Metagenomics: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Construction of Small-Insert and Large-Insert Metagenomic Libraries
1 Introduction
2 Materials
2.1 Metagenomic DNA
2.2 Generation of Small-Insert Metagenomic Libraries
2.3 Generation of Large-Insert Metagenomic Libraries
3 Methods
3.1 Generation of Small-Insert Metagenomic Libraries
3.1.1 Whole-Genome Amplification of Environmental DNA
3.1.2 Resolving Hyperbranched DNA Structures (See Note 2)
3.1.3 S1 Nuclease Treatment (See Note 2)
3.1.4 Shearing of Metagenomic DNA
3.1.5 End-Repair of Insert DNA
3.1.6 Size Fractionation of the Insert DNA
3.1.7 Addition of 3′ A-Overhangs to Blunt-Ended, Size-Fractionated DNA
3.1.8 Dephosphorylation of Insert DNA
3.1.9 TOPO Cloning
3.2 Generation of Large-Insert Metagenomic Libraries
3.2.1 Preparation of Host Cells
3.2.2 Shearing of Metagenomic DNA (See Note 8)
3.2.3 Size Fractionation of the Insert DNA (See Note 9)
3.2.4 End-Repair of Insert DNA
3.2.5 Ligation
3.2.6 Packaging of Fosmids
3.2.7 Transduction of Host Cells
4 Notes
References
Chapter 2: Extraction of Total DNA and RNA from Marine Filter Samples and Generation of a Universal cDNA as Universal Template...
1 Introduction
2 Materials
2.1 Bacterioplankton Samples
2.2 Simultaneous DNA and RNA Extraction
2.3 Purification of Extracted DNA and RNA
2.4 DNA Digestion and DNA-Contamination Control PCR
2.5 First- and Second-Strand Synthesis
3 Methods
3.1 Simultaneous DNA and RNA Extraction from Marine Filter Samples
3.1.1 Co-Extraction of DNA and RNA
3.1.2 RNA Isolation
3.1.3 DNA Isolation
3.1.4 Washing and Resuspension of RNA and DNA
3.2 DNA and RNA Purification
3.2.1 Removal of Residual RNA from DNA Samples
3.2.2 RNA Purification with the QIAGEN RNeasy Mini Kit
3.2.3 DNA Digestion
3.2.4 Control PCR for Residual DNA
3.3 First- and Second-Strand Synthesis
4 Notes
References
Chapter 3: Functional Metagenomics as a Tool to Tap into Natural Diversity of Valuable Biotechnological Compounds
1 Introduction
2 Materials
2.1 Sampling
2.1.1 Marine Water Sampling
2.1.2 Sampling from Marine Invertebrates
2.2 Isolation of Metagenomic DNA
2.3 Construction of a Metagenomic Large-Insert Library
2.4 Screening Metagenomic Libraries
2.4.1 Screening Metagenomic Libraries for Quorum Quenching Activities
2.4.2 Screening Metagenomic Libraries for Bioactive Peptides
3 Methods
3.1 Sampling Procedures
3.1.1 Marine Surface Water Sampling
3.1.2 Marine Deep Water Sampling
3.1.3 Sampling from Marine Invertebrates
3.2 Isolation of Metagenomic DNA
3.3 Construction of a Metagenomic Large-Insert Library
3.4 Function-Based Screens of Metagenomic Libraries
3.4.1 Screening Metagenomic Libraries for Natural Quorum Quenching Compounds
3.4.2 Screening Metagenomic Libraries for Bioactive Peptides
4 Notes
References
Chapter 4: Functional and Sequence-Specific Screening Protocols for the Detection of Novel Antimicrobial Resistance Genes in M...
