Download 2. Biotechnology Booklet [A2]

SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
Cloning in animals and plants
Learning objectives:
 Outline the differences between reproductive and non-reproductive cloning;
 Describe the production of natural clones in plants using the example of vegetative
propagation in elm trees;
 Describe the production of artificial clones of plants from tissue culture;
 Discuss the advantages and disadvantages of plant cloning in agriculture;
 Describe how artificial clones of animals can be produced;
 Discuss the advantages and disadvantages of cloning animals;
Key definitions:
Compile a glossary by writing your own definitions for the following key terms related to the
learning objectives above.
Key term
vegetative propagation
tissue culture
explant
callus
totipotent stem cells
cloned animal
Definition
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
Vegetative propagation of plants
Many flowering plants are able to reproduce asexually and spread quickly through vegetative
means e.g. through runners and suckers. Humans exploit this ability in the vegetative propagation
of plants by methods such as cutting and grafting. Such methods result in genetically identical
plants (clones) year after year. Clones are produced for several reasons, e.g. to obtain a uniform
plant performance (as in fruit trees), to multiply sterile or seedless species, or to propagate species
with flowers in which the stamens have changed to petals and there is no pollen produced. In
general, artificial propagation is a more efficient way to multiply certain kinds of plants because it
produces a larger plant faster than one raised from seed and it avoids seed dormancy. New varieties
can be developed by grafting, which combines the favourable characteristics of two existing
varieties.
Cutting: cutting is a method of propagation where a vegetative structure is removed from a
parent plant and grown as a new individual. Cuttings are successfully used to propagate
herbaceous plants, but can be used on woody plants with the use of hormones that promote
root growth.
Grafting: grafting is a procedure by which the structures of two or more plants are joined.
Typically a twig section (scion) from one plant is joined to the shoot of another (the
rootstock). Grafting is used for many fruit and landscape trees because it avoids juvenility,
and the special properties of the rootstock and the scion are able to be incorporated into
the same plant.
Plant tissue culture
Plant tissue culture, or micropropagation, is a method used for cloning plants. It is widely used for
the rapid multiplication of commercially important plant species with superior genotypes, as well as
in the recovery programmes for endangered plant species. Plant productivity and quality may be
rapidly improved, and resistance to disease, pollutants and insects increased. Continued culture of a
limited number of cloned varieties leads to a change in the genetic composition of the population
(genetic variation is reduced). New genetic stock may be introduced into cloned lines periodically to
prevent this reduction in genetic diversity.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
Micropropagation is possible because differentiated plant cells have the potential to give rise to all
the cells of an adult plant. It has considerable advantages over traditional methods of plant
propagation, but it is very labour intensive. In addition, the optimal conditions for growth and
regeneration must be determined and plants propagated in this way may be genetically unstable or
infertile, with chromosomes structurally altered or in unusual numbers. The success of tissue
culture is affected by factors such as selection of explant material, the composition of the culturing
media, plant hormone levels, lighting and temperature.
1. Stock plants are kept as free from pests and pathogens as possible.
2. Small pieces are cut (excised) from the plant. These pieces, called explants, may be stem
tissue with nodes, flower buds, leaves or tiny sections of shoot tip meristems.
3. The surfaces of explants are sterilised using solutions such as sodium hypochlorite.
4. The explants are transferred to a culture vessel under sterile conditions.
5. Incubation of culture vessels:
Duration: 3-9 weeks
Temperature: 15-30°C
Light regime: 10-14 hours per day
NOTE: Different kinds of hormones in culture media produce different growth responses. By
changing the relative levels of several plant hormones, the formation of callus, roots and
shoots can be initiated.
6. An undifferentiated mass of cells known as a callus develops.
Growth medium: contains nutrients and growth regulators (plant hormones such as auxins,
gibberellins and cytokinins) set in an agar gel.
7. New shoots that develop are removed from the explant and placed on new culture medium.
The process is repeated every few weeks so that a few plants can give rise to millions of
plants.
8. Tissue culture plants must be acclimatised in special glasshouses before they can be planted
outside.
9. Plant cell culture: if the callus is suspended in a liquid nutrient medium and broken up
mechanically into individual cells it forms a plant cell culture that can be maintained
indefinitely.
Advantages of tissue culture
Possible to create large numbers of clones from a single seed or explant.
Selection of desirable traits is possible directly from the culturing setup (in vitro), decreasing
the amount of space required for field trials.
Reproduction of plants is possible without having to wait for the onset of seed production.
Rapid propagation is possible for species that have long generation times, low levels of seed
production, or seeds that do not readily germinate.
Enables the preservation of pollen and cell collections from which from which plants may be
propagated (like a seed bank).
Allows the international exchange of sterilised plant materials (eliminating the need for
quarantine).
Helps to eliminate plant diseases through careful stock selection and sterile techniques
during propagation.
Overcomes seasonal restrictions for germination.
Enables cold storage of large numbers of viable plants in a small space.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
Cloning animals
Cloning by embryo splitting
Livestock breeds frequently produce only one individual per pregnancy and all individuals in a herd
will have different traits. Cloning (by embryo splitting or other means) makes it possible to produce
high value herds with identical traits more quickly. This technique also has applications in the
medical field e.g. in the cloning of embryonic stem cells. Such applications demonstrate the
advances made recently in cloning technology. Some of the most ambitious medical projects now
being considered involve the production of universal human donor cells. Scientists know how to
isolate undifferentiated stem cells from early embryos in mice. They are also learning how to force
stem cells to differentiate into different tissues. Such techniques may make it possible to
manufacture cells or replace tissues damaged by illness e.g. muscular dystrophy or diabetes.
Individually matched stem cells could be made by transferring the nucleus from one of the patient’s
cells into a human egg to create an embryo. The embryo would be allowed to develop only to a
stage where stem cells could be separated and cultured from it. Although the embryo would consist
of only a few hundred cells, there would be many ethical issues raised by this technique.
