1) Small piece of tissue is taken from plant to
be cloned, usually from shoot tip, called an
explant.
2) Cells divide but do not
differentiate, forming a mass of
undifferentiated cells called a Callus.
3) Single callus cells removed and
moved to a nutrient medium (rich in
minerals such as nitrates, etc.)
4) Clones moved to a
greenhouse, then
outdoors.
Example: English Elm - produces new growth in the
form of basal sprouts, which grow from meristem
tissue in the trunk close to the ground,
The production of new structures in an
organism that can grow into new individual
structures.
Artificial Cloning
Splitting embryos
1) In vitro fertilisation.
Grow in vitro to 16
cellembryo.
2) Split embryo into several segments.
3) Implant into surrogate mother.
4) All offspring are genetically
identical (clones) but not so to
their parents.
Nuclear transfer, using
enucleated eggs
1) Remove mammary
cells from udder and
ovum and enucleate
ovum.
2) Transplant nucleus from
mammary cell into ovum and
stimulate with electro-fusion.
3) Insert into surrogate
mother. Offspring will be
identical to sheep from
whom the mammary cell
(and therefore nucleus)
was taken.
Defintion: Totipotent Stem
Cells - e.g. embryonic cells,
capable to differentiating into
any type of adult cell found in
the organism.
Defintion: Biotechnology - The industrial
use of organisms/parts of them to
produce food, drugs, and other products.
Microorganisms in Biotechnology
Advantages
Grow rapidly in
favourable conditions
Can be genetically engineered to
produce specific products.
Grow well at relatively low temps.
Tend to produce products
more pure than chemical
processes.
Can be grown
on nutrients
otherwise toxic
or useless to
humans.
Batch Fermentation
Stirrer: Exposes reactants to
fresh O2 by homogenising the
mixture.
Water Jacket: Maintains
a steady, desired
temperature to stop
enzymes from
denaturing.
Acid/Alkali tube: Maintains pH
to prevent denaturing.
Continous Fermentation:
Substrates are constantly
added, product constantly
removed.
Example: Fusarium
- fungi that
produces Quorn
mycoprotein
Metabolites: products
of metabolic reactions.
Primary: normal product produced all the
time, e.g. CO2, lactic acid, urea.
Secondary: Not normally
produced. Produced during
stress.
Example: Penicillin - Produced by
Penicillium notatum to kill bacteria who are
competing for a limited resource. In
production, penicillium is allowed to
reproduce to a large mass and then
substrate is removed.
Definition: Aspepsis - absence of
unwanted microorganisms
Aseptic technique: Any
technique/measure taken to ensure
unwanted microorganisms do not
contaminate culture/products.
Immobilised enzymes
Definition: Immobilising
- A method by which to
separate the product
from the enzyme
because enemy could be
viewed as a
contaminant, and this
allows it to be reused.
Adsorption: Weak
attachment to an
immobilising support,
e.g. clay due to ionic
and hydrophobic
interactions. Active site
is not changed due to
bonding. Can be very
effective unless
leakage occurs.
Covalent Bonding:
Stronger covalent
bonding to a support
(e.g. clay particles).
Active site not affected
and a limited quantity
can be held. Leakage is
rare.
Entrapment: Enzyme trapped in a gel bead.
Active sites unaffected by less easily
accessible due to trapping barrier, thus
reaction rates can be lowered.No leakage.
Insulates the enzyme from denaturing due to
high temps or pH changes.
Membrane Seperation:
Enzyme and substrate
separated by a partially
permeable membrane,
across which the enzyme
cannot move, but the
substrate can. The
substrate diffuses over
and is catalysed to form
the product, and thus
diffuses back. Product is
washed away with
distilled water so all of
product will diffuse back.
Advantages
(enzyme) can be
re-used so reduces
cost
product, pure(r) /
uncontaminated
reduced downstream
processing costs
reaction, can be
faster / have higher
yield , because can be
done at higher
temperature
(immobilised
enzyme)
works at
high(er)
temperature
(immobilised
enzyme) works
in changed pH
The Genome
Definiton: Genomics - The
study of the whole set of
genetic information in the
form of DNA sequences
that occur in the cells of
organisms in a particular
species.
How DNA is
prepared for
sequencing.
1) Homogenise tissue
sample to increase
surface area.
2) Add
detergents to
break down cell
membranes.
3) Add protease to
remove histone
proteins and leave
'naked' DNA.
4) DNA is isolated
from mix by
precipitation with
ethanol.
5) DNA stands
re-suspended in aq.
pH buffered
medium.
6) DNA cut up
by restriction
enzymes.