1 Introduction
2 Materials
2.1 Saliva Sample Collection and DNA Extraction
2.2 PCR-Based Metagenomic Screening
2.3 DNA Quantification and Quality Control
2.4 Construction of a Metagenomic Library
2.5 Screening and Characterization of Antimicrobial Resistance Genes
2.6 Antimicrobial and Bacterial Cultures
2.7 Consumables, Equipment, and Instruments
3 Methods
3.1 Saliva Sample Collection and Metagenomic DNA Extraction
3.2 PCR-Based Detection for ARGs in the Oral Metagenomic DNA
3.3 Construction of Saliva Metagenomic DNA Library
3.3.1 Partial Digestion of the Extracted Oral Metagenomic DNA
3.3.2 Ligation of the Partial Digested Oral Metagenome into pCC1BAC Cloning Vector
3.3.3 Electroporation of Ligation Product into E. coli Surrogate Host
3.3.4 Library Size Determination
3.3.5 Preparation of Glycerol Stock of the Oral Metagenomic Library (See Note 22)
3.4 Screening Metagenomic Libraries Using Antimicrobial Compounds That Are Inactive in Agar
3.4.1 Determining whether Antimicrobials Are Active in Agar
3.4.2 Metagenomic Library Screening of Antimicrobial Compounds That Are Inactive in Agar
3.4.3 End-Point Monitoring
3.4.4 Continuous Monitoring
3.4.5 Secondary Screening
3.4.6 Tertiary Screening (See Note 26)
3.5 Characterization of Positive Hits from Metagenomic Screening
3.5.1 Genome Walking and Sequential Primer Design (Fig. 3)
3.5.2 Transposon Mutagenesis
3.5.3 Genome Sequencing
4 Notes
References
Chapter 5: Identification of PKS Gene Clusters from Metagenomic Libraries Using a Next-Generation Sequencing Approach
1 Introduction
1.1 Functional Metagenomics
1.2 Shotgun Metagenomics
2 Materials
2.1 Laboratory Equipment
2.2 Bacterial Culturing
2.3 Pooled Plasmid DNA Extraction
2.4 Pooled Plasmid DNA Library Preparation
2.5 Software
3 Methods
3.1 Metagenomic Library Pooling Design
3.2 Clone Culturing and Pooling
3.3 Pool Plasmid DNA Extraction
3.4 DNA Quality Check
3.5 Pooled DNA Multiplexed Sequencing
3.6 Demultiplexing Reads
3.7 Bioinformatic Analysis
3.7.1 Creating a Consistent PoolID System
3.7.2 Read Quality Check
3.7.3 Read Processing
3.7.4 Additional Quality Check
3.7.5 Filtering Vector Sequences and Possible Host Contamination
3.7.6 Pool Assembly
3.7.7 Renaming Contigs
3.7.8 Deconvolution from Contig to Clone
3.7.9 Identifying PKS Clusters
3.7.10 Comparing PKS Clusters
3.7.11 Taxonomic Origin of PKS Clusters
3.7.12 Conclusions
4 Notes
References
Chapter 6: Activity-Based Screening of Metagenomic Fosmid Libraries for Hydrogen-Uptake Enzymes
1 Introduction
2 Materials
2.1 Laboratory Equipment
2.2 Strains, Plasmids, and Growth Media
2.3 Kits and (Restriction) Enzymes
3 Methods
3.1 Construction of the Metagenomic Library with the Broad-Host-Range Vector pRS44
3.1.1 Preparation of the Broad-Host-Range Vector pRS44
3.1.2 Preparation of Metagenomic DNA
3.1.3 Ligation, Transduction, and Storage of the Metagenomic Library
3.2 Screening of Metagenomic Libraries for Hydrogen-Uptake Activities
3.2.1 Transfer of Metagenomic Fosmids into S. oneidensis ΔhyaB (See Notes 1 and 2)
3.2.2 Detection of Hydrogen-Uptake Active Clones (See Notes 3-5)
4 Notes
References
Chapter 7: Functional Metagenomics Approach for the Discovery of Novel Genes Encoding Phosphatase Activity
1 Introduction
2 Materials
