Inhalt
Contents
Manual
* Introduction
* UNIT 1 - HISTORICAL PERSPECTIVE
Development of Bacterial Genetics
The Genetic Material Is DNA: Transformation--The Transforming Principle, The Hershey-Chase Experiment; Conjugation-Recombination; Bacteriophage Studies; Deciphering the Genetic Code; Control Circuits; Manipulation of Genes; Transposable Genetic Elements; Mutagenesis; Mapping the Chromosome; Bacterial Genetics Today
* UNIT 2 - WORKING WITH BACTERIAL STRAINS
Introduction
Basic Bacterial Genetics Techniques: Isolation of Single Colonies, Growth of Bacteria, Replica Plating; Maintaining Bacterial Strains: Storing Strains, Sending Strains, Safety Considerations in Sending Strains, Maintaining Records of Strains, Resuscitating Strains
Experiment 1: Basic Bacterial Genetics Manipulations
Obtaining Single Colonies; Replica Plating
Procedures for Maintaining Bacterial Strains
Preparation of Stabs; Preparation of Frozen Glycerol and DMSO Cultures; Lyophilization Method I: Double-vial Method, Single-vial Manifold Method; Lyophilization Method II; Lyophilization Method III; Storage and Recovery of Lyophilized Strains; Sending Strains on Filter Paper
* UNIT 3 - THE lac SYSTEM
Introduction
The lac Operon Revisited: Negative Control in the lac System, Positive Control in the lac System, Physical Structure of the lac Operon; Applications of the lac System
Some Tools of the lac Geneticist
Selective Media; Indicator Media: Fermentation Indicators, Histochemical Indicators (for In Vivo Use); Inducers and Anti-inducers; Assays
Additional Systems Used to Monitor Gene Expression
Proteins Encoded by Reporter Genes
Experiment 2: Streaking on Indicator Plates
Procedures for Working with lac
Preparation of Indicator Plates; b-Galactosidase Assay: Assay, Opening Cells, Reaction, Specific Activity; Use of MUG as an Indicator: Application of MUG, Viewing or Recording Fluorescence; Quantitative Fluorometric Assay for b-Galactosidase Specific Activity; Xgal Staining of Cultured Cells; Xgal Histochemistry for Tissue: General Procedure for Any Tissue, Whole Mount Procedure
* UNIT 4 - MUTAGENESIS
Introduction
Nature of Mutations; Pathways of Mutagenesis: Spontaneous Point Mutations, Spontaneous Rearrangements, Programmed Gene Rearrangements, Induced Mutagenesis; Repair Mechanisms; Methods of Detecting Mutagenesis: Tester Systems, Reverse and Forward Selections, Shuttle Vectors; Specificity of Mutagens; Mutators; Applications of Mutagenesis
Experiments 3-6: Use of Mutagens
3. Mutagenesis with EMS; 4. Mutagenesis with MNNG; 5.Mutagenesis with UV; 6. Screening for Nonsense Mutations
Experiments 7-10: Reversion Tests
7. Mutagenesis with 2AP; 8. Mutagenesis with ICR 191; 9. Reversion Tests and Identification of Nonsense Suppressors; 10. Spot Tests for Reversions
Experiments 11-13: Mutator Strains
11. Characterization of Mutators; 12. Detection of Mutators; 13. Characterization of New Mutators
* UNIT 5 - GENE TRANSFER
Introduction
Methods of Gene Transfer: Transformation, Conjugation, Transduction
Experiment 14: Useful Genetic Markers
Experiments 15 -18: Hfr Mapping
15. Hfr Matings by Replica Plating; 16. Gradient of Transmission I; 17. Introducing Tetr and Kanr with Hfr Strains; 18. Gradient of Transmission II
Experiments 19 - 20: P1 Transduction
19. Preparation and Use of P1vir Lysate: Preparation of P1vir Lysate, Titering the P1vir Lysate, Transduction with P1vir Lysate; 20. Use of P1clr100: Construction of Lysogen, Preparation of Heat-induced Lysate
Experiment 21: Transformation of Bacteria with Plasmid DNA
Preparation of Plasmid DNA (Mini-prep); Preparation and Storage of Competent Cells; Simple Transformation
Experiments 22 - 23: Mutagenesis of Plasmids
22. In Vivo Mutagenesis of Plasmids with MNNG; 23. Treatment of Plasmid DNA with NH2OH: Large-scale Plasmid Preparation and CsCl Purification of DNA, NH2OH Mutagenesis
