Transcript III. Biotechnology

Biotechnology
Altering an organism's genetic code
so that it produces desired protein
A. A Brief History Lesson
1. Artificial Selection
The breeding of
plants and animals to
produce offspring
with desired traits has
been practiced for
thousands of years.
For example, faster
horses, cows that
produce more milk
and plants with
higher yield.
2. Contributions of Salvador Luria -1950
a) Early biochemistry work was conducted on organisms with small
genomes like E. coli and viruses that prey upon them
b) A plate with nutrient agar would
be inoculated with bacteria and
they would be allowed to grow until
they covered the plate
c) Later, phages were added and they
would attack and kill the bacteria
leaving empty spots on the plate called
plaques
Salvador Luria’s Observation and
Hypothesis
d) In 1950, Luria observed
some bacteria that were
unaffected when
exposed to phages
e) Luria hypothesized that
these bacteria had some
type of primitive
immune system that
restricted phage growth
3. Bacteria Evolved Restriction Enzymes
a) In order to reproduce,
viruses must attach to a
host cell
Phage
Viral DNA
Host Cell
Restriction Enzyme
Host Cell DNA
b) The virus then
injects it’s DNA into the
host cell
How Restriction Enzymes Protect Bacteria
c) Restriction enzymes
bind with the viral DNA at
specific base sequences
called recognition sites
d) The viral DNA is cut at
specific sites called restriction
sites which destroys it and
protects the bacteria from
infection
4. Contributions of Werner Arber,
Daniel Nathans and Hamilton Smith
In 1978 Arber, Nathans and Smith won the Nobel prize
for isolating the first restriction enzyme
5. Restriction Enzymes- enzymes that cut
DNA at a specific base sequence
6. Naming Restriction Enzymes
EcoR I
E
BamH I
genus
Echericia
B
genus
Co species
coli
am species
amyloliquefaciens
R
Strain
R
H
Strain
H
I
Order found 1st
I
Order found 1st
Hind III
H
genus
Haemophilous
in species
influenzea
d
d
Strain
III Order found 3rd
Bacillus
B. Contributions Daniel Nathans
and Joseph Sambrook
1. Nathans realized that DNA has a negative
charge and therefore restriction fragments could
be separated using an electric current
2. In 1970, Nathans used a polyacrylamide gel to
separate DNA restriction fragments by size
3. Sambrook introduced the use of agarose gel and
ethidium bromide staining used today
4. Gel Electrophoresis- separating cut
DNA fragments by size using an electric
current
C. Bacterial Transformation
1. Experiments of Frederick Griffith
2. Griffith was a British army doctor trying to find a
vaccine against Streptococcus pneumonie, a
bacterium that causes pneumonia in mammals.
He knew 3 things:
a) There are two strains of the bacteria: one
produces smooth colonies, S, and one that
produces rough colonies, R.
b) Cells of the smooth strain are covered with a
polysaccharide coat and the rough strains are not.
c) The alternative phenotypes (S&R) are inherited
3. Griffith preformed 4 experiments
Griffith injected live S strain into mice.
Results:
Conclusion: The S strain is pathogenic
Griffith injected mice with live R strain.
Results:
Conclusions: R strain is non pathogenic
Mice were injected with heat killed S strain
Results:
Conclusions: Heat killed bacteria are non pathogenic
Then for some reason known only to Griffith,
he decided to inject Heat killed S and live R
were injected into mice
Results:
Conclusion: The R strain acquired instructions to
make the polysaccharide coat from the dead S strain
Not only did the
mouse die, live S
strain was extracted
from its body. Griffith
could not explain
what had happened,
but he said the
bacteria had been
transformed
We now call the assimilation of external genetic
material by another cell Transformation
4. Morton Mandel and Akiko Higa
a) Discovered a method in 1970 to make
bacterial cell competent by treating them
with calcium ions.
b) Used a method involving a rapid change
in temperature called heat shock
D. Herbert Boyer & Stanley Cohen
collaboration occurred in spring 1973
1. In 1973 Herbert
Boyer, and Stanley
Cohen constructed the
first functional
organism that
combined and
replicated genetic
information from
H. Boyer
different species.
2. The science of genetic
engineering was born
S. Cohen
E. Other Techniques
1. Dr. Kary Mullis won the 1993 Nobel Prize for the
invention of PCR- Polymerase Chain Reactionallows specific DNA fragments to be copied
millions of times
2. RFLPs- Restriction Fragment
Length Polymorphism
a) Used to identify DNA when a mutation
adds or deletes a restriction site
b) Gel electrophoresis separates the DNA
fragments and mutations are identified by
an abnormal number of fragments
3. VNTRs & STRPs- similar to RFLP
analysis, but uses highly variable, noncoding sequences of DNA
4. DNA Sequencing
a) A piece of DNA with an unknown sequence is placed in 4
Eppendorf tube containing primase, nucleotides, and DNA
polymerase. Dideoxynucleotides (ddA, ddT, ddC & ddG) are added
to different tubes.
b) In the tube that contain ddT, sometimes deoxythymine will be
added, but sometimes ddT will be added. Because
dideoxynucleotides prevents the addition of the next base, the
chain will be terminated
c) The result will be fragments of DNA of various lengths. One
corresponding to each base in the chain
d) The tubes are incubated and the products are run through a gel
which results in fragments which can be read
dideoxynucleotides
The resulting
nucleotide
sequence is
complimentary to
the unknown
DNA sequence
5. Microinjection- thin needles
insert DNA into cells
6. Synthetic Organisms
Self replicating organisms whose genome is entirely constructed by
man
Craig Venter’s Institute manufactured the 1st entirely synthetic
organisms on the 21st of May of 2010
F. Uses of Recombinant DNA
Technology
1) Pharmaceuticals- Humilin, TPA, interferon,
TNF, Artificial hemoglobin, human growth
hormone
2) Agriculture- incide, Flavr-saver tomatoes, frost
resistance, salt resistance, insect resistance,
herbicide resistance, nitrogen fixation
3) Forensics- DNA finger printing
4) Medical- gene therapy
G.. Dangers of Genetic Engineering
1. Pathogens- disease causing organisms
2. Eugenics- should we control
or alter our own genome?
3. Stem Cells- growing new human
tissues from cell derived from fertilized
eggs
4. Legal Questions- can/should
we patent life?
5. Genetic Screening- who would get
the results of the tests and how could test
results be used?
6. GMOs- Genetically modified organisms