Transcript The Biotechnology Age: Issues and Impacts

ABE: Advances in Bioscience Education
A workshop bringing together students and faculty
to explore research in the molecular and
cellular biosciences.
Dr. Kabi Neupane, Coordinator (co-PI, LCC)
Faculty Partners
John Berestecky (KCC)
Ingelia White
(WCC)
Janice Ito (LCC)
Priscilla Millen (LCC)
Catherine Unabia (HPU)
David Christopher (PI, UH, Manoa)
Stepping into scientific research
 From the classroom to
the lab bench with intensity.
What are our goals?
What kind of research are we doing ?
What is an Arabidopsis plant?
What kind of results might you expect?
Research philosophy.
Goals
• Reach for new learning and
knowledge.
• To learn and grow - actively,
by doing.
• Move out of the comfort zone
and into the stretch zone.
• Take risk, try something new,
unfamiliar and break thru the fear barrier.
Research = Stretch Zone:
Willing to sail into
unchartered waters
To discover…
make mistakes…
…change direction.
Have FAITH that you can
learn from mistakes as you
move along.
Scientific Research:
Learn by Trial and error.
Embrace mistakes
Persistance is more
important than strength
Scientific Research: Discover new knowledge.
Use tools of molecular and cellular biology
to figure out the underlying biochemical
processes that control how living cells work.
From DNA of genes
 to LIFE !
From DNA sequence (chemical) to Life
GGTTCCAAAAGTTTATTGGATGCCG
TTTCAGTACATTTATCGTTTGCTTT
GGATGCCCTAATTAAAAGTGACCCT
TTCAAACTGAAATTCATGATACACC
AATGGATATCCTTAGTCGATAAAAT
TTGCGAGTACTTTCAAAGCCAAATG
AAATTATCTATGGTAGACAAAACAT
TGACCAATTTCATATCGATCCTCCT
GAATTTATTGGCGTTAGACACAGTT
GGTATATTTCAAGTGACAAGGACAA
TTACTTGGACCGTAATAGATTTTTT
GAGGCTCAGCAAAAAAGAAAATGGA
AATACGAGATTAATAATGTCATTAA
TAAATCAATTAATTTTGAAGTGCCA
TTGTTTTAGTGTTATTGATACGCTA
ATGCTTATAAAAGAAGCATGGAGTT
ACAACCTGACAATTGGCTGTACTTC
CAATGAGCTAGTACAAGACCAATTA
TCACTGTTTGATGTTATGTCAAGTG
AACTAATGAACCATAAACTTGGTCA
Tools of molecular and cellular biology
- Recombinant DNA technology
- Biochemistry
- Microscopy
- Molecular genetics
- Computers
Use these tools from an actual research project
National Sciences Foundation (NSF) funds ABE:
“Functional Genomics of Protein Disulfide Isomerase
Gene Family: Unraveling Protein Folding and
Redox Regulatory Networks”
•
Complicated name:
What does it mean?
“Functional Genomics of Protein Disulfide Isomerase
Gene Family: Unraveling Protein Folding and
Redox Regulatory Networks”
Functional Genomics:
• New field of biological science
• Rooted in Genetics
• Genome: all of the genes encoded in DNA in a
living organism.
• Function: Research to figure out what the genes
are doing.
• What proteins do they encode and what jobs in the
cell are they responsible for?
What jobs do proteins do in a cell?
• 1. Structure: hold things up
• 2. Enzymes: activity make and burn energy.
Stimulate growth and biomass production.
• 1000’s different enzymes -> unique activities
• Figure out their activities.
ENZ
A ----------->
B
• Where the enzyme is located in the cell?
• Do they need other protein partners to do their job?
“Functional Genomics of Protein Disulfide Isomerase
Gene Family: Unraveling Protein Folding and
Redox Regulatory Networks”
Making of a protein: Converting the code in
a polymer of nucleic acid to a polymer of amino acid
DNA

