Biotechnology
Unit 3: Biotechnology
Student Materials
[HIGHER]
Margot McKerrell
abc
The Scottish Qualifications Authority regularly reviews
the arrangements for National Qualifications. Users of all
NQ support materials, whether published by LT Scotland
or others, are reminded that it is their responsibility to
check that the support materials correspond to the
requirements of the current arrangements.
Acknowledgement
Learning and Teaching Scotland gratefully acknowledge this contribution to the National
Qualifications support programme for Biotechnology. The advice of Jim Stafford is
acknowledged with thanks.
First published 2005
© Learning and Teaching Scotland 2005
This publication may be reproduced in whole or in part for educational purposes by
educational establishments in Scotland provided that no profit accrues at any stage.
ISBN 1 84399 073 3
© Learning and Teaching Scotland
CONTENTS
5
Introduction
Section 1:
Biotechnological processing
Large-scale cell and tissue culture production
Comparison of batch and continuous flow processes
Downstream processing
Enzymes in production
Production of transgenic organisms
New breeding techniques
7
9
14
16
25
30
32
Section 2:
Biotechnology applications
Agriculture and horticulture
Clinical and forensic medicine
Environment
35
36
37
45
Bibliography
47
Appendix:
51
Advice for problem-solving outcomes
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INTRODUCTION
Biotechnology is the use of micro-organisms, cells or cell products (such
as proteins) in industrial and commercial processes. It is sometimes
thought that biotechnology is a recent technology but, in fact, some
biotechnology processes have been in existence for many centuries –
micro-organisms are used to produce bread, beer and cheese. In this
unit, you will find out about some of the modern biotechnology
processes used by industry for a wide range of commercial products.
You will also learn about some of the current applications of
biotechnology in agriculture, horticulture, clinical medicine and forensic
medicine, such as crops with ‘built-in’ pest resistance, genetically
engineered vaccines and the use of monoclonal antibodies in home
pregnancy kits.
While this is a stand-alone unit, it is highly recommended that you
complete the other two units comprising Higher Biotechnology
(Microbiology (Higher) and Microbiological Techniques (Higher))
beforehand, as the underpinning knowledge from the other two units is
assumed in this one.
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BIOTECHNOLOGICAL PROCESSING
SECTION 1
Biotechnological processing
This section introduces you to the many different ways in which
products are made using biotechnology processes. Some products, such
as antibiotics, are made by micro-organisms grown in large culture
vessels called fermenters. Other products are made using microbial
enzymes that have been immobilised and contained within a fermenter.
Another method is to genetically modify an animal, such as a sheep, so
that the product is present in its milk.
Industrial biotechnology processes are often illustrated as flow diagrams,
showing the stages in the process. A generalised flow diagram for a
biotechnological process is shown below:
Raw materials
Micro-organisms
V
Sterilisation
V
V
Fermentation in a
fermenter vessel
V
Separation of liquid
and solid waste
V
V
Extract liquid
Solid waste
V
V
Concentrate
product
Disposal
V
Purify product
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BIOTECHNOLOGICAL PROCESSING
The topics covered in this section are:
•
•
•
•
•
•
Large-scale cell and tissue culture production
Comparison of batch and continuous flow processes
Downstream processing
Enzymes in production
Production of transgenic organisms
New breeding techniques.
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Large-scale cell and tissue culture production
Laboratory models
Many biotechnology products are made by growing micro-organisms in
fermenters or bioreactors, for example penicillin (an antibiotic) and
beer are produced in large quantities in fermenters containing as much
as 100,000 litres of culture medium.
However, before a product is made on such a large scale, the optimum
conditions for growth of the micro-organism and formation of the
product have to be found. To do this, a laboratory model fermenter is
used. This type of fermenter is relatively small, containing only a few
litres of culture medium. By carrying out experiments in a laboratory
model, the costs involved with making culture media and sterilising it,
compared to full-scale production, are reduced. An added benefit of
these trials is that, if several laboratory model fermenters are available,
many different experiments can be run simultaneously, which reduces
the time needed to develop the optimal conditions for a new process in
an industrial-scale fermenter.
Laboratory model fermenters are used to find the optimum conditions
that ensure maximum growth of the micro-organism and maximum
product formation. Growth conditions that are investigated include:
• the range of
• the range of
organism
• the quantity
• the nutrient
pH required for maximum growth of the micro-organism
temperature needed for maximum growth of the microof oxygen needed by the micro-organism
supply needed by the micro-organism.
Laboratory models can also be used to investigate:
•
•
•
•
the
the
the
the
range of substrates that can be used
rate at which nutrients are used up
stage when useful products are produced
volume of gas consumed by cells or tissues as they grow.
