Download Plasmid

BIOTECHNOLOGY
 A.
Genetic engineering, also
known as recombinant DNA
technology, means altering the
genes in a living organism to
produce a Genetically Modified
Organism (GMO) with a new
genotype.
 B.
Various kinds of genetic
modification are possible:
1.inserting a foreign gene from
one species into another,
forming a transgenic organism
2.altering an existing gene so
that its product is changed
3.changing gene expression so
that it is translated more often
or not at all.
Humans are
made up of
trillions of cells
Each cell:
•46 human
chromosomes
•2 metres of DNA
•3 billion DNA
subunits (A,T,G,C)
•Approximately
22,000 genes
replication
DNA
genotype
transcript
RNA
translation
Protein
phenotype

A. It allows genes from one organism to be
inserted into a cell of a different organism
of a different species.
• Examples:
– Human genes can be inserted into a
bacterium
– Human genes can be inserted into cells
from other animals
– Bacterium genes can be inserted into
plant cells
1.
2.
3.
4.
Isolation-Isolate the gene
Restriction- Cutting
Transformation - Insert it in a host using a
vector and produce as many copies of the
host as possible
Expression-Separate and purify the product
of the gene
E. coli bacterium
Plasmid
Bacterial
chromosome
1
A cell with DNA
containing the gene
of interest
2
A plasmid
is isolated.
The cell’s DNA
is isolated.
Gene of
interest
3
DNA
The plasmid is cut
with an enzyme.
Examples of gene use
4
The cell’s DNA is cut
with the same enzyme.
Gene
of interest
5
6
The targeted fragment
and plasmid DNA
are combined.
DNA ligase is added,
which joins the two
DNA molecules.
Examples of protein use
Recombinant
DNA
plasmid
Gene
of interest
7
The recombinant plasmid
is taken up by a bacterium
through transformation.
Recombinant
bacterium
8
Clone
of cells
Genes may be inserted
into other organisms.
The bacterium
reproduces.
9
Harvested
proteins
may be
used
directly.
E. coli
bacterium
Bacterial
chromosome
A cell with DNA
containing the gene
of interest
Plasmid
1
2
A plasmid
is isolated.
Gene of
interest
The cell’s DNA
is isolated.
DNA
E. coli
bacterium
Bacterial
chromosome
A cell with DNA
containing the gene
of interest
Plasmid
1
2
A plasmid
is isolated.
The cell’s DNA
is isolated.
Gene of
interest
3
DNA
The plasmid is cut
with an enzyme.
4
The cell’s DNA is cut
with the same enzyme.
Gene
of interest
E. coli
bacterium
Bacterial
chromosome
A cell with DNA
containing the gene
of interest
Plasmid
1
2
A plasmid
is isolated.
The cell’s DNA
is isolated.
Gene of
interest
3
DNA
The plasmid is cut
with an enzyme.
4
The cell’s DNA is cut
with the same enzyme.
Gene
of interest
5
The targeted fragment
and plasmid DNA
are combined.
E. coli
bacterium
Bacterial
chromosome
A cell with DNA
containing the gene
of interest
Plasmid
1
2
A plasmid
is isolated.
The cell’s DNA
is isolated.
Gene of
interest
3
DNA
The plasmid is cut
with an enzyme.
4
The cell’s DNA is cut
with the same enzyme.
Gene
of interest
5
6
Recombinant
DNA
plasmid
The targeted fragment
and plasmid DNA
are combined.
DNA ligase is added,
which joins the two
DNA molecules.
Gene
of interest
Recombinant
DNA
plasmid
Gene
of interest
7
Recombinant
bacterium
The recombinant plasmid
is taken up by a bacterium
through transformation.
Recombinant
DNA
plasmid
Gene
of interest
7
The recombinant plasmid
is taken up by a bacterium
through transformation.
8
The bacterium
reproduces.
Recombinant
bacterium
Clone
of cells
Genes may be inserted
into other organisms.
Recombinant
DNA
plasmid
Gene
of interest
7
The recombinant plasmid
is taken up by a bacterium
through transformation.
Recombinant
bacterium
8
Clone
of cells
The bacterium
reproduces.
9
Harvested
proteins
may be
used
directly.
(a) Isolation of a specific gene from donor e.g.
human
•
Cells broken open
•
Genetic probe added
•
Reveals position of the gene of interest
Genetic probe
Position
of gene of
interest
Donor DNA
(b) Isolation of plasmid from a bacterial cell
Bacterial cell
Plasmid
www.sci.sdsu.edu



