Stable Nuclear Transformation of the diatom Phaeodactylum tricornutum

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Stable Nuclear Transformation of the diatom
Phaeodactylum tricornutum

• By
• Kirk E. Apt, Peter G. Kroth-Pancic, Arthur R.
  Grossman
• Presented By
• Keone Tyau and Joe Nelson

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Diatoms

• Eukaryotic microalgae
• Located in freshwater, marine and terrene
  enviroments.
• Primary productivity in water environments.
• Limited as experimental organisms.

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Phaeodactylum tricornutum

• A diatom that has been vastly studied.
• Was used to develop a stable nuclear
  transformation.

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Short and Long Term Goals

• The short term goal of the researchers was to
  develop a stable nuclear transformation of
  diatoms.

• The long term goal was to develop a way to
  regulate the genome of diatoms for both research
  and commercial uses.

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Zeocin and Sh ble

• Zeocin and Phleomycin kill P. tricornutum at low
  concentrations.
• Zeocin is chosen as the anitbiotic.
• Sh ble is resistant to the antibiotic Zeocin.
• Sh ble is chosen as marker gene.

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Sh ble PCR and Promoter

• fcpA promoter
• EcoRV and Hind III
• Sh ble replaces fcpA
• Plasmids are formed and ready for insertion.

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Bio-Rad Biolistic PDS-1000/He

• DNA was inserted using this -----?
• Tungsten M5 and M17 particles were used.
• M17 particles worked better at level 2 with
  super-coiled DNA.

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Gel electrophoresis Protein electrophoresis

• Gel electrophoresis is used when studying the
  amounts of DNA and RNA. Blots are performed.
  (Southern and Northern)

• Protein electrophoresis is used to identify
  presence of proteins. A western analysis is then
  performed.

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Southern Blot

• Southern blot for fcpA shows both original fcpA
  promoter and a second fcpA region after insertion
  of sh ble.
• Southern blot for sh ble shows the successful
  insertion of the gene to DNA.

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Southern blot continued.

• This southern blot shows that the sh ble gene has
  on been inserted into DNA from the nucleus (n)
  and not the organelles (o).

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Northern Blot

• The northern blot is done to prove that the DNA
  synthesized is capable of transcribing RNA.

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Western Analysis (Immunoblot)

• The western analysis shows protein that has been
  synthesized by the mRNA being transcribed from
  the DNA.

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Different fcp Promoters to Drive sh ble gene

• All of these promoter genes were tested to show
  which promoter produced the greatest amount of
  colonies per bombardment.
• As the table shows the fcpC promoter produced the
  most colonies.

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Further Studies

• The ability to insert a selectable marker into a
  diatom enables the manipulation of the gene
  structure and the regulation of the genome.
• With the ability to perform a nuclear
  transformation, a new door is open that allows
  diatoms to be mass produced in a laboratory
  making it more accessible for both researchers
  and commercial users.
• The commercial uses includes feeds in
  aquaculture, sources of specialty oil, and even
  potential sources in the pharmaceutical drug
  industry.

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Picture References

• NoE Marine Genomics Europe, provided by
  Coppermine Photo Gallery. http//www.marine-genomi
  cs-europe.org/gallery/albums/userpics/10001/normal
  _Cadoret2C20ifremer2C20Phaeodactylum20tricorn
  utum20exprimant20la20GFP.jpg
• http//www.botany.unimelb.edu.au/botanyunimelb/1pa
  ges/research/labs/EM/images/A882.jpg