2.1 Identification of Phosphatase Genes by Function-Based Screening of Metagenomic Libraries
2.1.1 Metagenomic Libraries
2.1.2 Media for Small-Insert and Large-Insert Metagenomic Library Screening
2.1.3 Function-Based Metagenomic Library Screening
2.1.4 Analysis of Metagenomic DNA Fragments Carried by Positive Clones
2.2 Heterologous Expression of Phosphatase Genes
2.2.1 Cloning of Putative Phosphatase-Encoding Genes into Expression Vector
2.2.2 Heterologous Expression of Genes Encoding Phosphatase Activity
2.2.3 Verification of Heterologous Target Gene Expression Based on Phosphatase Activity Test
3 Methods
3.1 Identification of Phosphatase Genes by Function-Based Screening of Metagenomic Libraries
3.1.1 Preparation of Screening Media
3.1.2 Function-Based Metagenomic Library Screening
3.1.3 Analysis of Metagenomic DNA Fragments Carried by Positive Clones
3.2 Efficient Heterologous Expression of Phosphatase Genes
3.2.1 Cloning of Putative Phosphatase-Encoding Genes into Expression Vector
3.2.2 Heterologous Expression of Genes Encoding Phosphatase Activity
3.2.3 Verification of Heterologous Target Gene Expression Based on Phosphatase Activity Test
4 Notes
References
Chapter 8: Isolation of Genes Encoding Carbon Metabolism Pathways from Complex Microbial Communities
1 Introduction
2 Materials
2.1 Bacterial Growth Media
2.2 Molecular Biology Reagents
3 Methods
3.1 Bacterial Growth and Storage Conditions
3.2 Construction of Metagenomic Libraries from Soil
3.3 Transfer of a Metagenomic Library into P. alloputida by Triparental Conjugation
3.4 Screening of Metagenomic Libraries for Clones That Complement P. alloputida´s Ability to Grow on Galactose
3.5 Transfer of Clones Back into E. coli for Subsequent Isolation of Cosmid DNA
3.6 Isolation of Cosmid DNA and Identification of Unique Clones via Restriction Enzyme Analysis
3.7 Sequencing and Analysis of Clones
3.8 Follow-Up Techniques
3.8.1 Subcloning
3.8.2 In Vitro Transposon Mutagenesis
4 Notes
References
Chapter 9: Screening Metagenomes for Algae Cell Wall Carbohydrates Degrading Hydrolases in Enrichment Cultures
1 Introduction
1.1 Algae Cell Wall Polymers: Structure and Enzymatic Degradation
2 Materials
2.1 Media and Consumables
2.2 Glucose Oxidase Assay Kit
2.3 Thin-Layer Chromatography
3 Methods
3.1 Enrichment of Highly Cell Wall-Hydrolyzing Microbial Communities
3.2 Enrichment Cultures
3.3 Characterize Initial Microbial Community
3.4 Meta-Omics Analyses
3.5 Degradation Quantification
3.6 Glucose Oxidase Assay
3.7 Thin-Layer Chromatography (TLC) Analyses for Lipopolysaccharides
4 Notes
References
Chapter 10: The PET-Degrading Potential of Global Metagenomes: From In Silico Mining to Active Enzymes
1 Introduction
2 Materials
2.1 Bioinformatic Analysis
2.2 Functional Screening on Agar Plates
2.3 UHPLC Measurements
3 Methods
3.1 Construction of a Profile HMM and Screening of a Protein Database for Putative PETases
3.2 Functional Screening on Polymer-Containing Indicator Plates
3.2.1 PET-Containing Plates
3.2.2 PCL-Containing Plates
3.2.3 BHET-Containing Plates
3.3 UHPLC Measurements
4 Notes
References
Chapter 11: High-Throughput Screening for Thermostable Polyester Hydrolases
1 Introduction
2 Materials
2.1 Screening Plates
2.2 Plasmid Recovery from Screening Plates
2.3 PCR Amplification of Plasmid Back Up
