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2290-Zab Unit.docx

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Western University
Biology 2290F/G

12/6/2012 10:08:00 AM 1. Transformation: some bacteria have a mechanism for actively taking up DNA from their environment; forms the basis for much of modern microbial and molecular genetics; plasmids are introduced into host cell and replicate and divide; E. coli needs to be artificially induced to become competence by subjecting them to incubation in cold calcium chloride and then heat shock; it is believed that the large DNA molecules move through adhesion zones where inner and outer membrane are fused to pores in the cell wall; chilling the cell stabilizes the lipid membranes and positively charged calcium ions chilled the negatively charged phosphates on the DNA backbone; heat shock may create a thermal imbalance physically pumping through DNA; once inside, plasmid mud my replicated in order or be stability inherited by daughter cells; pGreen has its own ori and can replicate independently of the host chromosome; if plasmid did not have own ori, it can replicate by recombination or transposition in host chromosome; a transformed cell carried a new trait (antibiotic resistance); the time from competence to expression is called phenotypic lag 2. Ampicillin: derived from penicillin which is a bactericide and interferes with cell wall biosynthesis; a transpeptidase enzyme necessary for cross linking new peptidoglycan chains in the growing cell wall is irreversibly inactivated by amp binding; only actively growing cells are affected; resistance is often conferred by genes that code for an enzyme that degrades the antibiotic; treats UTI, middle ear inflammation and meningitis Kanamycin: an amino glycoside antibiotic that kills bacterial cells by binding irreversibly to the 70 S subunit of ribosome’s causing misreading of mRNA; resistance is conferred by genes encoding for enzymes that modify the antibiotic molecule by adding phosphate or acetyl groups; the modified antibiotic can not longer bind to the ribosome; used to treat a wide variety of infections 3. Plasmids: typically extrachromosomal, circular, double stranded super coiled DNA molecules; some strains are known to contain up to a dozen different plasmids; widespread among natural bacterial populations; have functions such as conjugative genetic exchange, resistance to antibiotics and heave metals, metabolism of hydrocarbons, carbohydrates and proteins, and production of antibiotics, toxins, restriction enzymes and plant hormones; engineered plasmids are used in recombinant DNA research used y introduce cloned “passenger” DNA into a new host 4. See dilutions bellow 5. See examples Class Notes • TE buffer: weak solution used to dissolve DNA and is used in the control • Bacteria love LB • In 100 µL of pGreen, there would probably be 100 million bacteria • Confluent growth: so much growth you cant see individual colonies • There will be no growth on the LB+AMP plate + control; top film on plate is burst cells • AMP interferes with cell wall formation o Peptidoglycans: fibers that form cell wall; need to be joined one to another; AMP stops the cross-linking; cell wall gets weaker and weaker until cell bursts • Negative control: hoping to see no growth-control on LB+AMP o Testing to see if cells can become resistant on their own without treatment; if AMP can get in, maybe is can grow? • Positive control: control on LB plate; vitality control; hoping to see growth; want to make sure cells survived protocol • Positive and negative controls are called protocol controls • Controls are needed to compare experimental to normal situation • PGreen: GFP; under UV transformants glow; colony started as one cell picking up pGreen molecule, transcribing and translating to make AMP resistant protein • This happens in under 24 hours- went from 1 cell to 1 million • Transformant- implies transformation experiment has been completed and started from 1 cell • Not even 50 out of 100 million cells were transformed- it’s a rare event • Satellite colony: splattered white around transformant; come about because of transformant • The AMP resistant protein destroys the AMP on the plate-gets exported out of cell and still functions • Once cross-linking is complete in cells, it cannot be effected by AMP
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