Team:UCL/Project/Experiments

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<p class="minor_title">Creating Competent Bacteria</p>
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E. coli are not naturally transformable, which means they lack the ability to take up plasmids (competency). Competency is induced by divalent cations such as calcium. These alter the permeability of the membranes enveloping the bacterium to plasmids. Normally macromolecules on the outer surface of bacteria are negatively charged which means the negative charges of incoming DNA would be repelled. The addition of calcium chloride facilitates the movement of DNA into the cell.
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<p class="minor_title">Transformation</p>
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To insert foreign DNA into our competent cells we used the heat shock treatment. Our competent bacteria are stored in -80C. To transform, DNA with a selectable marker and competent bacteria are mixed together and kept on ice for thirty minutes to allow interactions between calcium ions and the negative charges on the bacterial envelope. The mixture is exposed to a brief period of 38C (heat shock). The rapid shift in temperature alters the fluidity of the membrane therefore allowing DNA to enter the cell.
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Afterwards, the bacteria containing the foreign DNA are streaked on selective plates. Bacteria containing the foreign DNA with the selectable marker, such as ampicillin resistance, would be the only bacteria growing on the selective plates.
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Revision as of 11:03, 27 September 2013

Creating Competent Bacteria

E. coli are not naturally transformable, which means they lack the ability to take up plasmids (competency). Competency is induced by divalent cations such as calcium. These alter the permeability of the membranes enveloping the bacterium to plasmids. Normally macromolecules on the outer surface of bacteria are negatively charged which means the negative charges of incoming DNA would be repelled. The addition of calcium chloride facilitates the movement of DNA into the cell.

Transformation

To insert foreign DNA into our competent cells we used the heat shock treatment. Our competent bacteria are stored in -80C. To transform, DNA with a selectable marker and competent bacteria are mixed together and kept on ice for thirty minutes to allow interactions between calcium ions and the negative charges on the bacterial envelope. The mixture is exposed to a brief period of 38C (heat shock). The rapid shift in temperature alters the fluidity of the membrane therefore allowing DNA to enter the cell. Afterwards, the bacteria containing the foreign DNA are streaked on selective plates. Bacteria containing the foreign DNA with the selectable marker, such as ampicillin resistance, would be the only bacteria growing on the selective plates.

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