Egg cells are removed from an animal and fertilised in a Petri dish.
At the first stage of development, one of these fertilised eggs divides into two. The cloning
process (forming the twin) begins here.
The zona pellucida is removed with an enzyme and the two cells are separated.
An artificial zona is added, allowing development to proceed.
The cells continue to divide, forming genetically identical embryos. These are implanted into
surrogates.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
Cloning by nuclear transfer
Clones are genetically identical individuals produced from one parent. Cloning is not new; it has
been used in plant breeding for years. In recent years clones have been produced from both
embryonic and non-embryonic cells using standard nuclear transfer techniques. In 2004, Australian
genetic researchers successfully cloned a cow (called Brandy) using serial nuclear transfer (SNT)
which involves an extra round of nuclear transfer to improve the reprogramming of the fused donor
cells. In animal reproductive technology, cloning has facilitated the rapid production of genetically
superior stock. These animals may then be dispersed among commercial herds. The primary focus
of the new cloning technologies is to provide an economically viable way to rapidly produce
transgenic animals with very precise genetic modification.
Creating Dolly using standard nuclear transfer
Dolly, the Finn Dorset lamb born at the Roslin Institute (near Edinburgh) in July 1996, was the first
mammal to be cloned from non-embryonic cells. Nuclear transfer has been used successfully to
clone cells from embryonic tissue, but Dolly was created from a fully differentiated udder cell from a
six year old ewe. This cell was made quiescent and then ‘tricked’ into re-entering an embryonic
state. Dolly’s birth was a breakthrough, because it showed that the processes leading to cell
specialisation are not irreversible; even specialised cells can be ‘reprogrammed’ into an embryonic
state.
While cloning seems relatively easy to achieve using this method, Dolly’s early death has raised
concerns that the techniques could have caused premature ageing. Although there is, as yet, no
evidence for this, the long term viability of animals cloned from non-embryonic cells has still to be
established.
Dolly the sheep was euthanased on February 14th, 2003 after examinations showed she had
developed progressive lung disease. Dolly was six years old; half the normal life expectancy of
sheep. A post-mortem examination showed that she succumbed to a viral infection, not uncommon
in older sheep, especially those housed inside. Despite the concerns of some scientists, there is no
evidence that cloning was a factor in Dolly contracting the disease.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
Donor cells taken from udder: cells from the udder of a Finn Dorset ewe were cultured in
low nutrient medium for a week. The nutrient deprived cells stopped dividing, switched off
their active genes, and became dormant.
Unfertilised egg has nucleus removed: in preparation for the nuclear transfer, an
unfertilised egg was taken from a Scottish Blackface ewe. Using micromanipulation
techniques, the nucleus containing the DNA, was removed. This left a recipient egg cell with
no nucleus, but an intact cytoplasm and the cellular machinery for producing an embryo.
Cells are fused: the two cells (the dormant donor cell and the recipient egg cell) were placed
next to each other and a gentle electric pulse causes them to fuse together (like soap
bubbles).
Cell division is triggered: a second electric pulse triggers cellular activity and cell division,
effectively jump-starting the cell into production of an embryo. This reaction can also be
triggered by chemical means.
After six days, the resulting embryo was surgically implanted into the uterus of the surrogate
mother; another Scottish Blackface ewe. Of the hundreds of reconstructed eggs, only 29
successfully formed embryos, and only one Dolly survived to birth.
Birth: after a gestation of 148 days, the pregnant blackface ewe gave birth to Dolly, the Finn
Dorset lamb that is genetically identical to the original donor.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
1. Human populations have herded cattle for milk for around 9000 years. Artificial
selection over this time has resulted in the modern dairy cow.
(a) State three phenotypic traits (characteristics) that have been selected for in
dairy cows.
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(b) Fig. 1.1 shows the pattern of variation of a phenotypic trait in a herd of dairy
cows. The shaded part of the graph indicates those cows that are chosen to
breed. Draw, on Fig. 1.1, a second curve to show the pattern of variation in
the next generation.
(c) In recent years, artificial selection of dairy cows has been helped by modern
reproductive technology.
Name two modern techniques or procedures that can be used in the
selective breeding of dairy cows.
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(d) Lactase is an enzyme that is necessary to digest lactose sugar in milk. In
some parts of the world, animals are not farmed for milk and no dairy
products are eaten. Adult humans that are native to these parts of the
world do not produce lactase.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
In areas where animals are farmed for milk, native adult humans do not
produce lactase. In these populations, a new allele has arisen by gene
mutation.
State what is meant by gene mutation.
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(e) Over time, the frequency of this new allele increased in the gene pool of the
human populations, whose diet included milk.
Name the process by which this increase occurred.
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(f) All human babies produce the enzyme lactase. The genetic change that
allows adults to produce this enzyme is thought to involve a mutation in a
regulatory gene. This mutation causes the structural gene to be expressed
in adults.
Distinguish between the terms ‘regulatory gene’ and ‘structural gene’.
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(g) Adult humans who cannot produce the enzyme lactase are described as
lactose intolerant and cannot drink milk without experiencing health
problems. However, lactose intolerant people can safely eat yoghurt.
Yoghurt is produced from milk that is fermented by bacteria. These bacteria
perform anaerobic respiration, using carbohydrate as their respiratory
substrate. Suggest why yoghurt is a suitable food for lactose-intolerant
people.
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
(h) The control of the expression of the lac operon genes, which allow uptake
and digestion of lactose in the bacterium Escherichia coli, is well known.
Fig. 1.2 shows the arrangement of the elements of the lac operon.
Describe how genes Z and Y are switched on in bacteria that are moved to a
nutrient medium that contains lactose.
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2. Molecular evidence has shown that all specimens of the English Elm tree,
Ulmus procera, form a genetically isolated clone. English elms developed from
a variety of elm brought to Britain from Rome in the first century A.D.
