march%2019%20transformation%20end%20ConjugationPosting

march%2019%20transformation%20end%20ConjugationPosting - We...

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We did: natural competence and mechanism of transformation We will do: Artificial competence and transformation And will continue with: Conjugation as a horizontal gene tranfer mechanism
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Transformation mechanism: The recipient cell must be “competent” The recipient must be in a special physiological state. The state is called “competence”. Some bacteria are always competent. Others become naturally competent at certain stages of growth. For example at high cell density (quorum sensing??) or when they are starved. When they are in the state of competence they make special membrane proteins that can transport DNA across the cell wall and membrane.
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Usually cells will be living near cells that are their “siblings”. So it’s likely that the DNA taken up from the medium will be mostly homologous. However, DNA is a stable molecule and persists in the environment. So cells could take up DNA from some other species. Transformation mechanism
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Not all Bacteria can take up DNA using ‘natural competence” (for example Gram negative cells like E. coli ). But if they are treated with divalent cations (like Calcium), this changes the membranes so they can be transformed. This process probably makes some sort of channel in the membrane. It’s still pretty mysterious. Chemically induced competent cells don’t degrade one DNA strand as it crosses the cell wall/membrane. So double stranded DNA can be taken up. For example a plasmid. Artificial Transformation
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Calcium Mediated in vitro Transformation Cell is treated with divalent cation (Ca ++ ). This alters the outer membrane making it more permeable. Cell is heated briefly at 42 o C. This causes membrane transition. Plasmid enters cell. Cells are then allowed to recover. Plasmid DNA is added. Cells incubated on ice.
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Transformation experiment 1 Grow trpB - mutant cells until competent. Split the sample. Grow wild type cells. Isolate DNA. DNA solution Spread cells on plates that do not have tryptophan. To one, add DNA from wild type. To other, no DNA is added
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Explanation: Transformation experiment 1 Add DNA from wild type Spread cells on plates that do not have tryptophan No DNA added trpB - trpB - trpE trpD trpC trpB - trpA trpE trpD trpC trpB trpA trpE trpD trpC trpB trpA transformants Donor Recipient So: How did trp B cells grow on plates without tryptophan?
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Grow trpA - cells. Isolate DNA. Grow trpB - mutant cells until competent. Compare two samples Add trpA - DNA to trpB - cells DNA solution Spread cells on plates that do not have tryptophan. What do you expect? No DNA
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march%2019%20transformation%20end%20ConjugationPosting - We...

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