* UNIT 6 - EXPERIMENTS WITH TRANSPOSABLE ELEMENTS
Introduction
Transposable Elements: Insertion Sequences, Trans-posons, Phage Mu; Genetic Rearrangements Stimulated by Transposable Elements; Uses of Transposable Elements
Experiment 24: Detection of Transposition by Conjugational Transfer
Experiments 25-26: Selection of Deletions and Mapping lacZ Mutations
25. Selection of Deletions Extending into lacZ; 26. Mapping lacZ Mutations against Deletions in lacZ
Experiment 27: Transposition of Tn10 from Phage lambda Vehicles
Preparation and Titering of l1098 and l1105 Lysates; Transposition with l1098 and l1105
Experiment 28: Use of P1 Transduction from Pools of Colonies to Obtain Transpositions Near a Gene of Interest
Experiment 29: Detection of Mutators after Insertion Mutagenesis
Determining Specificity of Mutators Detected by Insertion Mutagenesis
Experiment 30: Employing Transposable Elements for Localized Mutagenesis
Experiment 31: Detecting Tn10-promoted Adjacent Deletions
Experiments 32 - 34: Use of Mu Derivatives for Genetic Engineering In Vivo and Use of Transposons for Gene Fusions
32. In Vivo Cloning with Mini-Mu Plasmid Replicons: Preparation of a Heat-induced Lysate of Mu, Transduction of Mu/Mini-Mu Lysate; 33. Generalized Transduction and In Vivo Cloning with Mu Derivatives: Preparation of a Mucts Lysogen, Transduction with Mu; 34: Generating lac Fusions with MudX
Use of Transposable Elements for DNA Sequencing
Gamma Delta, IS1-promoted Deletions, Using the MudI Family of Phages, Transposon Tn5seq1 as a Mobile Source of Primer Sites, Tn5supF-based Reversed Genetics, Use of Recombinant M13mp Bacteriophages
* APPENDICES
CSH Strains
Formulas and Recipes
* Index
Handbook
Section 1. Escherichia coli Genetic Map
Section 2. Physical Maps of Escherichia coli and Their Correlation with the Genetic Map
A. Alignment of E. coli DNA Sequences to a Revised, Integrated Genomic Restriction Map; B. Location of Cosmid Clones on the Genetic Map of E. coli; C. Restriction Maps of E. coli K12; D. E. coli Proteins Identified on Two-dimensional Gels and a Plasmid Map of E. coli; E. Alignment of Genetic Maps of E. coli and S. typhimurium; F. Sizes of Bacterial Chromosomes
Section 3. Salmonella typhimurium Genetic Map
Section 4. Bacillus subtilis Genetic Map
Section 5. F Factor and F Derivatives
A. Genetic and Physical Map of the F Plasmid and Table of F Plasmid Genes; B. Hfr Points of Origins in E. coli K12 and Useful Hfr Strains; C. Hfr Strains of S. typhimurium and S. abony; D. Transduction Mapping Functions; E. Genetic Map Locations of Some Commonly Used F'Factors
Section 6. Bacterial Strains and How to Obtain Them
A. General Sources: 1. E. coli Genetic Stock Center, 2. American Type Culture Collection, 3. National Institute of Genetics (Japan) 4. Salmonella Genetic Stock Center, 5. Bacillus Genetic Stock Center; B. Strain Sets: 1. CSH Collection, 2. Singer/Gross Collection of Antibiotic-resistant Strains, 3. Singer/Gross Hfr Collection, 4. Wanner Collection of Hfr strains, 5. E. coli Genetic Stock Center Hfr Collection, 6. E. coli Genetic Stock Center F'Kit; C. Additional Useful Strains for Recombinant DNA Experiments
Section 7. Clone Banks and Libraries
1. Kohara et al. l Library of Entire E. coli Chromosome, 2. Blattner Library of Entire E. coli Chromosome, 3. Clarke-Carbon Bank of Hybrid Plasmids
Section 8. Databases
A. DNA Sequence Banks: 1. GenBank, 2. EMBL Data Library, 3. General; B. Gene-Protein Database of E. coli; C. Bibliographic Database
Section 9. Phage
A. Phage l Maps; B. Induction; C. Replication; D. E. coli Hosts; E. General Properties; F. Specialized Transducing Phage; G. Vectors
Section 10. Some Widely Used Plasmids
A. pBR322; B. pACYC177; C. pACYC184; D. pUC19/18; E. Replicons Carried by Currently Used Plasmid Vectors
Section 11. M13 Vectors