Different
enymes cut at
different base
sequences
called restriction
sites, of which
there are 2 main
types: Blunt
ends, and sticky
ends, which are
the ones used in
genetic
engineering.
Restriction enzymes: Also called
restriction endonucleases. They
are produced by bacteria to
break up virus DNA that is
injected into them.
PCR: Polymerase Chain Reaction
Used to
make
multiple
copies of a
(shorter)
section of
DNA. It is
artificial
DNA
replication
and is used
to amplify
DNA
samples.
Reaction mixture contains
Sample of
DNA to be
amplified
Excess of primers
(more chance of
bonding to unzipped
DNA strand)
DNA Polymerase
The 4 deoxyribose nucleotides
Definition: Primers - short, single
stranded sequences of DNA, around
10-20 bases, which bind to a section of
DNA, as DNA polymerase cannot bind
directly to single stranded DNA
fragments.
Process
1) Heated to 95 degrees,
breaking the H bonds and
separating the DNA
strands.
2) Cooled to 55 degrees - primers added
and they bond to complimentary site on
DNA (placed upstream and downstream
of target DNA section)
3) Heated to 72 degrees, the optimum temperature for Taq Polymerase to
build complimentary strands of DNA, binding to the primers and starting
from there, working along to the end of the DNA strand, This is repeated in
cycles.
Taq Polymerase: From
bacteria that naturally
exist in hot springs,
therefore their enzymes
have many disulphide
bridges, preventing
them from denaturing
at high temperatures
(known as thermophilic)
Sequencing using modified PCR: (Interrupted PCR and Electrophoresis)
Interrupted PCR
1) Some of the free deoxyribonucleotides have fluorescent dyes attached (one colour specific to each type).
2) These nucleotides are modified and if they are added to the growing
chain, the DNA polymerase is thrown off and the strand ends,
3) As the reaction proceeds, the fragments
produced vary in size. These strands are then run
through a machiene where a laser reads the colour
sequence from smallest to largest strand, allowing
the sequence of colours (and therefore the bases) to
be displayed.
Electrophoresis
Using electricity to separate differently
sized fragments of DNA.The samples of
DNA are placed on an agarose gel at the
negative anode end. Phosphate groups
have a charge of three minus, therefore
the DNa fragments will move to the
positive cathode. Smaller fragments are
more mobile and therefore move faster.
Researches add a tracking dye, giving
blue DNA.
Southern Blotting
To make a replica
of DNA fragments
of interest to do
further tests on
without destroying
the original.
An absorbant surface
(e.g. Nylon) is put on
the agarose gel after
electrophoresis to
draw up a small
amount of the
fragments.
DNA Probes
Single-stranded, short segments of
DNA with a known sequence. Bind to
complementary sections.
The DNA on an absorbent surface is put
into a basic solution to unzip it and make it
single stranded by disrupting the H bonds
between bases. DNA probes then used to
identify target sequences of DNA/Gene.
This can be used to diagnose diseases by
comparing healthy and unhealthy DNA
samples (Microarray).
Bacterial Artificial Chromosome (BACs)
Used to make 'clone libraries' -
multiple copies of (lager)
sections of DNA.
A fertility plasmid from bacterium,
which has been genetically modified
with DNA from another organism. It will
then make multiple copies by
replicating.
Definition: Recombiant Gene Technology -
Processes involving combining DNA from
different organisms/sources in a single
organism.
Obtaining the gene to be
engineered.
1) mRNA produced from transcription of
the gene can be obtained from cells
where that gene is expressed, e.g. insulin
from the B cells of the islet of
Langerhans.
2) Gene can be synthesised using an
automated polynucleotide
sequencer.
Placing gene in a vector.
The gene can be sealed into a
bacterial plasmid using the
enzyme DNA ligase. It can also
be sealed into virus genomes
or yeast cell chromosomes.
Getting the gene into the recipient cell
Once packaged in a
vector it is often part
of a large molecule
that doesn't easily
cross the membrane
to enter the recipient
cell.
Resulting DNA
is called
Recombiant
DNA
Methods
Electroportation: high voltage pulse applied to
disrupt the membrane.
Microinjection: DNA is injected using
a very fine micropipette into the host
cell nucleus
Viral Transfer: Uses virus's mechanism
for infecting cells by injecting DNA
directly.
Liposomes: DNA is injected in lipid molecules - fat soluble
so they can diffuse across lipid membrane.
Genetic engineering and bacteria
Any organism that
contains DNA added to
its cells as the result of
genetic engineering is
described as transgenic.
