Bacterial Transformation

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Bacterial Transformation

• RET Summer 2009

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Overall Picture

• Bio-Rad pGLO Transformation

• Need
• Good host
• Self-replicating vector to carry our gene
• Selection marker
• Insertion of GFP gene into HB101 E. coli

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Transformation

• The process of transferring foreign DNA fragments
  into a recipient (host) cell for growth and
  replication
• Our host cells HB101 E. coli
• Our foreign DNA GFP b-lactamase genes
  (contained in the pGLO plasmid)

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Plasmids

• Plasmids
• small (1-200,000 kb)
• circular
• extrachromosomal
• DNA
• Relaxed plasmids replicate independently of the
  hosts cell cycle amplification of gene product
• A type of cloning vector used to carry a gene not
  found in the bacterial hosts chromosome

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Overall Transformation Process

• The plasmid vector must be cut with a restriction
  endonuclease (aka restriction enzyme)
• DNA ligase joins the DNA fragment vector DNA
• Host cell is made competent so can plasmid can
  enter
• Transformed cells are grown on selection media

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Overall Transformation Process

• The plasmid vector must be cut with a restriction
  endonuclease (aka restriction enzyme)
• DNA ligase joins the DNA fragment vector DNA
• Host cell is made competent so can plasmid can
  enter
• Transformed cells are grown on selection media

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Restriction Enzymes

• Endonucleases
• protect bacteria from intruding DNA in nature
• cut up (restrict) the viral DNA
• can leave blunt or sticky ends
• cut only at very specific nucleotide sequences
• Restriction site
• recognition sequence for a particular restriction
  enzyme
• Restriction fragments
• segments of DNA cut by
• restriction enzymes in a reproducible way

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Restriction Enzymes

• Insertion of a gene
• Cut gene of interest with same restriction
  enzymes to make complementary sticky ends
• DNA ligase
• joins the sticky ends of DNA fragments
• Ideally, you would cut opposite ends of the gene
  with different restriction enzymes.

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Overall Transformation Process

• The plasmid vector must be cut with a restriction
  endonuclease (aka restriction enzyme)
• DNA ligase joins the DNA fragment vector DNA
• Host cell is made competent so can plasmid can
  enter
• Transformed cells are grown on selection media

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Transformation of Bacteria

• Generally occurs through heat shock addition of
  a divalent cation (Ca)
• Ca stabilizes negatively charged phosphates
• Heat shock briefly permeabilizes the membrane
• Competent cells are those capable of taking up
  the plasmid
• Cells most likely to become competent are in log
  growth phase

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Overall Transformation Process

• The plasmid vector must be cut with a restriction
  endonuclease (aka restriction enzyme)
• DNA ligase joins the DNA fragment vector DNA
• Host cell is made competent so can plasmid can
  enter
• Transformed cells are grown on selection media

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Selection

• Selective medium - used to determine which
  bacterial cells contain the antibiotic resistant
  plasmid insert which do not
• Example bacterium containing a plasmid with
  resistance to a particular antibiotic
  (ampicillin) will grow on medium that contains
  that antibiotic
• In addition, our plasmid contains a regulatory
  element that activates the GFP gene only in the
  presence of arabinose

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Selection Media

• LB plates
• LB amp
• LB amp ara

• Control (-pGLO)

Should contain only cells with the amp-resistant
pGLO plasmid colonies appear white (-pGLO,
pGLO)
Should contain only cells with the amp-resistant
pGLO plasmid colonies floresce green (pGLO)
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Factors that Affect Yield Quality of Plasmid DNA

• Plasmid copy number
• Host strain used, carbohydrate production
• Culture medium, selection, and culture time
• Want to harvest during log growth phase

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Transformation Applications
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GFP Uses

• Can be directed to specific subcellular
  compartments
• Can combine GFP coding region with the regulatory
  region for another gene and observe changes in
  gene expression
• Can be used to make a fusion protein to study
  localization, turnover intracellular
  associations of native protein
• GFP gene is switched on when cells are grown in
  the presence of arabinose

• Use as a reporter molecule to follow changes in
  gene expression over time
• Nondestructive, nontoxic
• Coding sequence can be cloned into a variety of
  vectors
• GFP keeps its fluorescence in cells from
  different species
• Can be tracked in living cells over to time to
  study development