A. M13mp18 and the M13mp Series of Vectors; B. Bluescript M13+, M13-
Section 12. Useful Expression Vectors
A. Factors Affecting Overall Product Yield of a Plasmid-directed Expression System in E. coli; B. Bacterial Strains with Mutations in Major Protease Systems; C. Gene Fusion Systems Used to Facilitate Protein Purification; D. Chemical and Enzymatic Agents That Have Been Used to Cleave Fusion Proteins Site-specifically; E. Some Representative Expression Vectors: 1. pKK177-3, 2. pET-3a, 3. pMON 5743, 4. pKC30; F. Some Improved Fusion Vectors
Section 13. Transposable Elements
A. Useful Transposable Elements and Structures of Representative Transposable Elements; B. Useful Tn10 Derivatives; C. Selected Restriction Sites in Tn10; D. Mini-Tn5 Transposon Derivatives; E. Copy Numbers of IS Elements in the Chromosomes of E. coli and Other Bacteria; F. Locations of Some IS Elements on the E. coli K12 Genetic Map
Section 14. Phage Mu
A. Genetic and Physical Maps of Phage Mu; B. Physical Maps of Useful Mu and Mini-Mu Derivatives: 1. Physical Maps of the Most Widely Used Plaque-forming Mu Derivatives That Carry Ampr and Kanr, 2. Physical Maps of the Mu Derivatives MudI (Ap,lac) and MudII (Ap,lac)That Allow the Generation of Gene (Operon) Fusions and Protein Fusions, 3. Physical Maps of the Most Widely Used Mini-Mu Phages, 4. Physical Maps of the Three Most Widely Used RP4::Mini-Mu; C. Phage Mu as a Genetic Tool: 1. Mini-Mu and Mini-Mu Replicons and Mini-Mu and Mu Fusion Elements, 2. placMu1; 3. Broad-host-range Plasmids with Mini-Mu or Mini-D108 Insertion
Section 15. Other Phages
A. P1: 1. Host Range of P1, 2. Genetic and Physical Maps of P1, 3. Genes of P1; B. P22: 1. Genetic Map of P22, 2. Genes and Proteins of P22; C. P2, 186, and P4: 1. Genetic Maps of P2, 186, and P4, 2. Genes and Gene Products of P2, 186, and P4; D. T4: 1. Genetic Map of T4, 2. Genes of T4; E. T7: 1. Genetic and Physical Map of T7, 2. Synthesis and Processing of T7 mRNAs; F. RNA Coli-phages: 1. Genetic Map of Group I-IV RNA Coliphages, 2. Properties of ssRNA Phages; G. fX174: 1. Genes of fX174, 2. Genetic Map of fX174
Section 16. Sequences in the lac System
A. Sequence of the lac Operon: 1. DNA Sequence from lacI to the Beginning of lacA, 2. Nucleotide Sequence of the lacA Gene; B. Restriction Enzyme Cleavage Sites in the lac Operon
Section 17. Regulation
A. Promoters: 1. Alignment of E. coli Promoter Sequences, 2. E. coli s70 Promoters, 3. Compilation of DNA Sequences for Promoters Utilizing Alternative s Factors; B. SOS System: 1. DNA Sequences of lexA-repressed Promoters, 2. SOS Genes, Map Positions, and Activities, 3. Global Regulatory Responses; C. Compilation of E. coli Ribosome Binding Sites; D. Transcription Termination
Section 18. Restriction Enzyme Specificities
Section 19. The Genetic Code and Codon Usage in Selected Organisms
Section 20. Natural and Synthetic Nonsense Suppressors
Section 21. Mutagen and Mutator Specificities
Section 22. Properties of Amino Acids
Section 23. Atomic Weights
Section 24. Additional Procedures
A. Transformation: 1. TFB-based Chemical Transformation Protocol, 2. FSB-based Frozen Storage of Competent Cells, 3. PEG/DMSO One-step Transformation Procedure, 4. Rapid Colony Transformation, 5. Electroshock Transformation of E. coli, 6. General Protocol for Electroshock Transformation of Gram-negative Microorganisms; B. Mutagenesis: 1. Mutagenesis with Aflatoxin B1, 2. Mutagenesis with 1,2-Dibromoethane
Section 25. Formulas and Recipes
A. Minimal Salts; B. Minimal Agar Plates; C. Rich Media; D. Indicator Plates; E. Antibiotics; F. Buffers; G. Soft Agar (Top Agar); H. Media for Storage of Strains
Section 26. Photography
Section 27. Commercial Suppliers
Index
Autoreninfo
Jeffrey H. Miller, University of California, Los Angeles
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