Although English Elm trees make pollen, they rarely produce seeds. Instead
they spread by developing structures known as suckers from their roots. Each
sucker can grow into a new tree.
This tendency of elms to create suckers has been exploited by humans, who
have separated the suckers, with roots attached, and used them to plant
hedges and establish new woodlands.
(a) Suggest a technique that could be used to provide molecular evidence that
all English Elm trees form a clone.
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
(b) State why the English Elm clone is genetically isolated from other varieties
of elm.
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(c) State the name given to the process in which plants reproduce asexually by
means such as suckers.
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(d) In 1967, a new, virulent strain of elm disease fungus arrived in Great Britain
on imported timber. Beetles that lived under the bark of elm trees spread
the fungus.
The saws used to cut down dead branches were not sterilised after use.
When the saws were used to prune healthy trees, these trees became
infected. Approximately 25 million elm trees, most of the English
population, died within a few years of the arrival of this fungus.
Explain why there was such a rapid loss of elm trees in Britain as a result of
this elm disease.
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
(e) Elm trees respond to fungal infection by plugging their xylem vessels. The
leaves on the upper branches of the tree then turn yellow and die. When
most of the branches have lost their leaves and died, the roots are
weakened and my also die.
Explain why the plugging of xylem vessels will result in the leaves of the
upper branches turning yellow.
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(f) Explain why the loss of leaves from the tree may result in the death of the
tree’s roots.
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(g) Many ornamental plants for gardens can be cloned by tissue culture.
Describe the process of cloning plants by tissue culture.
In your answer, you should use appropriate technical terms, spelled correctly
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
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3. Rhubarb, Rheum x hybridum, is a plant that is grown for its edible stems. In
spring, the stems and leaves grow from fleshy roots which have survived the
winter underground.
Growers have developed many new varieties of rhubarb by growing plants
from seed, choosing the best young plants and then asexually reproducing
them.
Seeds are produced by sexual reproduction and the rhubarb plants that grow
from seed show variation in characteristics such as stem colour, dormancy
period and the concentration of oxalic acid in their leaves.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
(a) Outline the events that lead to genetic variation in gametes and in the
plants grown from seed.
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(b) Traditionally rhubarb plants have been produced by vegetative propagation.
The best young rhubarb plants are allowed to grow for three seasons until
their underground root systems are large enough. Then they are dug up in
winter, the roots are cut into pieces and the pieces are replanted. Each
piece is then able to grow into a new rhubarb plant that is identical to the
parent.
State the biotechnological term for this type of vegetative propagation.
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
(c) A gardener wished to multiply his rhubarb plants using the traditional
method, but he discovered that his plants were infected by a virus.
Name the modern technique which allows commercial growers to produce
large numbers of genetically identical plants that are also virus-free.
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(d) Rhubarb plants must spend seven to nine weeks at a temperature below
3°C in order to break their winter dormancy and allow them to start growing
stems and leaves again.
The length of the cold period that is required depends on the variety of
rhubarb.
In the variety ‘Timperley Early’, the length of the cold period is shorter, so
the plants grow and produce a crop earlier in the year than the variety
‘Victoria’.
Suggest two ways in which the varieties may differ from one another
biochemically to account for the difference in the length of the cold period
required by each.
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(e) Rhubarb leaves contain oxalic acid, a relatively strong acid which is soluble
in water and in alcohol. High concentrations of oxalic acid makes rhubarb
leaves poisonous to humans and other animals.
The amount of oxalic acid in the leaves varies according to the variety of
rhubarb, the age of the plant and environmental factors.
Suggest and plan an experiment to compare how the variety of rhubarb
affects the amount of oxalic acid in rhubarb leaves.
Include in your plan:
 the variables that you could control
 an outline of the experimental procedure you would use
 any measurements that you would make
In your answer, you should use appropriate technical terms, spelled correctly
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
(f) As rhubarb leaves are poisonous, they are cut off when the stems are
harvested and may be left to decompose on the compost heap.
Outline the role of decomposers in the decomposition of leaves.
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(g) An early harvest of rhubarb stems can be obtained by placing an upturned
bin over the root when it comes out of dormancy, so the emerging shoots
are kept in the dark. The shoots then grow more quickly to a height suitable
for picking.
Use your knowledge of plant growth regulators (plant hormones) to
suggest why shoots kept in the dark grow taller than those left in the light.
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
Biotechnology
Learning objectives:
 State that biotechnology is the industrial use of living organisms (or parts of living
organisms) to produce food, drugs or other products;
 Explain why microorganisms are often used in biotechnological processes;
 Describe, with the aid of diagrams, and explain the standard growth curve of a
microorganism in a closed culture;
 Describe how enzymes can be immobilised;
 Explain why immobilised enzymes are used in large-scale production;
 Compare and contrast the processes of continuous culture and batch culture;
 Describe the differences between primary and secondary metabolites;
 Explain the importance of manipulating the growing conditions in a fermentation vessel in
order to maximise the yield of product required;
 Explain the importance of asepsis in the manipulation of microorganisms;
Key definitions:
Compile a glossary by writing your own definitions for the following key terms related to the
learning objectives above.
Key term
biotechnology
culture
primary metabolites
secondary metabolites
batch
continuous
asepsis
aseptic technique
Definition
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
The growth curve
A small number of organisms placed in a fresh ‘closed culture’ environment will undergo population
growth in a very predictable, standard way (see diagram above). A closed culture refers to the
growth of microorganisms in an environment where all conditions are fixed and contained. No new
materials are added and no waste products or organisms removed.
Lag phase: organisms are adjusting to the surrounding conditions. This may mean taking in
water, cell expansion, activating specific genes and synthesising specific enzymes. The cells
are active but not reproducing so population remains fairly constant. The length of this
period depends on the growing conditions.
