Transformation of Escherichia coli Using an Inducible Expression Vector Containing the Bioluminescent Vibrio fischeri Lux Operon

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Transformation of Escherichia coli Using an
Inducible Expression Vector Containing the
Bioluminescent Vibrio fischeri Lux Operon

• by
• Bryan Hart Crystal Harmon

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Bioluminescence

• biologically mediated synthesis of compounds that
  react to emit visible light energy
• found in diverse range of species
• fungi, insects, algae, free living bacteria,
  mollusks, crustaceans, and other animals in
  symbiosis with bioluminescent bacteria

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Evolutionarily speaking

• based upon reproductive communication and
  competition
• attract mates or advertise high fitness levels
  (remember energy allocation from EvoEco?)
• illumination for predation or protection
• ex. fireflies, cuttlefish, dragonfish
• or just to look cool

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Dragonfish
Comb Jelly
Panellus stypiticus
Firefly
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Vibrio fischeri

• common bioluminescent bacteria in photophores
  (light organs) of marine organisms
• Gram negative, f. Vibrionaceae
• pathogenic and symbiotic interactions with animal
  tissue
• virulent pathogens of crustaceans, also
  free living saprophytic cells in seawater
• symbiosis established by inoculation of juvenile
  animal hosts

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• V. fischeri streak plate

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the Lux operon

• gene group responsible for bioluminescence,
  synthesizes luciferase, key catalyst
• consists of 8 main genes
• three parts regulatory genes, fatty acid
  reductase polypeptides, and luciferase subunits

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(No Transcript)
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luxR luxI luxCDABEG
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Luciferase Cycle
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Protocol in a nutshell

• extract Vibrio fischeri DNA w/ DNeasy Tissue Kit
• create genomic library w shotgun cloning
• Sal I restriction digest of the chromosome
• ligate restriction fragments into inducible
  Promega pGEM -3Zf() vector
• transform BL21 (DE3) E. coli
  w/ cloned vectors
• select correctly transformed colonies by
  blue-white screening (and possibly
  bioluminescence)
• manipulate lux expression in successfully
  transformed cells

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Why Sal I?

• cleaves a six base pair palindromal sequence
  (GTCGAT) w/ sticky ends
• restriction fragment length of 4000 bp from
  average genome, but this may vary due to GC
  content
• but most importantly the lux operon exists on a
  Sal I restriction fragment of around 9kb

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Why pGEM -3Zf() ?

• T7 Sal I lacZ Amp

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Why BL21 (DE3) E. coli ?

• laboratory strain with the gene encoding T7 RNA
  polymerase conveniently under lac operon control
• induce/repress with carbs or analogs
• expression of lux operon through direction of lac
  operon- E. coli media
• compatible Shine-Dalgarno sequences

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Timeline

• Week of Sept 13th
• 15 pts
• Week of Sept 20th
• 15pts
• Week of Sept 27th
• 10pts
• Week of Oct 3rd
• 5pts
• Week of Oct 10th
• 5pts
• Until Nov 22nd-

• receive vector plasmid and DNeasy , begin DNA
  extraction
• chromosomal and vector digestion, gel
  verification
• ligation and gel verification
• prepare competent cells, transformation, and
  selection
• manipulation of operon
• possibly redoing steps

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Budget

• Promega pGEM -3Zf() vector
  66.00
• DNeasy Tissue Kit (50)
  110.00
• T4 DNA ligase 33.00
• Sal I 55.00
• Total 264.00

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References

• Altman, John. Autoinduction of Expression in the
  T7 Expression System. Altman Laboratory at Emory
  Vaccine Center. 3 Sept. 2004. http//www.microbiol
  ogy.emory.edu/altman/f_protocols/f_tetramers/autoi
  nd_annot.html
• Bluth, Brian J., Sarah E. Frew, and Brian
  McNally. Cell-Cell Communication and the lux
  operon in Vibrio fischeri. Carnegie Mellon
  University. 3 Sept. 2004. http//www.bio.cmu.edu
  /courses/03441/TermPapers/97TermPapers/lux/default
  .html
• Promega Bacterial Expression Vectors. Promega
  Corporation. 3 Sept. 2004. http//www.promega.com/
  vectors/bacterial_express_vectors.html
• Slock, James. Molecular Biology Experiments
  Utilizing the lux Genes of Vibrio fischeri and
  gfp Gene of Aequoria victoria. Kings College PA.
  3 Sept. 2004. lthttp//www.kings.edu/biology/lux/lu
  xbiolum.htmlgt
• Winfrey, Michael, Marc Rott, and Alan Wortman.
  Unraveling DNA Molecular Biology for the
  Laboratory. New Jersey Prentice Hall, 1997.