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.

Primary productivity in water environments.

 Limited as experimental organisms.

Phaeodactylum tricornutum

 A diatom that has been vastly studied.

 Was used to develop a stable nuclear transformation.

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.

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.

Compound

Antibiotics

Kanamycin Gentamicin Streptomycin Hygromycin Zeocin Phleomycin

Herbicides

Glyphosate Chlorsulfuron Bialaphos Growth + + + + + + Concentration 1 mg/ml 1 mg/ml 1 mg/ml 250 ug/ml 50 ug/ml 5 ug/ml 50 ug/ml 50 ug/ml 50 ug/ml

Sh ble PCR and Promoter

    fcp A promoter Eco RV and Hind III Sh ble replaces fcp A Plasmids are formed and ready for insertion.

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.

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.

Southern Blot

► ► Southern blot for fcp shows both original A fcp A promoter and a second fcp A region after insertion of sh ble .

Southern blot for sh ble shows the successful insertion of the gene to DNA.

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).

Northern Blot

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

Western Analysis (Immunoblot)

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

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.

Promoter Average # of colonies per bombardment fcpA 18.8(n = 4) fcpB fcpC fcpE Uhsp70 none 23.0(n = 4) 41.5(n = 4) 14.0(n = 4) 0(n = 4) 0(n = 4)

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.

Picture References

 NoE Marine Genomics Europe, provided by Coppermine Photo Gallery. http://www.marine genomics europe.org/gallery/albums/userpics/10001/norm al_Cadoret%2C%20ifremer%2C%20Phaeodactyl um%20tricornutum%20exprimant%20la%20GF P.jpg

 http://www.botany.unimelb.edu.au/botanyunime lb/1pages/research/labs/EM/images/A882.jpg