sequencing for Ribosome Profiling in Haloferax volcanii
1 Introduction
2 Materials
2.1 Equipment
2.2 Materials
2.3 Reagents
2.4 Solutions
2.4.1 Lysis Buffer and Sucrose Solutions
2.4.2 Gel Electrophoresis Buffers and Gels
2.4.3 Elution Buffers
2.5 Oligos
3 Methods
3.1 Harvest Cells
3.1.1 Whole Culture Freeze
3.1.2 Cell Lysis by Freezer Mill
3.2 Pellet Ribosomes on 1 M Sucrose Cushion
3.3 Footprint Ribosomes with MNase
3.4 Sucrose Gradient with Biocomp Gradient Maker
3.5 Fractionate Footprints to Collect Monosomes with the Biocomp Fractionator
3.6 Convert Footprints to cDNA
3.6.1 RNA Isolation from Sucrose Gradients
3.6.2 Size Selection with Polyacrylamide Gel Electrophoresis
3.7 Barcode Footprints and Sequence
3.7.1 Dephosphorylation
3.7.2 Linker Ligation
3.7.3 rRNA Depletion
3.7.4 Reverse Transcription (RT) Reaction and Gel Electrophoresis
3.7.5 Circularization
3.7.6 Pilot PCR
3.7.7 Pilot PCR Gel Electrophoresis
3.7.8 Preparative PCR
3.7.9 BioAnalyzer, Sequencing and Analysis
4 Notes
References
Chapter 15: Small RNA-Sequencing Library Preparation for the Halophilic Archaeon Haloferax volcanii
1 Introduction
2 Materials
2.1 PAGE Gel Electrophoresis
2.2 Enzymes
2.3 Buffers, Reagents, and Media
2.4 Oligos
2.5 Equipment
3 Methods
3.1 Cultures and Treatments
3.2 Total RNA Extraction
3.3 7% Polyacrylamide Gel Electrophoresis for Size Selection
3.4 Quantify Size-Selected RNA
3.5 Assess Quality of Size-Selected RNA
3.6 DNase I Treatment
3.7 RNA Cleaning and Concentration
3.8 Reverse Transcriptase (RT) DNA Check (to Assess DNA Contamination)
3.9 cDNA Synthesis
3.10 PCR Amplification for DNA Check
3.11 Library Preparation
3.12 Quality Checking
3.13 Illumina Sequencing
4 Notes
References
Chapter 16: Quantitative Mass Spectrometry by SILAC in Haloferax volcanii
1 Introduction
2 Materials
2.1 Haloferax volcanii Growth and Treatment
2.2 Protein Extraction, Solubilization, and Quantification
2.3 Processing of Tryptic Peptides
2.4 Reverse-Phase Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) Analysis
3 Methods
3.1 Culturing, Isotopic Incorporation, and Treatment
3.2 Protein Extraction by TRIzol According to Kirkland et al. with Modification
3.3 Solubilization and Quantification of Protein from TRIzol Extracted Pellets
3.4 Reduction, Alkylation and Trypsin Digestion of Proteins
3.5 Solubilization and Desalting of Tryptic Peptides
3.6 Strong Cation Exchange Chromatography of Peptides
3.7 Reverse-Phase Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Analysis
3.8 Data and Statistical Analyses
4 Notes
References
Chapter 17: Proteome Turnover Analysis in Haloferax volcanii by a Heavy Isotope Multilabeling Approach
1 Introduction
1.1 General Introduction
1.2 Experimental Design
2 Material
2.1 Equipment
2.2 Buffers and Solutions
2.2.1 H. volcanii Minimal Media (Hv-Min)
2.2.2 Cell Handling and Processing Solutions
2.2.3 Protein Sample Preparation and MS Analysis
3 Methods
3.1 Isotope Labeling of Cell Cultures
3.1.1 Pulse-Chase with 15N
3.1.2 Metabolic 13C Labeling for Internal Standard
3.2 Sample Preparation for Nano LC-MS/MS Analysis
3.2.1 Cells Processing
3.2.2 15N/14N Proteins Processing
3.2.3 14N/13C Protein Processing
3.2.4 Cleanup, Destaining, Reduction, and Alkylation of Protein Samples
3.2.5 Tryptic in-Gel Digestion
3.3 Nano LC-MS/MS Analysis
3.4 Bioinformatic Analysis
3.4.1 Protein Identification
3.4.2 Quantification of Stable-Isotope-Labeled Samples
Creating a New Project and Experiment
Importing Data and Setting Up the Experimental Design
