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Zabulionis Unit Quiz 1 Review

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Biology 2290F/G
Ray Zabulionis

Zabulionis Unit Quiz #1 Review -Putting the JM101 strain of E. coli species of bacteria in CaCl 2olution. TE buffer is a weak solution for dissolving DNA and acted as control for pGreen (dissolved in TE buffer), which was the experimental. Lysogenic Broth (LB) is food for bacteria plated. Spread 100μL on plate (10 bacterial cells); at least 100 million formed a mat of growth on plate overnight (confluent growth is the bacterial growth that covers the whole area of the plate). -A technical tip, when spreading keep the lid on top of the plate and move the spreader from edge-to- edge of the plate. -The pink plate had LB and ampicillin resulting in no colonies because JM101 strain is highly sensitive to all antibiotics. Ampicillin interferes with the cell wall fibres (peptidoglycans) by preventing their cross- linkage of the bacterial cell wall (the cell bursts from osmotic pressure). -Film on the plates could represent the guts (debris) of bacteria and the LB and ampicillin plate is referred to as a negative control (no growth). Kanamycin disrupts protein translation (binding to ribosomes) and resistance to Kanamycin is different than resistance to Ampicillin. -The favourable temperature of E. coli is 37°C, 45°C being a heat shock temperature. By putting the cells on ice (and whole protocol) without pGreen we test if cells can pick up new genetic material on their own. The TE buffer is the positive (vitality) control because we want to know if cells can make it through the protocol i.e. survive. -In the experimental control, after 100 seconds of heat pulse, unirradiated cells and compare the different times of heat pulse (0 seconds, 40 seconds, 100 seconds). In the pGreen/LB/Amp there were approximately 20 million cells in each (transformant) colony which arose due to a transformation event. Likewise do conjugation experiments yield conjugants and transduction experiments yield transductants. -It is a rare event, only few of millions of cells survive. Satellite colonies encircle transformants. Amp resistance protein cleaves the ring structure of Ampicillin molecules and destroys their antibiotic function. The amipicillin resistance enzyme is exported out of the cell and destroys Ampicillin in the media (Kanamycin resitance protein cannot do this). In transformation-yielding Kanamycin resistance plasmid (pKan), its resistance protein needs to be produced before plating. -Ampicillin only prevents cross-linking and it cannot reverse cross-linkage when it is already present on the cell wall. Ampicillin thus, only kills actively-growing bacteria (dormant cells are unaffected by Ampicillin). Most of th
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