Log (exponential) phase: the population size doubles each generation as every individual
has enough space and nutrients to reproduce. In some bacteria, for example, the population
can double every 20-30 minutes in these conditions. The length of this phase depends on
how quickly the organisms reproduce and take up the available nutrients and space.
Stationary phase: nutrient levels decrease and waste products like carbon dioxide and other
metabolites build up. Individual organisms die at the same rate at which new individuals are
being produced. Note: in an open system, this would be the carrying capacity of the
environment.
Decline or death phase: nutrient exhaustion and increased levels of toxic waste products
and metabolites lead to the death rate increasing above the reproduction rate. Eventually,
all organisms will die in a closed system.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
Industrial scale fermenters
An industrial-scale fermenter is essentially a huge tank which may have a capacity of tens of
thousands of litres. The growing conditions in it can be manipulated and controlled in order to
ensure the best possible yield of the product.
The precise growing conditions depend on the microorganisms being cultured, and on whether the
process is designed to produce a primary or secondary metabolite. They are:
temperature – too hot and enzymes will be denatured; too cool and growth will be slowed;
type and time of addition of nutrient – growth of microorganisms requires a nutrient supply,
including sources of carbon, nitrogen and any essential vitamins and minerals. The timing of
nutrient addition can be manipulated, depending on whether the process is designed to
produce a primary or secondary metabolite.
oxygen concentration – most commercial applications use the growth of organisms under
aerobic conditions, so sufficient oxygen must be made available. A lack of oxygen will lead to
the unwanted products of anaerobic respiration and a reduction in growth rate.
pH – changes in pH within the fermentation tank can reduce the activity of enzymes and so
reduce growth rates;
Such large cultures need large ‘starter’ populations of the microorganism. These are obtained by
taking a pure culture and growing it in sterile nutrient broth.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
A batch culture, where the microorganism starter population is mixed with a specific quantity of
nutrient solution, then allowed to grow for a fixed period with no further nutrient added. At the end
of this period, the products are removed and the fermentation tank is emptied. Penicillin is
produced using batch culture of Penicillium fungus.
A continuous culture, where nutrients are added to the fermentation tank and products removed
from the fermentation tank at regular intervals – or even, as the name suggests, continuously.
Human hormones such as insulin are produced from continuous culture of genetically modified
Escherichia coli bacteria.
The nutrient medium in which the microorganisms grow could also support the growth of many
unwanted microorganisms. Any unwanted microorganism is called a contaminant. Unwanted
microorganisms:
compete with the culture microorganisms for nutrients and space;
reduce the yield of useful products from the culture microorganisms;
may cause spoilage of the product;
may produce toxic chemicals;
may destroy the culture microorganisms and their products;
in processes where foods or medicinal chemicals are being produced, contamination means that all
products must be considered unsafe and so must be discarded.
The term aseptic technique refers to the measures taken to ensure asepsis, that is, that
contamination of the culture does not occur at any point from isolation of the initial culture,
through scaling up, fermentation and product harvesting.
Immobilising enzymes
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
Depending on the way in which the desired end-product is produced, enzymes may be used as
crude whole cell preparations or as cell-free enzyme extracts. Whole cell preparations are cost
effective, and appropriate when the processes involved in production of the end product are
complex, as in waste treatment and the production of semi-synthetic antibiotics. Cell free enzyme
extracts are more expensive to produce, but can be a more efficient option overall. To reduce costs
and improve the efficiency of product production, enzymes are sometimes immobilised within a
matrix of some kind and the reactants are passed over them.
Advantages of immobilised enzymes
The enzymes can be used repeatedly and recovered easily (this reduces costs).
The enzyme-free end-product is easily harvested.
The enzymes are more stable due to the protection of a matrix.
The life of some enzymes e.g. proteases, is extended by immobilisation.
Disadvantages of immobilised enzymes
The entrapment process may reduce the enzyme activity (more enzyme will be needed).
Some methods offering high stability(e.g. covalent bonding) are harder to achieve.
Immobilisation can be costly.
Methods of enzyme immobilisation
Micro-encapsulation: the enzyme is held within a membrane, or within alginate or
polyacrylamide capsules.
Lattice entrapment: enzyme is trapped in a gel lattice e.g. silica gel. The substrate and
reaction products diffuse in and out of the matrix.
Covalent attachment: enzyme is covalently bonded to a solid surface e.g. collagen or a
synthetic polymer.
Direct cross-linking: glutaraldehyde is used to cross-link the enzymes. They then precipitate
out and are immobilised without support.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
1. The antibiotic penicillin is produced by batch culture of the fungus Penicillium
chrysogenum.
(a) Fig. 4.1 shows the concentration of penicillin, lactose and ammonia as well
as the fungal biomass over time when penicillin is being produced by batch
culture.
With reference to Fig. 4.1, describe and explain the changes in
concentration of lactose and ammonia.
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
explanation ___________________________________________________
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(b) A student incorrectly suggested that penicillin might be produced by
continuous culture fermentation instead of by batch culture.
Suggest how the curves for lactose, ammonia and biomass on Fig. 4.1 might
differ in continuous culture.
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(c) A second student said that continuous culture would not be suitable, as
penicillin is a secondary metabolite.
What evidence is there in Fig. 4.1 that penicillin is a secondary metabolite?
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(d) Explain the importance of maintaining aseptic conditions in manufacturing
penicillin by fermentation.
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
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(e) State three physical or chemical factors within the fermenter, other than
nutrient levels, that need to be monitored and controlled.
For each factor, explain why it must be controlled.
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2. Microorganisms are often used in biotechnological processes.
Fig. 6.1 shows the standard growth curve for a culture of bacteria.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
(a) Identify the phases labelled P, Q and R in Fig. 6.1.
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(b) Metabolic processes taking place in bacteria grown in a batch culture
produce primary and secondary metabolites.
Explain what is meant by a primary metabolite.
