YEAST PROTOCOLS.

Description

Preface Contents Contributors Chapter 1: Use of Yeast Plasmids: Transformation and Inheritance Assays 1 Introduction 1.1 Low- and High-Copy Number Yeast Plasmids 1.2 Marker Genes 1.3 Yeast Expression Vectors 1.4 ARS/CEN and 2-μm YEp Plasmids Are Stable but Not Inherited with 100% Efficiency 1.5 Issues with 2-μm-Based Plasmids 1.6 Transformation of Yeast with Two Plasmids 1.7 Working with Transformed Yeast 2 Materials 2.1 Yeast Strains 2.2 Media 2.2.1 YPAD Medium 2.2.2 SD Medium 2.2.3 Amino Acid Dropout Mix (Lacks Histidine, Leucine, and Tryptophan) (See Note 3) 2.2.4 Amino Acid Stock Solutions 2.2.5 Nucleotide Base Stock Solutions 2.2.6 Media pH 2.3 Yeast Transformation 3 Methods 3.1 LiAc-Mediated Yeast Transformation 3.2 Plasmid Inheritance Assays 3.2.1 Assay for Percentage of Cells Containing Plasmid 3.2.2 Assay for Plasmid Stability 4 Notes References Chapter 2: Construction of Tight Conditional Mutants Using the Improved Auxin-Inducible Degron (iAID) Method in the Budding Yeast Saccharomyces cerevisiae 1 Introduction 2 Materials 2.1 Yeast Strains and Growth 2.2 Plasmids and Oligonucleotides 2.3 Antibodies 2.4 NAA and Doxycycline 3 Methods 3.1 Construction of the Host Strain for the iAID Mutants 3.2 PCR Amplification of the Transforming DNA for the Gene of Interest 3.3 Yeast Transformation and Verification of the Transformants 3.4 Examination of the Growth Conditions of the Obtained iAID Mutants 3.5 Example Usage of the iAID Mutant 4 Notes References Chapter 3: CRISPR Nickase-Mediated Base Editing in Yeast 1 Introduction 2 Materials 2.1 Media 2.2 Strains and Plasmid 2.3 Preparation of DNA Fragments for Gap Repair Cloning 2.4 Primers 2.5 Yeast Transformation 2.6 Isolation of Base-Edited Cells 3 Methods 3.1 Preparation of DNA Fragments Constituting the Plasmid for the CRISPR Nickase System 3.1.1 Fragment Amplification 3.1.2 Characterization of the Amplified Fragments 3.2 Gap Repair Cloning in Yeast Cells 3.3 Base Editing by Single Cas9 Nickase 3.4 Isolation of Base-Edited (CAN1 Mutagenesis) Cells 4 Notes References Chapter 4: Genomic Promoter Shuffling by Using Recyclable Cassettes 1 Introduction 2 Materials 2.1 Strains 2.2 Plasmids 2.3 Culture Media 2.4 Yeast Transformation 2.5 Yeast Genomic DNA Extraction 3 Method 3.1 Make Promoter Shuffling Strains 3.1.1 Integration Primer Design 3.1.2 Amplify Recyclable Cassettes by PCR 3.1.3 Genomic Integration by Transformation 3.1.4 Selection of Pop-Out Strains 3.1.5 Confirmation of Strains by Genomic DNA PCR 3.2 Creation of a tTA Strain 3.2.1 Obtaining Integration Cassette 3.2.2 Genomic Integration by Transformation 3.2.3 Selection of Pop-Out Strains on 5-FOA Plates 3.3 Construction of a Custom-Made Promoter Shuffling Cassette 3.3.1 Amplify Cassette Elements 3.3.2 Clone the URA3 Marker Gene into pBluescript to Form pBS-URA3 (See Note 9) 3.3.3 Clone Both Upstream and Downstream Promoter Elements into pBS-URA3 to Form pXUX 4 Notes References Chapter 5: Study Essential Gene Functions by Plasmid Shuffling 1 Introduction 2 Materials 2.1 Strains 2.2 Plasmids 2.3 Culture Media 2.4 Yeast Transformation and Lysis Reagents 3 Methods 3.1 Construction of a YCp-URA3-YFEG Plasmid 3.2 Transformation of YCp-URA3-YFEG into a Selected Yeast Strain (See Note 2) 3.3 Deletion of YFEG by a Selectable Marker Other Than URA3 3.4 