Pulse Chase (Synthesis) or Dual Labeling (Degradation) Quantification
3.4.3 Analysis of the Quantification Results
4 Notes
References
Chapter 18: Proteomic Sample Preparation and Data Analysis in Line with the Archaeal Proteome Project
1 Introduction
2 Materials
2.1 Buffer Exchange and Protein Concentration Components
2.2 Protein Solubilization and Digest Components
2.3 HPLC-MS/MS Analysis Components
2.4 Software
3 Methods
3.1 Buffer Exchange and Protein Concentration
3.2 Protein Solubilization and Digest Using Suspension Trapping
3.3 HPLC-MS/MS Analysis
3.4 Bioinformatic Analysis Using Multiple Search Engines and Facilitating Integration of Results in ArcPP
3.5 Interactive Visualization of ArcPP Results
4 Notes
References
Chapter 19: In Vivo Protein Cross-Linking and Coimmunoprecipitation in Haloferax volcanii
1 Introduction
2 Materials
2.1 Equipment
2.2 Haloferax volcanii Hv-Min Medium
2.3 Processing Solutions and MS Analysis
3 Methods
3.1 H. volcanii Cell Culture
3.2 In Vivo Protein Cross-Linking
3.2.1 Using DSP
3.2.2 Using Formaldehyde
3.2.3 Checking Cross-Linking Reactions
3.3 Cell Lysate and Separation of Membrane Fraction
3.4 Coimmunoprecipitation of Protein Complexes
3.4.1 Preparation of the Bead Slurry
3.4.2 Solubilization of Membrane Proteins
3.4.3 Immunoprecipitation: Incubation of Protein Samples with Antibody and Beads (See Note 21)
3.4.4 Checking the Efficiency of Immunoprecipitation
3.5 On-Bead Digestion with Trypsin
3.6 Cleaning the Samples
3.7 Nano LC-MS/MS Analysis
3.8 Protein Identification, Quantification, and Statistical Analysis
4 Notes
References
Chapter 20: Proteolytic Activity Assays in Haloarchaea
1 Introduction
2 Materials
2.1 In Vivo Extracellular Protease Detection on Casein Plates
2.1.1 Halophilic Medium
2.1.2 Hv-YPC Medium
2.2 Ethanol Precipitation of Extracellular Proteolytic Activity
2.3 Azocasein Assay
2.4 SDS-Polyacrylamide Gels Containing Glycine-Betaine
3 Methods
3.1 In Vivo Extracellular Proteolytic Activity Detection
3.1.1 Screening Protease Positive Environmental Isolates of Haloarchaea
3.1.2 Screening of H. volcanii Strains Expressing the Recombinant Halophilic Protease NEP
3.2 Ethanol Precipitation of Extracellular Proteolytic Activity
3.3 Azocasein Assay for Quantification of Proteolytic Activity
3.4 Electrophoresis of Halophilic Peptidases in Polyacrylamide Gels Containing Betaine for Detection of Active Bands
4 Notes
References
Chapter 21: Carotenoids from Haloarchaea: Extraction, Fractionation, and Characterization
1 Introduction
2 Materials
2.1 Haloferax volcanii Minimal Medium (Hv-Min)
2.2 Carotenoid Extraction, Fractionation, and Characterization
2.3 DPPH Radical Scavenging Activity: EPR Assay
3 Methods
3.1 Haloarchaeal Strains, Growth Medium, and Cultivation Conditions
3.2 Carotenoids Extraction
3.3 Carotenoid Quantification
3.4 Carotenoid Fractionation and Identification
3.4.1 UV-VIS Absorption Spectrum of Total CE
3.4.2 Thin-Layer Chromatography (TLC) Analysis of the CE
3.4.3 HPLC Analysis of the CE
3.5 Antioxidant Activity Determination
3.5.1 DPPH Radical Scavenging Activity: EPR Assay
3.5.2 EC50 Determination
4 Notes
References
Chapter 22: Metal nanoparticles Biosynthesis Using the Halophilic Archaeon Haloferax volcanii
1 Introduction
2 Materials
2.1 Chemicals
2.2 Equipment
2.3 Strain
2.4 H. volcanii MGM Medium
2.5 Solutions
3 Methods
3.1 H. volcanii Cell Culture
3.2 Biosynthesis of Ag NPs and Au NPs
4 Notes
References
Chapter 23: Simplified Enzymatic Synthesis of 2-Keto-3-Deoxy-D-Gluconate from D-Gluconate Using the Gluconate Dehydratase from...