The growth rate of the micro-organism is monitored during these
investigations by measuring the mean generation time of the microorganism. This is the time taken for the micro-organism to double in
numbers. Also, laboratory fermenters are used to find out the growth
phase when useful product is formed.
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BIOTECHNOLOGICAL PROCESSING
Scaling up
After laboratory-scale fermenters have been used to work out the
optimum growth conditions, the process is next scaled up to a pilot
plant fermenter, containing several hundred litres of culture medium.
The pilot plant fermenter is used to trial the industrial process and
production, as described below.
•
It is used to confirm that the growth conditions that were optimal
in the laboratory-scale fermenter are the same when the process is
scaled up.
•
The control systems in the pilot plant fermenter are fine tuned to
ensure that the optimum temperature, pH and volume of the
culture medium are maintained.
•
It is used to ensure that aseptic conditions can be maintained (by
ensuring both that contaminant micro-organisms are prevented
from entering the fermenter, and that the micro-organisms are
contained within the fermenter and are not escaping into the
surrounding environment).
•
The pilot plant fermenter is used to study the best way of
recovering the product from the culture medium (a process
known as downstream processing).
•
Sufficient product is formed to allow initial safety trials to be
carried out. This is to ensure that the product is harmless to the
production workers and those who will eventually buy and use it.
After the pilot plant systems have been developed, the cost of scaling up
to an industrial plant fermenter is worked out. Assuming that it is not
prohibitive (and large custom-built fermenters are expensive), the
process can be further scaled up to full industrial size.
Industrial fermenters
Industrial fermenters, or bioreactors, are used for large-scale
fermentations. They are used for growing bacteria, fungi and for animal
and plant cells. Fermenters are used for the production of a wide range
of substances such as vaccines, enzymes, organic acids, amino acids and
antibiotics.
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Industrial fermenters range in capacity from about 20 litres up to 1
million litres. In general, fermenters designed for the growth of animal
cells tend to be smaller, due to the high cost of the culture medium
needed to grow these cells.
Fermenters can be designed to operate under aerobic conditions
(where oxygen is added) or anaerobic conditions (where oxygen is
excluded from the fermentation process). Fermenters designed to work
under anaerobic conditions are known as anaerobic digesters.
Many aerobic fermentation processes take place in a stirred tank
bioreactor (Figure 1). This is a cylindrical vessel made of stainless steel
that has mechanisms for stirring the culture medium, monitoring the
conditions within the bioreactor, cooling the culture medium and
harvesting the product.
Figure 1: A typical stirred tank bioreactor
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Some of the features of a typical bioreactor are described below:
• Stainless steel container
Most industrial fermentations take place under sterile conditions and
it is essential that the bioreactor is sterilised before it is inoculated,
and that sterile conditions are maintained while the fermentation
process is taking place. Many bioreactors are made of stainless steel,
as this allows them to be sterilised in situ (this means without
dismantling them) using saturated steam produced under high
pressure. The pressure is monitored using a pressure gauge situated
on the outside of the bioreactor. If the pressure should exceed the
safety limit, a safety valve is positioned on top of the bioreactor to
allow steam and liquid to escape.
The internal surface of a bioreactor is smooth, to help maintain
aseptic conditions within the bioreactor.
• Paddles
Within the bioreactor is a motor-driven central shaft that supports the
paddles. They turn around and create turbulence so that the microorganism and the nutrients are mixed together. The speed at which
the paddles are turned depends on the type of micro-organism being
cultivated. In general, the stirring rates are higher for bacteria than
that for fungi or for animal cells. This is because high stirring rates
may damage the fungal mycelium and animal cells.
• Baffles
These are found in bioreactors used for growing bacteria and fungi.
There are normally four baffles projecting into the bioreactor from
the inner walls. Their function is to prevent swirling and vortexing of
the culture medium.
• Sparger
This introduces air into the bioreactor. The air is sterilised before
entering the bioreactor by passing through an air filter. The sparger
can have a single hole through which the air is introduced, or it can
have multiple perforations. The size of the perforations affects the
size of the air bubbles. The smaller the air bubble, the more
efficiently air is introduced into the bioreactor.
Air that is sparged into the bioreactor is used to provide a source of
oxygen for the micro-organisms, so that they grow under aerobic
conditions.
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It has been found that the use of spargers damages fragile animal
cells, so alternative approaches are used in bioreactors designed for
animal-cell culture.
• Anti-foaming agents
Aeration and agitation of the culture medium causes foam to be
produced, which can interfere with the production process. To
prevent this, anti-foaming agents are added to the culture medium.
• Electrically operated probes
The temperature and pH of the culture medium are monitored using
electrically operated probes.