Construction of Plasmid YCp-LEU2-YFEG and Creation of Desired Mutations in YFEG 3.5 Counterselection Against Cells Carrying Plasmid YCp-URA3-YFEG 3.6 Phenotypic Analysis of YFEG Mutations 4 Notes References Chapter 6: Scarless Genomic Protein Labeling in Saccharomyces cerevisiae 1 Introduction 2 Materials 2.1 Yeast Strains 2.2 Plasmids 2.3 Yeast Media 2.4 Yeast Transformation Solutions 2.5 Yeast Genomic DNA Extraction Buffer 3 Methods 3.1 Generate Scarless Protein-Labeled Strains 3.1.1 Integration Primer Design 3.1.2 Amplify Recyclable Cassettes by PCR 3.1.3 Genomic Integration by Yeast Transformation 3.1.4 Screen for Cassette Integration (or Pop-In) Isolates by Genomic DNA PCR 3.1.5 Obtain and Screen for Pop-Out Isolates by Genomic DNA PCR 3.2 Construction of a Custom-Made Protein Labeling Cassette 3.2.1 PCR Primer Design for Protein Labeling Cassette 3.2.2 Amplify Cassette Elements 3.2.3 Linearize the Vector 3.2.4 Build In-Fusion Reaction System 3.2.5 Transformation 3.2.6 Screen for Positive Clones 4 Notes References Chapter 7: High-Copy Yeast Library Construction and High-Copy Rescue Genetic Screen in Saccharomyces cerevisiae 1 Introduction 2 Materials 2.1 Media 2.2 Reagents 3 Methods 3.1 pRS425 Based High-Copy Library Generation 3.2 Yeast Library Transformation and Screen 3.3 Yeast Plasmid Extraction and Identification 4 Notes References Chapter 8: Genome-Wide Imaging-Based Phenomic Screening Using Yeast (Saccharomyces cerevisiae) Strain Collections 1 Introduction 2 Materials 2.1 Equipment 2.2 Yeast Strains 2.3 Plates 2.4 Chemicals and Media 3 Methods 3.1 Query Strain Construction 3.2 Preparing a 1536-Density Yeast GFP Collection Array 3.3 Introducing the Hsp104-mRuby2 Marker into the GFP Collection Using the SGA Method 3.4 Store the Constructed Yeast Collection in 384-Well Glycerol Stock Plates 3.5 Inoculate and Revive the Yeast from Frozen Plates for Imaging-Based Screening 3.6 Sample Treatment, Image Acquisition, and Image Analysis 3.7 Hit Confirmation and Data Analysis 4 Notes References Chapter 9: Applications of Oxford Nanopore Sequencing in Schizosaccharomyces pombe 1 Introduction 2 Materials 2.1 Long Fragment DNA Library Preparation 2.2 Direct RNA Sequencing Library Preparation 2.3 Equipment 3 Methods 3.1 Nucleic Acid Sequencing Library Preparation 3.1.1 Preparation of Large Fragment Genomic DNA (gDNA) 1D Library 3.1.2 Direct RNA Sequencing Library Preparation 3.2 Sample Loading onto the Flow Cell Followed by Sequencing 3.2.1 Loading DNA Sample (SQK-LSK109) 3.2.2 Loading RNA Sample (SQK-RNA002) 3.2.3 Sequencing Run 3.3 Raw Data Analysis 3.4 Oxford Nanopore Technologies Applications 3.4.1 Application One: De Novo Assembly of a Complete Genome 3.4.2 Application Two: Prediction of Large Structural Variations 3.4.3 Application Three: RNA Direct Sequencing 4 Notes References Chapter 10: Measuring Genome-Wide Nascent Nucleosome Assembly Using ReIN-Map 1 Introduction 2 Materials 2.1 Yeast Cell Culture and In Vivo BrdU Incorporation 2.2 Zymolyase Treatment Lysis and MNase Digestion of the Lysed Cells 2.3 Bead-Beating Lysis and Sonication Treatment of the Lysed Cells 2.4 Monitor the Cell Synchronization and Cell Cycle Progression 2.5 Check MNase Digestion Pattern and Sonication Treatment Efficiency 2.6 Reversing Cross-Links and DNA Extraction 2.7 Immuno-precipitation of BrdU-Labeled DNA 