1 Introduction
2 Materials
2.1 Cloning Procedures
2.2 Heterologous Gene Expression, Cell Harvest and Lysis
2.3 Protein Purification
2.4 GAD Activity Determination
2.5 KDG Determination
2.5.1 Thin Layer Chromatography
2.5.2 Thiobarbituric Acid (TBA) Assay
2.6 Enzymatic Preparation of KDG from D-Gluconate
3 Methods
3.1 Gene Cloning
3.2 Heterologous Overexpression, Cell Harvest and Lysis
3.3 Rapid Protein Purification Via Heat Precipitation and Fractionated Ammonium Sulfate Precipitation (See Note 5)
3.4 GAD Activity Measurement
3.5 Determination of KDG
3.5.1 Thin Layer Chromatography (TLC)
3.5.2 Thiobarbituric Acid (TBA) Assay
3.6 Enzymatic Preparation of KDG from D-Gluconate
4 Notes
References
Part III: Cellular Biology of Archaea
Chapter 24: Progress and Challenges in Archaeal Cell Biology
References
Chapter 25: Methods to Analyze Motility in Eury- and Crenarchaea
1 Introduction
2 Materials
2.1 Materials for Haloferax volcanii
2.2 Materials Sulfolobus acidocaldarius
3 Methods
3.1 Analysis of Motility of Haloferax Cells by Time-Lapse Light Microscopy
3.2 Analysis of Motility of Haloferax Cells on Semi-Solid Agar Plates
3.3 Analysis of Swimming Behavior of Sulfolobus acidocaldarius Via Time-Lapse Imaging
3.4 Analysis of Motility on Semi-Solid Gelrite Plate for Sulfolobus acidocaldarius
4 Notes
References
Chapter 26: Immersed Liquid Biofilm and Honeycomb Pattern Formations in Haloferax volcanii
1 Introduction
2 Materials
2.1 Media (Semi-Defined Hv-Cab Medium) (See Note 1)
2.2 Culture Preparation
2.3 Immersed Liquid Biofilm and Honeycomb Pattern Observation
2.4 Quantitative Image Analysis
3 Methods
3.1 Preparing Solid Semi-Defined Hv-Cab Plates Containing 1.5% (Wt/Vol) Agar (See Note 1)
3.2 Preparing Liquid Semi-Defined Hv-Cab Medium (See Note 1)
3.3 Preparing Cultures for Immersed Liquid Biofilm Formation
3.4 Observing Immersed Liquid Biofilms and Honeycomb Patterns
3.5 Documenting and Quantifying Immersed Liquid Biofilm Coverage
3.6 Documenting and Quantifying Honeycomb Pattern Formations
4 Notes
References
Chapter 27: Cost-Effective and Versatile Analysis of Archaeal Surface Adhesion Under Shaking and Standing Conditions
1 Introduction
2 Materials
2.1 Semi-Defined Hv-Cab Medium
2.2 Culture Preparation
2.3 mPAD Mount
2.4 Adhesion Assay under Shaking Conditions
2.5 Adhesion Assay Under Standing Conditions
2.6 Staining and Imaging
2.7 Miscellaneous
3 Methods
3.1 Preparing Solid Semi-Defined Hv-Cab Plates Containing 1.5% (Wt/Vol) Agar
3.2 Preparing Liquid Semi-Defined Hv-Cab Medium
3.3 Preparing Cultures for Adhesion Assays
3.4 Setting Up the mPAD Mount for Adhesion Assays Under Shaking Conditions
3.5 Setting Up 12-Well Plate for Adhesion Assays Under Standing Conditions
3.6 Sampling and Fixing Coverslips at Different Stages of Biofilm Formation
3.7 Staining Coverslips
3.8 Observing Biofilm Formation
4 Notes
References
Chapter 28: Cell Adhesion and Biofilm Formation Analysis
1 Introduction
2 Materials
2.1 Microbiological Culture
2.1.1 Saline Water Based Medium (SWB)
2.1.2 Strains
2.2 Chemical Reagents
2.2.1 Glass Cleaning Solutions
2.3 Biofilm Growth Setup
2.3.1 The Growth Chamber and the Liquid and Gas Flow Systems
2.3.2 Sample Materials
3 Methods
3.1 Starter Cultures
3.2 Sample Material Preparation
3.3 Liquid Flow System Assembly
3.4 Thin Film Reactor Assembly
3.5 Adhesion Assays
3.6 Biofilm Growth Assay
3.7 Microscopic Observation
4 Notes
References
Chapter 29: BrdU Incorporation and Labeling of Nascent DNA to Investigate Archaeal Replication Using Super-Resolution Imaging