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(c) With reference to the information in Fig. 6.1, state the phase or phases, P,
Q, R or S, when:
 primary metabolite production is at its highest rate:
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 most secondary metabolites are produced:
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 the concentration of secondary metabolites reach a maximum:
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(d) Some aerobic recombinant bacteria were grown in a fermenter. They
synthesised the protein human growth hormone (HGH).
Suggest two ways in which named factors inside the fermenter could be
adjusted in order to maximise the yield of HGH.
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
2 ____________________________________________________________
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(e) HGH made in this way is given by injection to some children who have a
genetic mutation. The mutation means that they do not produce enough
HGH to enable them to grow at the normal rate.
Explain why injecting recombinant HGH in this way is not an example of
gene therapy.
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3. Microorganisms include fungi and bacteria. Fungi are eukaryotes. Bacteria are
prokaryotes.
(a) Describe one distinctive feature of the cell structure of each of these
microorganisms.
fungal cell _____________________________________________________
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bacterial cell ___________________________________________________
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
(b) The use of microorganisms in biotechnology involves aseptic technique.
Aseptic technique prevents pathogens contaminating products.
What is meant by the term pathogen?
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(c) State what is meant by biotechnology using suitable examples from
different areas of biotechnology and explain why microorganisms are used
in biotechnological processes.
In your answer, you should give examples of products and the microorganisms
used to make them, as well as the advantages of using microorganisms.
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4. Fig. 5.1 is a crossword that should contain five words relating to the use of
microorganisms by humans.
Use the clues below to write the five appropriate words in the correct spaces
on Fig. 5.1.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
5. Enzyme immobilisation is an important technique in biotechnology.
Figs 1.1 and 1.2 show two stages in making a bioreactor to remove lactose
sugar from milk.
In Fig. 1.1 the enzyme lactase is immobilised in alginate beads.
In Fig. 1.2 milk flows over the beads and the lactose sugar is hydrolysed to two
other sugars.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
(a) Suggest and explain how you might use the method shown in Fig. 1.2 to
obtain milk that was lactose-free.
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(b) Fig. 1.1 and Fig. 1.2 show that alginate beads can be used to immobilise an
enzyme.
Outline two other methods of immobilising enzymes.
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(c) Enzyme immobilisation is used in the biotechnology industry for the largescale production of materials.
Discuss the benefits of using immobilised enzymes for large-scale
production.
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
Genomes and gene technologies
Learning objectives:
 Outline the steps involved in sequencing the genome of an organism;
 Outline how gene sequencing allows for genome-wide comparisons between individuals and
between species;
 Define the term recombinant DNA;
 Explain that genetic engineering involves the extraction of genes from one organism, or the
manufacture of genes, in order to place them in another organism (often of a different
species) such that the receiving organism expresses the gene product;
 Describe how sections of DNA containing a desired gene can be extracted from a donor
organism using restriction enzymes;
 Outline how DNA fragments can be separated by size using electrophoresis;
 Describe how DNA probes can be used to identify fragments containing specific sequences;
 Outline how the polymerase chain reaction (PCR) can be used to make multiple copies of
DNA fragments;
 Explain how isolated DNA fragments can be placed in plasmids, with reference to the role of
ligase;
 State other vectors into which fragments of DNA may be incorporated;
 Explain how plasmids may be taken up by bacterial cells in order to produce a transgenic
microorganism that can express a desired gene product;
 Describe the advantage to microorganisms of the capacity to take up plasmid DNA from the
environment;
 Outline how genetic markers in plasmids can be used to identify the bacteria that have taken
up a recombinant plasmid;
 Outline the process involved in the genetic engineering of bacteria to produce human
insulin;
 Outline the process involved in the genetic engineering of ‘Golden Rice TM’;
 Outline how animals can be genetically engineered for xenotransplantation;
 Explain the term gene therapy;
 Explain the difference between somatic cell gene therapy and germ line cell gene therapy;
 Discuss the ethical concerns raised by the genetic manipulation of animals (including
humans), plants and microorganisms;
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
Key definitions:
Compile a glossary by writing your own definitions for the following key terms related to the
learning objectives above.
Key term
downstream processing
immobilisation
genomics
DNA profiling (genetic
fingerprinting)
genomic sequencing
genetic engineering
gene therapy
coding DNA
non-coding DNA
clone libraries
electrophoresis
annealing
primers
amplification
recombinant DNA
technology
Definition
SACKVILLE SCIENCE DEPARTMENT
Key term
vector
restriction enzymes
restriction site
sticky end
DNA ligase
recombinant DNA
transgenic
recombinant plasmid
replica plating
gene therapy
genetically modified
organism (GMO)
liposomes
xenotransplantation
Definition
A2 BIOLOGY
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
Gel electrophoresis
Gel electrophoresis is a method that separates large molecules (including nucleic acids or proteins)
on the basis of size, electric charge, and other physical properties. Such molecules possess a slight
electric charge. To prepare DNA for gel electrophoresis the DNA is often cut up into smaller pieces.
This is done by mixing DNA with restriction enzymes in controlled conditions for about an hour.
Called restriction digestion, it produces a range of DNA fragments of different lengths. During
electrophoresis, molecules are forced to move through the pores of a gel (a jelly-like material),
when the electrical current is applied. Active electrodes at each end of the gel provide the driving
force.
The electrical current from one electrode repels the molecules while the other electrode
simultaneously attracts the molecules. The frictional force of the gel resists the flow of the
molecules, separating them by size. Their rate of migration through the gel depends on the strength
of the electric field, size and shape of the molecules, and on the ionic strength and temperature of
the buffer in which the molecules are moving. After staining, the separated molecules in each lane
can be seen as a series of bands spread from one end of the gel to the other.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
The polymerase chain reaction – PCR
Many procedures in DNA technology (such as DNA sequencing and DNA profiling) requires
substantial amounts of DNA to work with. Some samples, such as those from a crime scene or
fragments of DNA from a long extinct organism, may be difficult to get in any quantity. The diagram
below shows the laboratory process called polymerase chain reaction (PCR).