Definiton: Bacterial
Conjugation - a
process where
genetic material may
be exchanged (N.B.
only plasmids).
If large quantities of
recombinant plasmids are added
to a mix of bacteria, some will
take them up. The addition of
calcium salts and heat shock
increases the rate at which they
are taken up. However it is very
inefficient; less than 0.25% take
up the plasmid.
Heat shock: Temperature is lowered
to freezing, then quickly raised to
forty degrees.
Bacteria are
known as
transformed
bacteria after
taking up
recombinant
plasmid.
Example: Insulin
1) Insulin: a polypeptide containing
51 amino acids. Scientists used
specialised centrifugation to obtain
the mRNA for it.
2) Used reverse transcriptase to
synthesise a complimentary
DNA strand. Adding DNA
polymerase and nucleotides to
this produced a second strand,
thus giving us a copy of the
original gene, called a cDNA
gene.
3) Plasmids cut upon
with restriction
enzyme. Some take
up the gene, DNA
ligase seals the
plasmid, giving us a
recombinant plasmid.
Bacteria is then put
onto an agar plate.
4) Plasmids
with the insulin
gene are
identified using
radio-labelled
antibodies that
bind specifically
to insulin.
N.B. Modern
methods work
differently, using
genetic markers.
1) Original plasmids are chosen
because they contain genes that
make them resistant to two
different antibiotic chemicals.
These genes are genetic markers.
2) Plasmids are cut by restriction
enzyme that has its restriction site
on one of the resistance genes. If
required gene is taken up, one of
the resistance genes will be broken
up and won't work, whilst the other
will.
3) Replica plating is then
used. This is where the
bacteria grow and form
colonies. These are tested
on materials made with
the chemicals tied into the
resistant genes.
4) By seeing which bacteria colonies
grow on one growth material and not
the other, we know which have taken up
the required genes.
5) The transformed
bacteria are then
produced on a large
scale.
Example: Golden Rice.
Vitamin A deficiency can cause
irreversible blindness, and only comes
from animal sources in the diet.
However, it can be derived from a
precursor called beta-carotene, which
is converted into Vitamin A in the gut.
Rice plants (Oryza Sativa)
contain genes that come
for Beta-carotene in the
grain. However, these
genes are switched off.
Thus genetic engineering is
used to insert 2 genes into
the rice genome to
activate the metabolic
pathway in the endosperm
cells.
These genes code for two enzymes.
Phytopene
Synthetase
(extracted from
daffodil plants)
Converts
precursor
molecules into
Phytopene.
Crt 1 enzyme
(extracted from
soil bacterium
Erwinia
Uredova)
Converts the Phytopene
produced by Phytopene
Synthetase into Lycopene.
Enzymes already present into the rice
endosperm then convert Lycopene to a
range of carotenoid molecules, including
Beta-carotene.
Golden Rice
(TM) is said to be
biofortified
because it
contains higher
concentrations
of a particular
nutrient. Rice
was then
cross-bred with
natural rice
varieties to
increase
beta-carotene
conc.s further.
Gene Therapy
Somatic Cell Gene Therapy (Body Cells)
Gene therapy by adding genes
(augmentation). Some disorders
are caused by faulty alleles; by
engineering a functional copy into
the relevant specialised cells, the
functional gene product can be
produced.
N.B. Only lasts as long as cell lives.
Can also kill
cells by making
specific cells
(e.g. cancerous
ones) express
genes that
produce
antigens that
lead to their
being attacked
by the immune
system.
Can also silence cells, by
iIntroducing interference
RNA (RNAi) into cells. this
is complimentary to
mRNA of a specific gene,
which it binds to, making
it double-stranded and
therefore unable to be
translated, causing the
mRNA to be broken
down.
Germline Cell Gene Therapy (sperm/egg/zygote)
Means all body cells will contain new gene. There are
concerns about it inadvertently causing a new disease
and the lack of possible consent and interference
with evolution. Currently illegal.
Defintion: Xenotransplant - surgical
procedure in which tissue/whole organs
are transferred from one species to
another.
Example: Pig hearts
Genetically modified to prevent rejection: lack
the enzyme a-1,3-transferase, which is needed
to make antigens on the pig cells that the
antibodies involved in hyper-acute rejection
bind to.
Also insert the human
gene for nucleotidase
enzyme - regulates the
immune response.
Problems
Size of Organ
Animal Rights
Lifespan of organ/animal
Religious beliefs
Body Temperature of pigs is 39 degrees.
Possible disease transfer
Defintion:
Allotransplantation -
refers to
transplantation
between animals of
the same sepcies.