1 Introduction
2 Materials
2.1 Haloferax volcanii Strain
2.2 Cell Cultures
2.3 Cells Chemical Fixation
2.4 BrdU Revelation
2.5 Sample Mounting (for SIM and/or STORM Imaging)
2.6 Microscopy Components and Setup for STORM Imaging (See Note 9)
2.7 Microscopy Components and Setup for SIM Imaging (See Note 12)
3 Methods
3.1 5′-Bromo-2′-deoxyuridine Incorporation in Replicating Cells
3.2 Cell Fixation
3.3 Dehydration and Formamide DNA Denaturation
3.4 5′-Bromo-2′-deoxyuridine Revelation
3.5 Sample Mounting (STORM and/or 3D-SIM)
3.6 STORM Imaging
3.7 Wide-Field Imaging (See Note 12)
3.8 3D-SIM Imaging (See Note 12)
3.9 STORM Image Reconstruction
4 Notes
References
Chapter 30: Isolation, Purification, and Characterization of Membrane Vesicles from Haloarchaea
1 Introduction
2 Materials
2.1 Microbiological Cultures
2.2 Harvesting MVs from Culture Supernatant
2.3 Purification of MVs
2.4 Downstream Analysis of MVs
3 Methods
3.1 Growing Cell Cultures for MV Production
3.2 Harvesting MVs from Culture Supernatant
3.3 Purification of MVs
3.4 Downstream Analyses of MVs
3.4.1 Preparation of MVs for Transmission Electron Microscopy (TEM)
3.4.2 Nucleic Acid Extraction
3.4.3 Proteomic Analysis
4 Notes
References
Chapter 31: Archaeal Viruses: Production of Virus Particles and Vesicle-like Viruses and Purification Using Asymmetrical Flow ...
1 Introduction
2 Materials
2.1 Archaeal Strains
2.2 Archaeal Viruses
2.3 Media and Buffers
2.4 AF4 Instrumentation
2.5 Others
3 Methods
3.1 Virus Growth by Double-Layer Method (Virus Agar Stock)
3.2 Plaque Assay to Determine the Number of Infectious Viruses (Titration)
3.3 Virus Production in Liquid Culture
3.4 Virus Purification by AF4
3.4.1 Setup of the AF4 Instrument
3.4.2 Performing an AF4 Experiment
4 Notes
References
Part IV: Environmental Biology
Chapter 32: Progress and Challenges in Studying the Ecophysiology of Archaea
1 Microscopy
2 Metagenomics
3 Other Omics of Archaea
4 Phylogenomics
5 Biochemistry and Structural Biology
6 Case Studies
6.1 Anaerobic Methane and Alkane Oxidizers (AMO, AAO)
6.2 Altiarchaeota and DPANN
7 Conclusion
References
Chapter 33: Reconstruction of Archaeal Genomes from Short-Read Metagenomes
1 Introduction
2 Materials
2.1 Generating Sequencing Reads
2.2 Basic Bioinformatics Knowledge
2.3 Desktop Computer
2.4 Server Requirements
2.5 Code Writing Conventions
2.6 Data and Data Structure
2.6.1 FASTA Format with Nucleotide Sequences
2.6.2 FASTQ Format with Read Sequences
2.6.3 Paired Reads in Separate FASTQ Files
2.6.4 Shuffled FASTQ File
2.7 Software
2.7.1 Creation, Loading, and Installation of python3 conda Environment and Software
2.7.2 Creation of ruby 2.3 and Installation of ruby gem Dependencies Using rvm
2.7.3 Creation, Loading, and Installation of the python2 conda Environment and Software
2.8 Databases
2.8.1 Formatting of the FunTaxDB as a DIAMOND BLAST Database
3 Methods
3.1 Read Quality Control
3.1.1 Read Quality Control and Contaminant Removal
3.2 Assembly
3.2.1 Basic Execution of MetaSPAdes Command
3.2.2 Basic Execution of MEGAHIT Command
3.2.3 Scaffold/Contig Renaming
3.3 Processing of Metagenomic Assemblies
3.3.1 Execution of the uBin Wrapper
3.3.2 Mapping
3.3.3 ORF Prediction and Annotation
3.3.4 Collection of Scaffold Information
3.3.5 Prediction of Single Copy Genes per Scaffold
3.4 Binning of Metagenome-Assembled Genomes (MAGs)
3.4.1 Calculation of 4-mer Frequencies
Copying of the GitHub Directory ``Binning´´
Calculation of 4-mer Frequencies.