Using this technique, vast quantities of DNA identical to trace samples can be created. This process
is often termed DNA amplification. PCR can be used to make billions of copies in only a few hours.
It is repeated for about 25 cycles.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
1. A DNA sample (called target DNA) is obtained. It is denatured (DNA strands are separated)
by heating at 98°C for 5 minutes.
2. The sample is cooled to 60°C. Primers are annealed (bonded) to each DNA strand. In PCR,
the primers are short strands of DNA; they provide the starting sequence for DNA extension.
3. Free nucleotides and the enzyme DNA polymerase are added. DNA polymerase binds to the
primers and, using the free nucleotides, synthesises complementary strands of DNA.
4. After one cycle, there are now two copies of the original DNA.
5. This is repeated for about 25 cycles – repeat cycle of heating and cooling until enough copies
of the target DNA have been produced.
Restriction enzymes
One of the essential tools of genetic engineering is a group of special restriction enzymes (also
known as restriction endonucleases). These have the ability to cut DNA molecules at very precise
sequences of 4 to 8 base pairs called recognition sites. These enzymes are the ‘molecular scalpels’
that allow genetic engineers to cut up DNA in a controlled way. Although first isolated in 1970,
these enzymes were discovered earlier in many bacteria. The purified forms of these bacterial
restriction enzymes are used today as tools to cut DNA.
Enzymes are named according to the bacterial species from which they were first isolated. By using
a ‘tool kit’ of over 400 restriction enzymes recognising about 100 recognition sites, genetic
engineers can isolate, sequence, and manipulate individual genes derived from any type of
organism. The sites at which the fragments of DNA are cut may result in overhanging ‘sticky ends’ or
non-overhanging ‘blunt ends’. Pieces may later be joined together using an enzyme called DNA
ligase in a process called ligation.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
‘Sticky end’ restriction enzymes
 A restriction enzyme cuts the double-stranded DNA molecule at its specific recognition site.
 The cuts produce a DNA fragment with two sticky ends (ends with exposed nucleotide bases
at each end). The piece it is removed from is also left with sticky ends.
Restriction enzymes may cut DNA leaving an overhang or sticky end, without its complementary
sequence opposite. DNA cut in such a way is able to be joined to other exposed end fragments of
DNA with matching sticky ends. Such joins are specific to their recognition sites.
‘Blunt end’ restriction enzymes
 A restriction enzyme cuts the double-stranded DNA molecule at its specific recognition site.
 The cuts produce a DNA fragment with two blunt ends (ends with no exposed nucleotide
bases at each end). The piece it is removed from is also left with blunt ends.
It is possible to use restriction enzymes that cut leaving no overhang. DNA cut in such a way is able
to be joined to any other blunt end fragment, but tends to be non-specific because there are no
sticky ends as recognition sites.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
Ligation
DNA fragments produced using restriction enzymes may be reassembled by a process called
ligation. Pieces are joined together using an enzyme called DNA ligase. DNA of different origins
produced in this way is called recombinant DNA (because it is DNA that has been recombined from
different sources). The combined techniques of using restriction enzymes and ligation are the basic
tools of genetic engineering (also known as recombinant DNA technology).
Creating a recombinant DNA plasmid
If two pieces of DNA are cut by the same restriction enzyme, they will produce fragments
with matching sticky ends (ends with exposed nucleotide bases at each end).
When two such matching sticky ends come together, they can join by base-pairing. This
process is called annealing. This can allow DNA fragments from a different source, perhaps a
plasmid, to be joined to the DNA fragment.
The joined fragments will usually form either a linear molecule or a circular one, as shown in
the diagram above for a plasmid. However, other combinations of fragments can occur.
The fragments of DNA are joined together by the enzyme DNA ligase, producing a molecule
of recombinant DNA.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
Gene cloning using plasmids
Gene cloning is a process of making large quantities of a desired piece of DNA once it has been
isolated. The purpose of this process is often to yield large quantities of either an individual gene or
its protein product when the gene is expressed. Methods have been developed to insert a DNA
fragment of interest e.g. a human gene for a desired protein into the DNA of a vector, resulting in a
recombinant DNA molecule or molecular clone.
A vector is a self-replicating DNA molecule e.g. plasmid or viral DNA used to transmit a gene from
one organism into another. To be useful, all vectors must be able to replicate inside their host
organism, they must have one or more sites at which a restriction enzyme can cut, and they must
have some kind of genetic marker that allows them to be easily identified. Organisms such as
bacteria, viruses and yeasts have DNA that behaves in this way.
Large quantities of the desired gene can be obtained if the recombinant molecule is allowed to
replicate in an appropriate host. The host e.g. bacterium may then go on to express the gene and
produce the desired protein. Two types of vector are plasmids and bacteriophages (viruses that
infect bacteria).
Cloning a human gene
1. A gene of interest (DNA fragment) is isolated from cells that have been grown in laboratory
culture.
2. An appropriate plasmid vector is isolated from a bacterial cell.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
3. Both the human DNA and the plasmid are treated with the same restriction enzyme to
produce identical sticky ends.
4. The restriction enzyme cuts the plasmid DNA at its single recognition sequence, disrupting
the tetracycline resistance gene.
Antibiotic resistant marker genes may be used to identify the bacteria that have taken up
the foreign e.g. human DNA. The plasmid used often carries two genes that provide the
bacteria with resistance to the antibiotics ampicillin and tetracycline. Without this plasmid,
the bacteria have no antibiotic resistance genes.
A single restriction enzyme recognition sequence lies within the tetracycline resistance gene.
A foreign gene, spliced into this position, will disrupt the tetracycline resistance gene,
leaving the bacteria vulnerable to this antibiotic. It is possible to identify the bacteria that
successfully take up the recombinant plasmid by growing the bacteria on media containing
ampicillin, and transferring colonies to media with both antibiotics.