3 Methods
3.1 Applying a Mild Expression System Using Gibson Assembly
3.2 Preparing Screening Plates
3.3 Inoculation and Incubation of a Screening Plate
3.4 Plasmid Recovery from Screening Plates
3.5 Plasmid Recovery from Extract Solution by PCR Amplification
4 Notes
References
Chapter 12: Metagenomic Screening of a Novel PET Esterase via In Vitro Expression System
1 Introduction
2 Materials
2.1 Preparation of DNA Template
2.2 In Vitro Transcription
2.3 Preparation of Cell Extract
2.4 In Vitro Translation
2.5 BHET/TPA Assay
3 Methods
3.1 Preparation of DNA Template
3.2 In Vitro Transcription (ivTx)
3.3 Preparation of Cell Extract
3.4 In Vitro Translation (ivTT)
3.5 BHET/TPA Assay
4 Notes
References
Chapter 13: Assigning Functions of Unknown Enzymes by High-Throughput Enzyme Characterization
1 Introduction
2 Materials
2.1 Working Solutions
2.2 Ester Stock Solution (See Note 3)
2.3 Triglyceride Solution (See Note 4)
2.4 4-Nitrophenyl Ester Stock Solution (See Note 6)
2.5 α-Naphthyl Ester Solution
2.6 Enzyme-Containing Solution
2.7 Ester-Hydrolase Assays in Liquid Format
2.8 Ester-Hydrolase Assays in Agar Plate Format
3 Methods
3.1 Ester-Hydrolase Assays in Liquid Format (See Notes 7 and 8)
3.1.1 Using Phenol Red pH 8.0 as a pH Indicator (See Note 9)
3.1.2 Using p-Nitrophenol pH 7.2 as a pH Indicator (See Note 11)
3.1.3 Using Nitrazine Yellow pH 7.2 as a pH Indicator (See Note 13)
3.1.4 Using Fluorescein pH 7.0 as pH Indicator
3.1.5 Using p-Nitrophenyl Esters
3.2 Ester-Hydrolases Assays in Agar Plate Format
3.2.1 Using α-Naphthyl Esters
3.2.2 Using Tributyrin
3.2.3 Using Coconut Oil
3.2.4 Using Olive Oil
3.2.5 Using Egg Yolk
3.2.6 Using Polylactic Acid (PLA) Emulsion
3.2.7 Using Polycaprolactone Diol (PCDMn530)
3.2.8 Using Polycaprolactone (PCL) Nanoparticles
3.2.9 Using Polyethylene Terephthalate (PET) Nanoparticles (Adapted from)
4 Notes
References
Chapter 14: Isolation of a Host-Confined Phage Metagenome Allows the Detection of Phages Both Capable and Incapable of Plaque ...
1 Introduction
2 Materials
2.1 Bacterial Host Strain
2.2 Media and Working Solutions
2.3 Consumables
3 Methods
3.1 Preparation of the Host B. pondensis LVF1T
3.2 Preparation of Sewage Water for Bacteriophage Enrichment
3.3 Enrichment of Host-Specific Bacteriophages via a Plaque Assay
3.4 Total Nucleic Acids Purification
3.5 Preparation of Viral dsDNA, ssDNA, dsRNA, and ssRNA
3.6 Preparation of dsDNA, dsRNA, and ssRNA Virome
3.7 Transcription of Viral ssDNA to dsDNA for NGS Sequencing
3.8 PCR-Based Screening for Phages Incapable of Plaque Formation
4 Notes
References
Chapter 15: CRISPR-Cas9 Shaped Viral Metagenomes Associated with Bacillus subtilis
1 Introduction
2 Materials
2.1 Protospacer Identification
2.2 The pRH030 Vector System
2.3 Cas9 Targets
2.4 Cloning Host
2.5 Bacillus Host
2.6 Phage Source
2.7 Media
2.8 Chemicals
2.9 Enzymes
2.10 Consumables
3 Methods
3.1 Identification of Suitable Protospacer Sequences
3.2 Primer Design for CRISPR Array Construction
3.3 Constructing an Artificial CRISPR Array
3.4 Setting Up the B. subtilis TS01 Host System
3.5 Prepare a Phage Suspension from Sewage
4 Notes
References
Chapter 16: Cloning and Expression of Metagenomic DNA in Streptomyces lividans and Its Subsequent Fermentation for Optimized P...
