E. coli double in this amount of time 20 to 60 minutes Why E. coli is used as a model organism Easy to grow on defined medium; Grows quickly (20-60 minute doubling time); Relatively small genome (4.7 * 10^6 bp); Grows as clone; Genetic transfer is convenient Size of E. coli's genome 4.7 * 10^6 bp Number of genes in E.coli's genome 4,000 Why Yeast is used as a model organism Simplest eukaryotic system; small genome (14 * 10^6 bp); Genetic system can be manipulated; Grows quickly (2 hr doubling time) Simplest eukaryotic model organism Yeast Simplest system for differentiation and development Dictyostelium discoidium (cellular slime mold) Why Dictyostelium discoidium is used as a model organism Simplest system for differentiation and development; Aggregates into multicellular organism when food is limited; Great for studying cell motility Why C. elegans is used as a model organism Multicellular; most complete system for differentiation and development (100 * 10^6 bp); 19,000 genes; Embryonic origin and lineage of every cell is known This organism has 959 somatic cells and 1000-2000 germ cells C. elegans Number of genes in C. elegans' genome 19,000 Why Drosophila melanogaster is used as a model organism Excellent genetic model, also good for development; Short life cycle for a multicellular eukaryote (2 weeks) Number of genes in Drosophila melanogaster's genome 14,000 Why Arabidopsis thaliana is used as a model organism Plant; Important for agricultural development; easy to grow in lab; genetic system is well-known Number of genes in Arabidopsis thaliana's genome 15,000 Why Xenopus laevis is used as a model organism Vertebrate; eggs are unusually large cells (1 mm) which develop outside of the mother Examples of vertebrate model organisms Xenopus laevis (frog), Zebrafish, Mouse Two critical parameters of microscopy Resolution, contrast Resolution formula D = 0.61 y / (N sin A) [D = min. resolving dist.; y = wavelength; N = refractive index; A = angular aperture] Advantages of fluorescence microscopy Supplies contrast with the fluorescence, but can do so on living cells Advantages of confocal microscopy Focuses on a specific depth; can be used to reconstruct 3D images Types of electron microscopy Transmission (most common), and Scanning How immunofluorescence works Raise an Antibody to a specific molecule; Label the Ab with a fluorescent tag; Detect with fluorescence microscopy Fluor A molecule that absorbs light at one wavelength and emits light of a different wavelength Limitations of staining Kills the cells; May alter the cellular structures; Cannot see the cells in normal living state Darkfield Microscopy Only detects diffracted light Phase Contrast Microscopy Converts differences in density or thickness to differences in contrast Cell Fractionation Used to isolate cellular structures Steps of cell fractionation Collect large amount of cells; Lyse cells (osmotically, or homogenize with blender/french press/chemical lysis); Fractionate cellular contents (centrifuge) Flow cytometry A technique for counting, examining and sorting microscopic particles suspended in a stream of fluid. FACS Fluorescence-activated Cell Sorting. Tag cell w/ fluor (antibody); Impart charge to marked cells; Separate by charge 5 stages of viral cycle Adsorption; Penetration; Replication; Assembly; Escape Recombinant DNA is also known as Genetic Engineering Features of a plasmid vector Origin of replication, multiple cloning site, a selectable marker (resis. to antibiotic) An example of a restriction endonuclease EcoRI How plasmids are introduced into E. coli cells Transformation: treated with CaCl2 to make membrane more permeable to DNA Competent cells Cells that have been treated with a transformation process -- ready to accept new DNA Shuttle vectors Vectors that can replicate in more than one species The DNA polymerase used in PCR Taq polymerase (from Thermus aquaticus) Used to separate DNA molecules based upon their size Gel Electrophoresis Hybridization Reannealing a DNA probe (complementary strand with isotope or fluor marker) Probe Complementary DNA fragment Southern Blot Digest genomic DNA into fragments with restriction endonuclease; Separate fragments on agarose gel; Transfer fragments to nitrocellulose paper (blot); Hybridize probe to detect gene SDS-PAGE Sodium dodecyl sulfate Polyacrylamide Gel Electrophoresis; used to separate proteins by size. Western Blot Detects proteins expressed. SDS-PAGE, Blot, Probe with Ab, Detect Microarrays Compares expression of thousands of genes at once (i.e. normal v. cancerous, uninfected v. infected). Creates arrays of microspots on glass slides. Gene Transfer to Animal Cells Transfection, Electroporation, Liposome Fusion, Microinjection, Viral Vectors Transfection Calcium Phosphate with target DNA used to ppt small particles which are taken into the cell Transgenics Organisms with modified/added genes