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Stable Nuclear Transformation of the diatom Phaeodactylum tricornutum

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. Diatoms. Eukaryotic microalgae Located in freshwater, marine and terrene enviroments.

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Stable Nuclear Transformation of the diatom Phaeodactylum tricornutum

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  1. 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

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

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

  4. 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. Short and Long Term Goals

  5. 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.

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

  7. 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.

  8. 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. Gel electrophoresis & Protein electrophoresis

  9. 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.

  10. 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).

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

  12. Western Analysis (Immunoblot) • The western analysis shows protein that has been synthesized by the mRNA being transcribed from the DNA.

  13. 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.

  14. 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.

  15. Picture References • NoE Marine Genomics Europe, provided by Coppermine Photo Gallery. http://www.marine-genomics-europe.org/gallery/albums/userpics/10001/normal_Cadoret%2C%20ifremer%2C%20Phaeodactylum%20tricornutum%20exprimant%20la%20GFP.jpg • http://www.botany.unimelb.edu.au/botanyunimelb/1pages/research/labs/EM/images/A882.jpg

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