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Bacterial and Phage Genetics Chapter 15 meiosis Why study Bacteria and Viruses Many cause human diseases, or affect agriculture. Many are beneficial to human health and mankind in general. Because they can be manipulated so easily, they are fantastic research tools. Used in nearly all labs. They can be genetically engineered to produce drugs, plastics, or other useful products. Bacterial and Phage Genetics • Very short doubling time. Human: 25 years; Mice: 3-4 months; Drosophila: 12-20 days; Bacteria: 20 min-24 hrs; Phage: 100x-1000x per infection cycle • Number of organisms examined is several orders of magnitude larger than with plants or flies • Genetically pure cultures - asexual reproduction yields clones (binary fission). • Haploid, genotype = phenotype. • Small Genome Size Ecoli: 4 X 106 bp; Drosophila: 1.65 X 108 bp; Human: 2.9 X 109 bp Methods of growing bacteria in the laboratory Selectable genetic markers in bacteria • Nutritional markers Prototrophic Bacteria can grow on minimal medium, which only contains inorganic salts, a carbon source and water (wild type) . Auxotrophic Bacteria will not grow on minimal medium, but can grow on minimal medium plus one or more specific nutrients or supplements, or on rich media (mutant). Bacterial nomenclature Genes are indicated using three letters (not capitalized, and italic), followed by a + for wild type or a - for mutant. phe+ Wild type phe gene. phe- Mutant phe gene. Phenotypes are indicated the same way, but not italic and first letter capitalized. Phe+ Does not require phenylalanine added as a supplement to minimal medium Phe- Requires phenylalanine added as a supplement to minimal medium • Other nutritional markers Anything the bacteria needs to synthesize for its growth, such as Adenine, Biotin, Thiamine,..... AdeAde+ Requires adenine added as a supplement to minimal medium Does not require adenine added as a supplement to minimal medium • Carbon Source Lactose For energy, such as: galactose, glucose, lactose, maltose, ....... lacZ enzyme Lac- Lac+ Cannot grow on lactose as its sole carbon source. Can can grow on lactose as its sole carbon source. • Antibiotic Selections Bacteria will not grow colonies on the medium containing certain antibiotics Sensitive Str s Resistant Str - streptomycin is an antibiotic that interferes with the correct initiation of protein synthesis by binding the 30S subunit of the ribosomal RNA. Bacteria will grow colonies on the medium containing certain antibiotics r - A mutation in a protein subunit of the 30S unit confers resistance by preventing binding by the antibiotic. The antibiotic is unable to exert its action upon the substrate, and the bacteria continues to grow. Selection vs screen. Selection: Establish conditions in which only the desired mutant will grow. Example: Select for StrR mutants on streptomycin. Genetic screen: an examination of each colony in a population for its phenotype. Example: screening for auxotrophs. Ways to enrich for auxotrophs in a genetic screen. 1. Add a mutagen to increase mutation rate. UV light Proflavin EMS etc………….. 2. Replica plating to quickly look at lots of colonies. 3. Penicillin enrichment scheme. Replica Plating A method to quickly transfer colonies from one plate to another. The original plate is called the Master Plate. It is pressed on a piece of velvet, transferring about 50% of the cells from each colony. A second plate, replica 1, is then pressed on the velvet, to leave an imprint. Identification of Arg- auxotroph using replica plating. Penicillin enrichment scheme. • Penicillin only kills bacteria cells that are actively dividing, since it affects cell wall synthesis. • Auxotrophs get selected since they stop dividing. Three ways to get exchange of genetic information in bacteria • Transformation A process in which recipient cells acquire genes from free DNA molecules in the surrounding medium. • Conjugation A process in which DNA is transferred from a bacteria donor cell to a recipient cell by cell-to-cell contact. • Transduction A process in which a bacterial DNA fragment is transferred from one bacterial cell to another by a phage particlecontaining the bacterial DNA. Do bacteria undergo sexual mating and recombine their genes? Joshua Lederberg and Edward Tatum, 1946 Strain A met-bio-thr+leu+thi+ Strains A+B Strain B met+bio+thr-leu-thi- A wash cells AB wash cells Plate 108 cells Plate 108 cells min. media min. media No colonies B wash cells Plate 108 cells min. media met+bio+thr+leu+thi+ prototrophic colonies No colonies U- tube experiment (Bernard Davis) Do bacteria have to touch each other to exchange? Now there was no growth on minimal media. Bacteria have to touch each other to have genetic exchange. Genes are transferred in one direction William Hayes, 1953 Strain A (met-thr+leu+thi+) streptomycin (antibiotic) kills cells slowly, but will die. wash out strep. MIX Strain B (met+thr-leu-thi-) Plate on minimal media (colonies grew) Strain B (met+thrleu-thi-) streptomycin (kills slowly) wash out strep. Strain A (met-thr+leu+thi+) MIX Plate on minimal media (no colonies) Conclusions • Genetic transfer is not reciprocal • In this experiment, transfer occurs from Strain A to Strain B only, not B to A. They came up with some terminology Donor cells (male): • Hayes experiment: A strain • Cells that can donate DNA to other cells • F+ Recipient cells (female): • Hayes Experiment: B strain • Cells that can receive DNA from other cells • F- Years later it was found that there are two types of bacteria: F+ carry the F factor in the cytoplasm contain Pili on the surface can transfer the F factor to F- Bacteria Cannot receive DNA from other strains. F- do not carry the F factor in the cytoplasm do not contain pili on the surface cannot transfer DNA by conjugation can receive DNA transferred from F+ bacteria Overview of Conjugation F factor is a small plasmid. Explains unidirectionality. F+ x FF+ F+ F- F+