Transcript Stable Nuclear Transformation of the diatom Phaeodactylum

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
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Eukaryotic microalgae
Located in freshwater,
marine and terrene
enviroments.
Primary productivity in
water environments.
Limited as
experimental
organisms.
Phaeodactylum tricornutum
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A diatom that has
been vastly studied.
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Was used to develop
a stable nuclear
transformation.
Short and Long Term Goals
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The short term goal
of the researchers
was to develop a
stable nuclear
transformation of
diatoms.
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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
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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
Growth
Concentration
Antibiotics
Kanamycin
Gentamicin
Streptomycin
Hygromycin
Zeocin
Phleomycin
+
+
+
-
1 mg/ml
1 mg/ml
1 mg/ml
250 ug/ml
50 ug/ml
5 ug/ml
Herbicides
Glyphosate
Chlorsulfuron
Bialaphos
+
+
+
50 ug/ml
50 ug/ml
50 ug/ml
Sh ble PCR and Promoter
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fcpA promoter
EcoRV and Hind III
Sh ble replaces fcpA
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 supercoiled DNA.
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Gel electrophoresis & Protein
electrophoresis
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Gel electrophoresis
is used when
studying the
amounts of DNA
and RNA. Blots are
performed.
(Southern and
Northern)
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Protein
electrophoresis is
used to identify
presence of
proteins. A
western analysis is
then performed.
Southern Blot
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.
► Southern
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
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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.
Average # of
Promoter colonies per
bombardment
fcpA
18.8(n = 4)
fcpB
23.0(n = 4)
fcpC
41.5(n = 4)
fcpE
14.0(n = 4)
Uhsp70 0(n = 4)
none
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.
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Picture References
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NoE Marine Genomics Europe, provided by
Coppermine Photo Gallery. http://www.marinegenomicseurope.org/gallery/albums/userpics/10001/norm
al_Cadoret%2C%20ifremer%2C%20Phaeodactyl
um%20tricornutum%20exprimant%20la%20GF
P.jpg
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http://www.botany.unimelb.edu.au/botanyunime
lb/1pages/research/labs/EM/images/A882.jpg