1 Introduction
2 Transformation of S. lividans
2.1 Materials and Growth Media
2.1.1 General Growth Media
2.1.2 Preparation of S. lividans Spore Suspension
2.1.3 Plasmid Conjugation Between E. coli and S. lividans
2.1.4 Triparental Mating
2.1.5 Preparation of Streptomyces Protoplasts
2.1.6 Protoplast Transformation
2.2 Methods
2.2.1 Growth of S. lividans
2.2.2 Preparation of S. lividans Spore Suspension
2.2.3 Plasmid/Cosmid Conjugation from E. coli to S. lividans
2.2.4 Triparental Mating
2.2.5 Preparation of Streptomyces Protoplasts
2.2.6 Protoplast Transformation
3 Streptomyces Vectors and Their Features
3.1 Materials
3.1.1 Isolation of pDNA by Alkaline Lysis with Potassium Acetate Precipitation
3.1.2 Isolation of Low-Copy Number Plasmids from S. lividans Using the QIAGEN Plasmid Midi Kit
3.1.3 Miniprep Isolation of Total DNA with Potassium Acetate Precipitation
3.1.4 Salting Out Protocol for Isolation of Genomic DNA
3.2 Methods
3.2.1 Plasmid DNA Purification
Isolation of pDNA by Alkaline Lysis with Potassium Acetate Precipitation
Isolation of Low-Copy Number Plasmids from S. lividans Using the QIAGEN Plasmid Midi Kit
3.2.2 Total DNA Isolation from S. lividans
Quick Isolation of Total DNA with Potassium Acetate Precipitation
3.2.3 High-Quality Genomic DNA Isolation
4 Gene Expression Control Elements for Use in Streptomyces
4.1 Materials
4.1.1 Cloning of Target Gene Under Control of Synthetic Promoter of Choice
4.1.2 Modification of Large Clones by Insertion of Promoter Cassettes
Amplification of Activation Cassette
Recombination Procedure
Marker Removal
Marker-Less Integration into S. lividans Chromosome Using pAMR23A-Based System
4.2 Methods
4.2.1 Cloning of Target Gene Under Control of Synthetic Promoter of Choice
4.2.2 Modification of Large Clones by Insertion of Promoter Cassettes
Amplification of Activation Cassette
Recombination Procedure
Removal of the Marker
Marker-Less Integration into S. lividans Chromosome via Homologous Recombination Using pAMR23A-Based System
5 Fermentations with Recombinant S. lividans
5.1 Materials
5.1.1 Fermentation Media
5.1.2 Bioreactor
5.2 Methods
5.2.1 Inoculum Preparation
5.2.2 Medium Selection
5.2.3 Small-Scale Batch Fermentations
5.2.4 Biomass Determination
5.2.5 Upscaling Strategies
6 Notes
References
Chapter 17: DNA-, RNA-, and Protein-Based Stable-Isotope Probing for High-Throughput Biomarker Analysis of Active Microorganis...
1 Introduction
2 Materials
2.1 General Reagents and Equipment for DNA-SIP, RNA-SIP, and Protein-SIP
2.2 Biomarker Purification (DNA, RNA, Protein)
2.2.1 Materials for Manual Purification of DNA and RNA
2.2.2 Materials for Purification of Protein During DNA/RNA Isolation
2.2.3 Materials for In-Gel Tryptic Digestion of Proteins
2.3 Materials for DNA/RNA Ultracentrifugation
2.4 Materials for Quantitative PCR of 16S rRNA
2.5 Materials for Reverse Transcription of RNA to cDNA
2.6 Metagenome/Metatranscriptome Sequencing
3 Methods
3.1 Biomarker Purification from Labelled Soil or Sediment Samples
3.1.1 Manual Purification of DNA and RNA
3.1.2 Protein Purification: Adaptation of DNA/RNA Purification Method
3.2 In-Gel Tryptic Digestion of Proteins
3.3 Separation of Labelled DNA by CsCl Gradient Centrifugation
3.4 Separation of Labelled RNA by CsTFA Gradient Centrifugation
3.5 Quantitative PCR of 16S rRNA
3.5.1 Reverse Transcription of RNA to cDNA
3.5.2 Quantitative PCR of 16S rRNA
3.6 Analysis of 16S rRNA Gene Data
3.7 Analysis of Metagenomes and Metatranscriptomes
3.8 SIP-Proteomics Analysis by High-Resolution MS
4 Notes
References
Index