Genetic Transformation

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Genetic Transformation

• and DNA Isolation

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Two Procedures

• 1. Isolate DNA from onion using a Woolite NaCl
  solution.
• 2. Experiment involving genetic transformation.
  This will be the basis of your third lab report.

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History

• 1928Frederick Griffith, an English
  bacteriologist, studies a disease causing
  bacterium Streptococcus pneumoniae and a harmless
  variant

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Escherichia coli(E. coli)

• Grows rapidly- a doubling time of only 20 minutes
• Today we will be genetically transforming E. coli
  by giving it a new gene.

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Bacterial DNA

• In addition to a single circular chromosome,
  bacteria often have rings of DNA called Plasmids.
• Transformation allows bacteria to pick up free
  DNA from the environment which may come from the
  chromosome of another bacterium or a plasmid.

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Plasmids

• Small circular self-replicating DNA molecule that
  is separate from the bacterial chromosome.
• The bacterium can pass on its plasmids to its
  offspring.

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Transformation requires

• Competence- the ability for a cell to take up a
  plasmid.
• We will increase this by adding CaCl2 and by
  heat shocking.

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pUC 18

• pUC 18 is a plasmid which contains an
  ampicillin-resistance gene called ß lactamase
• ß lactamase allows E. coli to grow in the
  presence of ampicillin.

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pUC 18
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Genetic Transformation Procedure

• Genetically transform E. coli with pUC 18
• Will E. coli grow in the presence of ampicillin?

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Plus with AMP With No Amp
Plus with AMP With Amp
Plus with AMP - With No Amp
Plus with AMP - With Amp
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Sterile Procedure

• Wash hands before and after lab.
• Disinfect your lab bench before and after lab.
• Expose the petri dishes and media tubes to air as
  little as possible.
• Keep sterile objects off the lab bench.