5. The DNA fragments are mixed together and the complementary sticky ends are attracted to
each other by base-pairing. The enzyme DNA ligase is added to bond the sticky ends.
6. The recombinant plasmid, or molecular clone, is introduced into a bacterial cell by adding
the DNA to a bacterial culture. Under the right conditions, some bacteria will take up the
plasmid from solution by the process of transformation.
Preparation of the clone up to this point
Cloning the gene starts here
7. The actual gene cloning process (making multiple copies of the human gene) occurs when
the bacterium with the recombinant plasmid is allowed to reproduce.
8. Colonies of bacteria that carry the recombinant plasmid can be identified by the fact that
they are resistant to ampicillin but sensitive to tetracycline.
Most often today, another gene plays the role of the tetracycline resistance gene, but the
principle remains the same; the inserted DNA disrupts the activity of the gene whose activity
is easily determined.
Preparing a gene for cloning
Double stranded DNA of a gene from a eukaryotic organism e.g. human containing introns.
As a normal part of the cell process of gene expression, transcription creates a primary RNA
molecule.
The introns are removed by splicing enzymes to form a mature mRNA (now excluding the
introns) that codes for the making of a single protein.
The introns are removed as it makes the DNA (the human gene) shorter, and therefore
easier to insert into plasmids. It also allows the bacterial enzymes to properly translate the
human gene from the ‘reassembled’ DNA (bacterial enzymes cannot cope with the introns).
Reverse transcriptase is added which synthesises a single stranded DNA molecule
complementary to the mRNA.
The second DNA strand is made by using the first as a template, and adding the enzyme DNA
polymerase.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
Gene therapy
Gene therapy refers to the application of gene technology to correct or replace defective genes. It
was first envisioned as a treatment, or even a cure, for genetic disorders, but it could also be used
to treat a wide range of diseases, including those that resist conventional treatments. Gene therapy
may operate by providing a correctly working version of the faulty gene or by adding a novel gene
to perform a corrective role. In other cases, gene expression may be blocked in order to control
cellular (or viral) activity. About two thirds of currently approved gene therapy procedures are
targeting cancer, about one quarter aim to treat genetic disorders, such as cystic fibrosis, and the
remainder are attempting to provide relief for infectious diseases.
Gene therapy requires a gene delivery system; a way to transfer the gene to the patient’s cells. This
may be achieved using an infectious agent such as a virus; a technique called transfection. A
promising development has been the recent approval for gene therapy to be used in treating
tumours in cancer patients. Severe combined immune deficiency syndrome (SCIDS) has also shown
improvement after gene therapy. Infants treated for this inherited, normally lethal condition have
become healthy young adults.
Gene therapy involving somatic cells may be therapeutic, but the genetic changes are not inherited.
The transfection of stem cells, rather than mature somatic cells, achieves a longer persistence of
therapy in patients. In the future, the introduction of corrective genes into germline cells will
enable genetic corrections to be inherited.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
Gene delivery using extracted cells
1. Body cells from patient are isolated. These cells are homozygous for the defective allele.
2. A copy of the normal human allele is inserted into the DNA of a viral vector using restriction
enzymes and DNA ligase.
3. Isolated body (somatic) cells are infected with virus containing the recombinant DNA.
4. Viral DNA carrying the normal allele inserts itself into the patient’s somatic cell
chromosome.
5. Somatic cells containing the introduced normal allele are cultured in a nutrient medium. In
this way, the desired gene is cloned.
6. Cultured cells are injected into the patient.
7. Symptoms are relieved in the patient by the expression of the normal allele.
By using the techniques of recombinant DNA technology, medical researchers insert a functional
gene into a patient’s body (somatic) cells. This should make the patient capable of producing the
protein encoded in that allele.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
1. Fig. 1.1 is a flow diagram showing the main stages involved in making
cheese. The starting material is milk, which contains the protein, casein.
(a) Explain why making cheese can be described as a biotechnological process.
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
(b) Suggest two benefits of the pasteurisation stage.
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(c) Rennin is a protein that can be obtained from the stomach lining of calves. It
is used in the cheese-making process in the ratio one part rennin to 10 000
parts milk.
Suggest what type of protein rennin is and explain how a very small quantity
of rennin is able to convert a large quantity of milk.
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(d) Rennin could, in theory, be immobilised for use in cheese-making.
List two potential advantages of this.
1 ____________________________________________________________
2 _________________________________________________________ [2]
(e) Rennin can now be made by genetically modified microorganisms.
Outline the process by which bacteria can be genetically modified to
produce rennin.
In your answer, you should make clear how the steps in the process are
sequenced.
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
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2. This question is about genetic engineering and the techniques used for
making multiple copies of genes (gene cloning).
(a) Genetic engineering uses the following:
A an enzyme that synthesises new DNA
B an enzyme that cuts DNA at specific sequences
C an enzyme that reseals cut ends of DNA
D small circular pieces of DNA found in bacteria; these pieces of DNA have
antibiotic resistance genes
E an enzyme found in some viruses with an RNA genome; this enzyme
converts RNA into DNA
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
Name A to E.
A ____________________________________________________________
B ____________________________________________________________
C ____________________________________________________________
D ____________________________________________________________
E _________________________________________________________ [5]
(b) Genes are cloned for a number of reasons. For example:
 one group of research scientists at a hospital wanted to sequence a
disease-causing mutation to learn more about a human disease; these
scientists started their research using white blood cells;
 another group of scientists at a biotechnology company wanted to clone
the insulin gene in order to manufacture its protein product to treat
diabetes; these scientists started their research using cells from the
pancreas;
Suggest and explain the biological reasons why the two groups each started
with a different cell.
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
(c) A gene can be cloned in vitro (in a test tube) by the polymerase chain
reaction (PCR). Alternatively, a gene can be cloned in vivo (in living cells) by
introducing the gene into bacterial host cells.