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 Transcribed
RNA

PROTEIN
 Translated
ENZYME: Protein Disulfide Isomerase = PDI
• Chain of amino acids representing a PDI.
• Disulfide: “Two sulfurs”
The amino acid containing sulfur is cysteine
Bond with 2 cysteines
|
SH
|
SH
|
SH
ENZYME: Protein Disulfide Isomerase
• Isomer: Different molecules with same chemical
formula.
• Alter chemical bonding --> different “shapes” -->
activities and functions.
•Isomerase: an enzyme that can make different
molecular shapes out of the same substance.
•Disulfide Isomerase: emzyme that alters molecular
shape by acting on the disulfide bonds.
PDI can make different protein shapes based on
altered disulfide bonding
|
SH
|
SH
2 isomers with
new activity !
OR
-S S-
|
SH
|
SH
-S-S-
|
SH
“Functional Genomics of Protein Disulfide Isomerase
Gene Family: Unraveling Protein Folding and
Redox Regulatory Networks
Proteins do not do their job unless
they are folded correctly
• So, PDIs fold other proteins correctly in cells.
• A major responsibility for keeping cells normal,
development, metabolism and growth.
Protein Disulfide Isomerase (PDI)
Gene Family
• Study all the PDIs in the genome of a small plant.
•All the PDIs in the same related family.
• but they go off and have different jobs at various
locations in the cell.
PDI Protein folding- oxidoreductase
PDI
= cys
Inactive state
Active state
All proteins have to fold to proper states
Chemical Mechanism REDOX
• Oxidation Remove 2 electrons and 2 H+
2 cysteine sulfhydryls --> make disulfide bridge
• Reduction --> breaks bridge -->
– Add 2 electrons and 2 H+ to the 2 sulfhydryls
In Yeast and humans - PDIs located in the
endoplasmic reticulum (ER)
• But what about plants????
Arabidopsis thaliana Plants
Arabidopsis
• First plant to have its genome completely sequenced
– Smallest Plant Genome known
-> 26,000 genes but makes a plant!!
– 40 days from seed to seed.
– Easily genetically engineered.
– Easy to knock out genes to see what they do.
Primary structure of a generic PDI
ER retention motif
C--C
C--C
Signal sequence
Thioredoxin domains
Directs a protein
to a specific
location
Contains 2 cysteines and
active catalytic site for
oxidation-reduction and
folding of proteins
KDEL
Research activities of workshop
Learn some recombinant DNA methods
Map genes that have been tagged by a T-DNA
Learn PCR and RT-PCR
Isolate proteins from leaves and detect proteins
using antibodies
Use a microscope to find where PDIs and green
fluorescent protein are located in the cell.
What kind of results might you expect?
Any kind of result is a success
Learn by doing !!!
Have fun while you learn !
Nothing has to work perfectly to be a
valuable learning experience.
T-DNA Mapping
M
W.T
1-a
1-b
3-Ⅰ 3-Ⅱ 3-Ⅲ
4-Ⅰ 4-Ⅱ
4-Ⅲ
Fluorescence microscopy uses high energy
blue - UV light to excite the sample, which
emits light at a lower energy
Chlorophyll
fluoresces
red
Green fluorescent chemical attached to an
antibody that binds to a chloroplast PDI
My first attempts at preserving tissue
for transmission electron microscopy
Sabbatical at University of Colorado
2005-2006
High pressure freezing:
Plant tissue is flash frozen in a pressure bomb
-197 C
Water in the tissue is replaced with acetone
over 5 day period
Acetone saturated tissue is embedded in resin
Resin is cut in thin sections, 80 nm thick
Add antibody immunolabeling
Electron microscope
Very
Wrinkled
Chloroplast
Carnage
Pretty bad
fixation
2nd time: procedures were done poorly, but there is hope…
Back to the
drawing board
to start over.
But what to
correct?
What to do
different?
Will it
improve?
When things do not work right, keep moving forward
and ignore doubt, pressure and negativism.
3rd time
A charm
Excellent preservation
And
Immunolabeling
the 3rd TIME
HIGH
MAG
RE-search Not search
Must be repeated
Research time is spent:
70% trouble-shooting
15% success
15% communicating success.
ROOT
HOOK-o-PLASM
PDI in
Vacuole
CNGC in Golgi Apparatus
g
200 nm
PDI in Golgi Apparatus
c
G
200 nm
Dividing mitochondria
Channel located to the plasma membrane
Channel located to the plasma membrane -plasmolysis
We learn more from mistakes than successes…