2.8 ssDNA Library Preparation for Illumina HiSeq Systems 3 Methods 3.1 Yeast Cell Culture and In Vivo BrdU Incorporation 3.2 Chromatin Fragmentation 3.2.1 Zymolyase Treatment Lysis and MNase Digestion of the Lysed Cells [9] 3.2.2 Bead-Beating Lysis and Sonication Treatment of the Lysed Cells [10, 11] 3.3 Quality Control Before BrdU-IP 3.3.1 Monitor the Cell Synchronization and Cell Cycle Progression 3.3.2 Check MNase Digestion Pattern and Sonication Treatment Efficiency [9] 3.4 Reversing Cross-Links and DNA Extraction [10, 11] 3.5 Immuno-precipitation of BrdU-Labeled DNA [6, 11] 3.6 ssDNA Library Preparation for Illumina HiSeq Systems [11, 12] 3.6.1 Dephosphorylation and Heat Denaturation of ssDNA Sample 3.6.2 Ligation of the First Adapter at 3′-End (See Note 11) 3.6.3 Immobilization of Ligation Products on Streptavidin Beads 3.6.4 Primer Annealing and Extension 3.6.5 Blunt-End Repair 3.6.6 Ligation of the Second Adaptor at the 5′-End 3.6.7 Library Elution 3.6.8 Determine the Library Preparation Efficiency and Library Quantities by qPCR 3.6.9 Library Amplification and Indexing 3.7 Sequencing and Data Analysis 4 Notes References Chapter 11: Targeted Integration of Complex Genetic Elements at Multi-Copy Loci by Golden Gate Assembly 1 Introduction 2 Materials 2.1 Rapid Plasmid Construction by Golden Gate Assembly 2.2 Medium Preparation 2.3 Reagents for Yeast Transformation 3 Method 3.1 Site-Specific Mutagenesis in the pUC Vector 3.2 Preparation of Intermediate Cloning Vector 3.3 Sub-Module Design 3.4 Sub-Module Assembly 3.5 Integration Constructs 3.6 Yeast Transformation [15, 16] 4 Notes References Chapter 12: Chromosomal Rearrangements of Synthetic Yeast by SCRaMbLE 1 Introduction 2 Materials 2.1 Media Used 2.2 Reagents Used 2.3 Primers Used 3 Methods 3.1 Integrate the ReSCuES Cassette into the HO Locus of a Synthetic Yeast Strain 3.2 Induce SCRaMbLE in a Synthetic Strain 3.3 Dissect the SCRaMbLEd Genome 4 Notes References Chapter 13: Use YeastFab to Construct Genetic Parts and Multicomponent Pathways for Metabolic Engineering 1 Introduction 2 Materials 2.1 Polymerase Chain Reaction 2.2 Golden Gate Assembly in HCKan Vectors 2.3 E. coli Transformation and Verification 2.4 Golden Gate Assembly in POT Vectors 2.5 Assembly of Pathways 2.6 Yeast Transformation 3 Methods 3.1 Amplify and Clone Standard Genetic Parts into HCKan Vectors 3.1.1 Primer Design 3.1.2 Amplify the Genetic Parts from the Yeast Genome 3.1.3 Golden Gate Assembly 3.1.4 Bacterial Transformation 3.1.5 Verification of Assembled Clones 3.2 Assembly of Standard Genetic Parts into Transcription Units 3.2.1 Golden Gate Assembly 3.2.2 Bacterial Transformation 3.2.3 Verification of Assembled Clones 3.3 Assembly of Transcription Units into Pathways 3.3.1 Releasing URR1, URR2, and LEU2 3.3.2 Releasing all Transcription Units in POT Vectors 3.3.3 Ligation 3.3.4 Yeast Transformation 4 Notes References Chapter 14: A Versatile Protocol to Generate Translocations in Yeast Genomes Using CRISPR/Cas9 1 Introduction 2 Materials 2.1 Bacterial Media 2.2 Yeast Media 2.3 Bacterial and Yeast Growth Culture 2.4 Microbiological Manipulation 2.5 Agarose Gel Electrophoresis 2.6 PCR Amplification and Purification 2.7 Cloning the gRNA Sequences in pAEF5 2.8 Bacterial Transformation 2.9 Yeast Transformations 2.10 PFGE 2.11 Strain Storage 3 Methods 3.1 