This manual encompasses an integrated series of molecular biology laboratory exercises that involve the cloning and analysis of the bioluminescence(lux) genes from the marine bacteriumVibrio fischeri. The manual is divided into discrete units with each demonstrating one or more aspects of the cloning project. The manual is based on one of nature's most fascinating biological phenomenon: the biological production of light. This results in a recurrent theme of interest and makes the project very relevant to interdisciplinary topics such as fish symbiosis, biochemistry, biophysics, etc. Includes instruction in the basic techniques of modern molecular biology: DNA isolation and analysis, DNA restriction, agarose gel electrophoresis, ligations, transformation of recombinant DNA, preparation and screening a genomic library, restriction mapping, Southern blotting, hybridization, DNA sequencing, pulsed field gel electrophoresis. Designed for a one semester course in Molecular Biology. Also appropriate for a molecular biology component of Microbial Genetics, Genetics, Biochemistry, or Advanced Microbiology courses.
I. INTRODUCTORY TECHNIQUES.
1. Introduction to the Laboratory: Basic Equipment and Bacteriological Techniques.
2. Preparation of Media and Reagents Used in Molecular Biology.
3. Isolation of Luminescent Bacteria from Natural Sources.
4. Restriction Digestion and Agarose Gel Electrophoresis of DNA.
II. DNA ISOLATION AND ANALYSIS.
5. Isolation of Chromosomal DNA from Vibrio fischeri.
6. Large-Scale Purification of Plasmid DNA.
7. Spectrophotometric Analysis of DNA.
III. CLONING THE LUX OPERON
8. Restriction Digestion of Vibrio fischeri Genomic DNA and Plasmid Vector.
9. Quantification of Genomic DNA by Fluorometry and Agarose Plate Fluorescence.
10. Ligation of Restriction Fragments of Vibrio fischeri DNA to Plasmid Vector.
11. Preparation of Competent Escherichia coli DH5.
12. Transformation of Competent Escherichia coli DH5 with Recombinant Plasmids.
13. Screening the Vibrio fischeri Genomic Library for Light Producing Clones.
IV. RESTRICTION MAPPING AND SOUTHERN BLOTTING
14. Small-Scale Plasmid Isolations (Mini-preps) from Bioluminescent Clones.
15. Restriction Mapping of Plasmids from Bioluminescent Clones.
16. Southern Blotting and Hybridization to Detect the luxA Gene.
V. SUBCLONING THE LUX A GENE
17. Restriction Digestion of lux Plasmids and Cloning Vector for Subcloning luxA.
18. Gel Purification of DNA Restriction Fragments Containing luxA.
19. Subcloning luxA into A Plasmid Vector.
20. Transformation of Competent Escherichia coli DH5 with Subcloned DNA.
21. Colony Hybridization to Screen for luxA Subclones.
22. Small-Scale Plasmid Isolations (Mini-Preps) from luxA Clones.
VI. ADVANCED TECHNIQUES.
23. Amplification of luxA from Natural Isolates by the Polymerase Chain Reaction (PCR).
24. Southern Blotting and Hybridization of PCR Products.
25. DNA Sequencing of lux Genes from Plasmid Templates.
26. Computer Analysis of DNA Sequences Using the World Wide Web.
27. Mapping the Vibrio fischeri Genome by Pulsed Field Gel Electrophoresis.
28. Independent Projects in Molecular Biology.
1. The Metric System and Units of Measure.
4. Agarose and Polyacrylamide Gel Electrophoresis.
5. Methylene Blue Staining of Agarose Gels.
6. Nucleic Acid Hybridization.
7. Alcohol Precipitation of Nucleic Acids.
8. Care and Handling of Enzymes.
9. Restriction Endonucleases.
10. Enzymes Used in Molecular Biology.
11. Maps of Cloning Vectors and Bacteriophage Lambda.
12. Proper Handling and Disposal of Hazardous Materials.
13. Procedures and Precautions for the Use of Radioisotopes.
14. Equipment and Supplies.
15. Media and Reagents.
16. Bacterial Strains.
17. Lists of Suppliers.
18. Recommended References.
19. Restriction Mapping Problems.