Table 5.1 identifies some of the key steps in each process.
Compare the two processes of gene cloning by explaining the advantages of
each.
In your answer, you should ensure that clear comparisons between the two
processes are made and explained.
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
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3. Describe the differences between: somatic cell gene therapy germ line cell
gene therapy
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SACKVILLE SCIENCE DEPARTMENT
4.

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A2 BIOLOGY
Genetic modification of organisms uses a ‘toolkit’ that includes:
enzymes that cut DNA
enzymes that join sections of DNA together
vectors that introduce DNA into new host cells
(a) Some of the enzymes and vectors that are important in genetic modification
are given an identifying letter in Table 4.1.
Select one correct letter from Table 4.1 to fit each of the following
statements:
An enzyme that cuts DNA
________
An enzyme that joins sections of DNA together
________
A vector to introduce foreign DNA into bacteria
________
A vector to introduce foreign DNA into plant cells
________
A vector to introduce foreign DNA into animal cells
________
[5]
(b) Discuss the potential benefits to mankind and the ethical concerns raised
by the following examples of genetically modified organisms:
 rice modified for increased vitamin A content (‘Golden RiceTM’)
 humans having somatic gene therapy treatment for a genetic disease
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
In your answer, you should give a balanced account of the benefits and concerns
for each example of genetic modification.
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
5. Transgenic goats, containing a gene from a spider that codes for spider web silk
protein, have been produced by genetic modification. The silk protein can be
harvested from the milk of the female transgenic goats.
Spider silk protein is lightweight but has very high tensile strength. It is used to
make items such as bullet-proof vests.
(a) A vector containing recombinant DNA is needed to produce transgenic
goats.
Define the term recombinant DNA.
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(b) Complete Table 3.1 by suggesting one example of a suitable vector for each
of the following applications of genetic modification.
[4]
(c) In order to make spider silk protein on a commercial basis, many transgenic
goats will be needed.
Outline the process by which an animal, such as the first transgenic goat,
may be cloned to produce a population.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
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(d) An alternative method for producing a population of more transgenic goats
is to breed the transgenic goat with normal goats.
Discuss the advantages and disadvantages of cloning the transgenic goat
compared with breeding the transgenic goat with normal goats.
advantages ____________________________________________________
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disadvantages __________________________________________________
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
6. A number of new techniques for manipulating cells and genomes are now
available, and it is hoped this manipulation will allow cures for diseases to be
developed.
(a) Five goals that scientists would like to achieve are described below and are
listed A to E:
A
producing large numbers of genetically identical ‘model’
transgenic mice that show symptoms of diabetes
B
growing a replacement kidney identically tissue-matched to an
individual patient
C
obtaining replacement hearts from transgenic pigs, partially
tissue-matched to humans
D
genetically manipulating cells of one adult to cure a genetic
disease in that individual
E
altering a prokaryotic pathogen for use as a vaccine
The names of the procedures corresponding to four of the five goals A to E
are written below.
Match the correct letters to the names. No letter should be used more
than once.
xenotransplantation
__________
somatic gene therapy
__________
non-reproductive cloning
__________
animal reproductive cloning__________
(b) Table 7.1 shows four different combinations of techniques used to achieve
goals A to E.
Write the letters A, B, C, D or E in the first column of the table to match
each goal to the appropriate combination of techniques needed to achieve
it.
Use each letter only once.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
7. The Oxford Botanic Garden was founded in 1621 to grow plants for the
teaching of medicine. Since that time it has seen many changes. When the
ideas of Linnaeus were adopted in the 18th Century, the plants were dug up and
re-planted in family groups according to his new system of taxonomy.
(a) Recently the plants have once again had to be reorganised:
 DNA sequencing techniques, together with cladistic analysis, have
provided a radical new view of plant evolutionary relationships.
 The same techniques have also improved the ability of researchers to
pinpoint new cures for diseases, by examining the closest relatives of
plants already known to have medicinal properties.
Comment on what the different arrangements of plants in the Oxford
Botanic Garden over time tell us about the nature of scientific knowledge.
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(b) Suggest two purposes of a plant collection in a modern botanic garden.
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
(c) DNA sequencing techniques have provided new information about plant
relationships.
Outline the roles of each of the following procedures in sequencing a
genome:
the polymerase chain reaction (PCR)
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electrophoresis
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digestion of DNA by restriction enzymes
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(d) Suggest why a genome has to be fragmented before sequencing
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
8. Table 5.1 lists some plants considered for genome sequencing by the ‘Floral
Genome Project’. The chromosome numbers and genome sizes in mega base
pairs (Mbp) are shown.
One Mbp is equal to 1 000 000 base pairs of DNA.
(a) The sequencing method that will be used is only able to sequence
fragments of DNA with a maximum length of 750 base pairs.
Calculate the minimum number of DNA fragments that would need to be
sequenced to read the genome of Amborella.
Show your working.
Answer = ____________________ [2]
(b) Monkey flower and blueberry belong to the same taxonomic group within
the plant kingdom. Only one pair was chosen for further sequencing work.
Using the data in Table 5.1, suggest reasons why monkey flower was
chosen instead of blueberry.
SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
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(c) Use your knowledge of the effects of polyploidy in bread wheat to suggest
one way in which the fruit of a hexaploid (6n) blueberry might differ in
appearance from that of a diploid (2n) blueberry.
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(d) DNA sequence information is most useful when used with the phylogenetic
(cladistic) approach to classification.
How does the phylogenetic approach to classifying species differ from the
biological species concept?
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9. Steroid hormones are one example of molecules that can switch genes on and
off in mammalian cells.
Other molecules involved in genetic control have been studied in eukaryotes
and prokaryotes.
Describe one other example of genes being switched on or being switched off
by a molecule that binds directly to DNA.
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SACKVILLE SCIENCE DEPARTMENT
A2 BIOLOGY
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