Design of the gRNA Sequence 3.1.1 Targeted Translocations 3.1.2 Multiple Translocations 3.2 Construction of the gRNA Sequences 3.2.1 Targeted Translocations 3.2.2 Multiple Translocations 3.3 Cloning gRNA Sequences 3.4 Validation of the Clones 3.5 Design of the Donor DNA Fragments 3.5.1 Targeted Translocations 3.5.2 Multiple Translocations 3.6 Yeast Transformation 3.6.1 Targeted Translocation 3.6.2 Multiple Translocations 3.6.3 Lithium Acetate Yeast Transformation 3.7 Validation of the Transformants 3.8 Loss of the Plasmid 3.9 Storage of the Clones 4 Notes References Chapter 15: Yeast Nucleoplasmic Extracts and an Application to Visualize Chromatin Assembly on Single Molecules of DNA 1 Introduction 2 Materials 2.1 Preparation of the Yeast Nucleoplasmic Extracts (YNPE) 2.2 Preparation of the Flow Cell 2.3 λDNA Biotinylation 2.4 Single-Molecule Visualization 2.5 Chromatin Assembly on Single Molecules of DNA 3 Methods 3.1 Preparation of the Yeast Nucleoplasmic Extracts (YNPE) 3.2 Preparation of the Flow Cell 3.3 λDNA Biotinylation 3.4 Single-Molecule Visualization 3.5 Chromatin Assembly on the Single DNA Molecule and Data Analysis 4 Notes References Chapter 16: Assays for Autophagy III: Observing Dynamic Protein Trafficking 1 Introduction 1.1 Non-radioactive Pulse-Chase Assay of Ape1 Maturation 1.2 Dynamics of Atg8 During Autophagosome Formation 1.3 Bidirectional Trafficking of Atg9 Vesicles 2 Materials 2.1 Plasmids 2.2 Media and Additives 2.3 Protein Extraction and Western Blotting 3 Methods 3.1 Non-radioactive Pulse-Chase Assay of Ape1 Maturation 3.1.1 Strain Construction 3.1.2 Pulse-Chase Labeling of Ape1 3.1.3 Sample Preparation for Western Blotting 3.2 Dynamics of Atg8 During Autophagosome Formation 3.2.1 Strain Construction 3.2.2 Time-Lapse Imaging of GFP-Atg8 3.3 Bidirectional Trafficking of Atg9 Vesicles 3.3.1 Strain Construction 3.3.2 Accumulation and Dispersal of Atg9 Vesicles in atg1ts Cells 4 Notes References Chapter 17: Isolation of Aged Yeast Cells Using Biotin-Streptavidin Affinity Purification 1 Introduction 2 Materials 2.1 Media and Reagents 2.2 Lab Materials and Instruments 3 Methods 3.1 Culture and Biotinylate Yeast Cells 3.2 Isolation of Biotinylated Mother Cells 3.3 Determining Replicative Age 4 Notes References Chapter 18: High-Throughput Rapid Yeast Chronological Lifespan Assay 1 Introduction 2 Materials 3 Methods 3.1 Chronological Lifespan Assay 3.2 Data Analysis 4 Notes References Chapter 19: Long-Term Imaging and Dynamic Analysis of Cytoophidia in Yeast 1 Introduction 2 Materials 2.1 Yeast Culture 2.2 Agarose Gel Preparation 2.3 Microscopy 3 Methods 3.1 Yeast Culture 3.2 Sample Preparation 3.3 Fluorescence Imaging 3.4 Data Analysis 3.4.1 Quantification of Cytoophidium Abundance 3.4.2 Quantification of Cytoophidium Length 3.4.3 Particle Tracking of Cytoophidium 3.4.4 Dynamic Analysis 4 Notes References Chapter 20: Monitoring 5′-End Resection at Site-Specific Double-Strand Breaks by Southern Blot Analysis 1 Introduction 2 Materials 2.1 Medium 2.2 Genomic DNA Extraction and Enzyme Digestion 2.3 Agarose Gel 2.4 Southern Blotting 2.5 DNA Hybridization 2.6 Scanning Phosphor Screen 3 Methods 3.1 Sample Preparation 3.1.1 Genomic DNA Extraction 3.1.2 RNase Digestion 3.1.3 Restriction Digestion 3.2 Southern Blotting 3.3 Probe Labeling and Hybridization 4 Notes References Index