3.4.2 ABAWACA Using
Execution of the Automatic Binner ABAWACA
3.4.3 MaxBin2
Execution of the Automatic Binner MaxBin2
3.4.4 CONCOCT
3.4.5 Manual Binning Tools
Extraction of Bins in FASTA Format from esomana Output
3.4.6 Additional Binning Tools
3.4.7 Generation of scaffold2bin Tables
Generation of scaffold2bin Tables for the DAS Tool Input
Renaming Bin Names to Make Them Unique
3.4.8 Aggregation of Bin Sets
Execution of DAS Tool for the Aggregation of Individual Bin Sets
3.5 Manual Curation of Metagenomic Bins
3.5.1 Addition of Bin Information
3.5.2 Transfer of Files to the Local Machine
scp-based Transfer of Files from Remote Location to the Local Computer
3.5.3 Manual Curation of Genomes with uBin
3.5.4 Identification and Curation of Archaeal Bins in uBin
3.5.5 Export of Bins from uBin
3.6 Branch-Specific Completeness and Contamination Prediction
3.7 Filtering Out Low-Quality Genomes
3.8 Phylogenomic Placement
3.8.1 Phylogenomic Placement of Genomes Using GTDK-tk
3.9 Beyond Binning
3.9.1 Extending and Completing Genomes
3.9.2 ORF Prediction on Genomes
3.9.3 Dereplication of Genomes
3.9.4 Prediction of Minimal Generation Time
3.9.5 Prediction of Metabolic Potential
3.9.6 Virus-Host Matching
3.9.7 Single-Nucleotide-Polymorphism (SNP) and Strain Analysis
3.9.8 Assembly error correction
4 Notes
References
Part V: Outreach Protocol
Chapter 34: Accessible and Insightful Scientific Learning Experiences Using the Microorganism Haloferax volcanii
1 Introduction
2 Materials
2.1 Media
2.1.1 Solid H. volcanii Agar (1.5% wt/vol) Plates and Liquid Media (See Note 1): Laboratory-Grade Recipe Components (Makes 1 L...
2.1.2 Recipe Components Using Reagents that Can Mostly be Obtained at a Grocery Store (Makes 1 L of Medium)
2.2 Growth
2.3 Storage
2.3.1 Short-Term Storage (Up to a Year)
2.3.2 Long-Term Storage (See Note 9)
2.4 Antibiotic Plate Preparation
2.5 Optional: E. coli Preparation (See Note 12)
2.5.1 Media (See Note 13): Solid E. coli Agar 1.5% (wt/vol) Plates and Liquid Media (See Note 1) (Makes 1 L of Medium)
2.5.2 Growth (See Note 10)
2.5.3 Storage
3 Methods
3.1 H. volcanii Media
3.1.1 Laboratory-Grade Recipe for Agar Plates
3.1.2 Laboratory-Grade Recipe for Liquid Medium
3.1.3 Recipe Using Reagents that Can be Obtained at a Grocery Store
3.2 Growing H. volcanii on Agar Plates and in Liquid Medium
3.2.1 Restreak on a New Plate from the Original H. volcanii Agar Plate or from a Frozen Stock
3.2.2 Grow in Liquid Medium for Freezing
3.3 Storing H. volcanii on Agar Plates and Freezing
3.4 Preparing Plates for Antibiotic Experiment
3.5 Plating Cultures for Antibiotic Experiment
3.6 Placement of Antibiotic Disks and Subsequent Observation for Antibiotic Experiment
3.7 Optional: E. coli Preparation (See Note 12)
3.7.1 Media (See Note 13)
3.7.2 Growth on Plate (See Note 10)
3.7.3 Growth in Liquid Culture for Freezing
3.7.4 Storage
4 Notes
References
Index