Bacterial Transformation

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Bacterial Transformation
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Central Dogma of Biology

• DNA ? RNA ? Protein
• The flow of information in a cell.

The instructions
The messenger-sent to the ribosome
The product Protein, made by ribosome using the
instructions
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• A section of DNA that has enough information to
  make one protein is called a gene
• The gene is copied into RNA instructions to be
  sent to the ribosome.

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• The ribosome takes the instructions and uses it
  to make the correct sequence of amino acids for
  the protein

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What is transformation?

• Changing the proteins produced by bacteria by
  giving it new instructions or DNA!
• Lets review bacterial DNA first
• Do you remember anything about what it looks like
  or where it is?

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Bacterial genome

• Bacteria are prokaryotesno nucleus.
• The area where DNA is located is called the
  nucleoid
• DNA is organized in one double stranded circular
  molecule
• Kind of cool when replication happens

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Plasmids
Plasmid

• Bacteria can also have a plasmid
• Plasmid a small, circular, self-replicating DNA
  molecule separate from chromosome
• Bacteria can pass their plasmid on

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What is carried on the Plasmid?

• The plasmid contains genes necessary for survival
  and can be passed from one bacteria to another
• Antibiotic Resistance Some bacteria have genes
  coding for enzymes that destroy certain
  antibiotics!

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The transformation lab

• Our plasmidpBlu plasmid
• Into E. coli (scary?no!)
• Our plasmid contains genes for
• AMP ampicillin (an antibiotic) resistance
• Beta-galactosidase-an enzyme that converts X-Gal
  ? Indo Blu

RNA
Protein that allows for antibiotic resistance
Enzyme that breaks down X-Gal to make Indo Blu
RNA
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How do we get the plasmid inside of the bacteria?
2. pBlu plasmid

• Obtain E. Coli bacteria cells Add to CaCl2
  (helps plasmid attach to bacteria)
• Add plasmid to same microtube

1. E. Coli
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How do we get the plasmid inside the bacteria?

• Waitand then
• 3. Heat shock! This temporarily opens pores to
  allow the plasmid to enter the bacteriatiming is
  critical!!!

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Growing the bacteria

• After they have received the plasmid
• Placed on a growth media and allowed to grow.

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How will we know if the bacteria actually got the
plasmid??

• Any ideas?
• We can grow the bacteria on a plate
• That contains ampicillin and X-Gal
• Regular bacterial medium
• What do you predict will happen in each?

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Predict
What will we observe???
Amp X-Gal

• pBlu
• pBlu
• Control
• Control

Regular
Amp X-Gal
Regular
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Applications

• What could transformation be used for out there
  in the real world?

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Applications

• Recombinant DNA Inserting new DNA into a
  plasmid.
• We can construct plasmids to contain whatever
  genes we want to mass produce
• Insulin
• BST
• Genetically modified foods
• Bioremediation

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How?

• Use enzymes to cut plasmid DNA
• Insert a DNA sequence for a desired protein
• Insert plasmid into bacteria and growgrows fast!
• Isolate protein made by bacteria

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