Restriction enzymes (also called endonuclease) act
as molecular scissors and cut DNA at specific sites
called restriction sites
This makes a staggered
cut with sticky ends
ex: EcoRI
Restri
ction
site
Clipartguide.com
Restriction
site
Restriction
ezymes
Restriction site
Restriction site
Donor DNA
Restriction
sites
Restriction
enzymes
Plasmid
Donor DNA
Plasmid
Sticky Ends
Plasmid
DNA with
sticky ends
Enzyme DNA ligase
bonds sticky ends
together

A. Vector – molecule of DNA which is used
to carry a foreign gene into a host cell
◦ Most common vectors are bacteria and viruses
B. A vector has to have certain properties:
1. It is big enough to hold the gene we
want
2. It is circular (or more accurately a
closed loop), so that it is less
likely to be broken down
3. It contains control sequences, such
as a transcription promoter, so
that the gene will be replicated or
expressed.
4. It contain marker genes, so that
cells containing the vector can be
identified
Enzyme DNA Ligase
bonds sticky ends
together
Plasmid
DNA
Recombinant DNA
Donor
DNA
Next slide
for
animation
www.gch.ulaval.ca

D. Recombinant DNA introduced into
bacterial cell
Bacterial
cell
Bacterial
chromosome
Recombinant
DNA

Bacterial cell reproduces by Binary Fisson
• Bacterial cell produces the polypeptide
• Coded for by the donor DNA
Donor DNA
Plasmid
1. Cut with
restriction enzymes
Donor DNA
Sticky
Ends
2. Ligase
bonds sticky
ends together
Recombinant
DNA
www.encarta.msn.com
Genetically Engineered
Products


Insulin regulates
the blood sugar
level.
The gene for
human insulin is
inserted into yeast
or bacteria, from
which large
quantities of the
human insulin are
manufactured
Production of humulin
https://www2.vaxserve.com/ index.cfm?event=get...


Undernourished people in poorer countries
may have blindness caused by a lack of
vitamin A
Contains beta-carotene, which forms
Vitamin A

rBGH is a genetically
engineered version of a
hormone, which is
found in the pituitary
gland of cows and
controls milk
production
http://www.healingdaily.com/detoxification-diet/rBGH.

rBGH can increase
cows’ milk production
by as much as 20-30%
http://simple.wikipedia.org/wiki/Image:Milk_glass.jpg


Elastic, light weight fiber 5 times stronger than
steel
One method involves inserting the gene from a
weaving spider into a fertilized goat egg. The
resultant “spider-goat” produces milk that can be
manufactured into strong fibers.
http://www.chm.bris.ac.uk/motm/spider/goat.jpg

To make flak
jackets, rope,
textiles, sutures,
artificial tendons,
and bandages for
burn victims


Genes that make jellyfish glow are inserted
into other organisms
The organism glows under UV light


Bt is a bacterial toxin that is
toxic to some insects. In the
lab, the gene that produces
the toxic effect is inserted
into the DNA of plants
Bt crops produce an
insecticide protein thousands
of times more powerful than
the chemical spray
insecticides
Genetically
modified corn
www.farmbureaulaporteco.org
Can resist
infection by an
insect European
corn borer

Transgenic plants and farm animals
pest-resistant (reduce use of pesticides),
increase yield
Increase storage time
e.g. green tomato
tomato with beef genes

Clonal propagation:
a source of tissue or organ for transplantation
avoid all problems of immunoincompatibility.

Gene therapy:
Artificially replace the disease-causing
gene with a normal allele.
The normal allele can be carried by a virus
vector to the target tissues.
e.g. treatment of cystic fibrosis

Production of pharmaceuticals for
treatment of diseases e.g. human insulin,
interferons
Production of pharmaceuticals for
disease prevention e.g. vaccine
(hepatitis B vaccine)

Use of GM microorganisms to make stonewash jeans
Use of GM microorganisms to produce
enzymes e.g. detergents

GM E. coli possesses gene to break down
cellulose, speeding up recycling of the
most abundant biomass on earth
GM microorganisms with enhanced
ability to break down environmental
pollutants





Dangerous pathogens formed in the
course
New tools for militarists and terrorists
Triggering of catastrophic ecological
imbalance
Moral problems in the use of GM
techniques in man e.g. germ cell gene
therapy
Unknown effect of GM food on men



Main concerns are in Genetically Modified
Foods.
Effects on Food Chains of genetically
modified plants and animals.
Also conflicts in relation to human cloning
and stem cell research.