The pH probe is linked to a control panel and, if it detects any
changes in the culture medium, the control panel activates a pump
that allows alkali or acid to be added to the bioreactor to restore the
optimum pH.
• Water jacket
The temperature probe is also linked to the control panel and, if a
change in temperature is detected, a pump is activated that allows
cooler or warmer water to flow around the water jacket situated
around the outer part of the bioreactor. Therefore, the water jacket is
involved in the regulation of the temperature of the bioreactor.
• Pressure gauge
As mentioned previously, the pressure gauge is used when the
bioreactor is being sterilised under pressure. It is also used when a
fermentation is being carried out, as it can indicate the presence of a
blockage in any of the pipelines leading out of the bioreactor.
• Inoculation/sampling port
This is a port that is used to introduce the starter culture to the
bioreactor. It is also used to remove samples of the culture medium
during the fermentation process, to allow analysis such as monitoring
the growth phases and finding out how much product has formed.
• Harvest pipe
This is for the collection of culture media from the bioreactor at the
end of the fermentation process. Media collected from the bioreactor
will contain cells, as well as substances secreted from the cells.
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Comparison of batch and continuous flow processes
Processes occurring in industrial fermenters can either be batch culture
or continuous culture. Bacterial cells, fungi and animal cells can be
grown in either type of culture.
Batch culture
In a batch culture, or closed system, sterile nutrients are added to the
fermenter and they are brought to the correct operating conditions
(temperature, level of oxygen). The cells are inoculated and the
fermentation process is allowed to proceed. Nothing is added to or
removed from the closed system during this time, except small samples
that are removed for analysis (to measure growth rate, concentrations of
nutrients remaining and rate of product forming).
Figure 2 shows a typical graph of the growth curve of the cells in the
fermenter, and the changes in concentration of nutrients and product
over the time period of the batch culture.
Figure 2: Batch culture
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The cells in the fermenter show a typical growth curve with a lag phase,
a log (or exponential) phase, stationary phase and death phase. The
cells are growing at their fastest during the log phase, but as they use up
the available nutrients and produce waste, the conditions within the
fermenter become unfavourable and the cells enter the stationary phase.
Batch cultures are often used for the production of secondary
metabolites, such as penicillin. A secondary metabolite is one
produced during the stationary phase that is a by-product of
metabolism, and is not critical for the functioning of the cells. Other
secondary metabolites include vitamins and steroids.
Advantages of batch culture include the following:
• It is useful for the production of secondary metabolites.
• The fermentation time is short, so product can be made relatively
quickly.
• It is easy to control.
• All stages of growth of the cells is possible.
Continuous flow culture
Continuous flow culture is an open system that involves a continuous
feed of nutrients (or substrate) into the fermenter and the continuous
removal of product from it. Temperature and pH are monitored and any
changes are corrected, so that these conditions remain constant
throughout the process. In this type of culture, the continuous
replenishment of nutrients and removal of waste allows the cells to
reach a higher density than in a batch culture.
In some continuous flow culture systems, the cells are removed from
the fermenter continuously, whereas in other systems the cells remain
within the fermenter.
Continuous flow culture is used for the production of metabolites, such
as lactic acid (a primary metabolite produced during the log phase)
and vitamin C (a secondary metabolite produced during the stationary
phase).
Figure 3 shows a typical graph of the growth curve of the cells (which
have remained within the fermenter), and the concentration of
nutrients and product over the time period of the continuous culture.
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Figure 3: Continuous culture
Product (1° metabolite)
Advantages of continuous culture include:
• increased productivity
• continuous supply of product.
Downstream processing
After fermentation, the desired product must be extracted from the
culture medium and purified. The extraction and purification of a
product is known as downstream processing because, in a flow
diagram, it occurs after the fermentation process.
The following flow diagram shows some of the downstream processing
steps that may follow fermentation.
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Figure 4: Steps involved in downstream processing
Fermentation
V
Separation of cells
from liquid medium
V
V
Cells
Liquid medium
V
V
Disruption
of cells
V
V
Freeze drying
Extraction of
product
V
V
Dried cells
Liquid
product
V
V
Purification of
product
Drying
V
Solid
product
The types of products that are extracted from the culture medium and
further purified include whole cells, such as yeast; organic acids, such as
lactic acid and citric acid; antibiotics, such as penicillin; and alcohol,
amino acids, enzymes and vaccines. The final product can either be in
liquid or solid form.
The way that the products are extracted and purified depends on the
chemical nature of the product. Some products (such as proteins) are
heat sensitive, and so techniques must be used that will not destroy
them. Also, it is important to minimise the number of steps used in
downstream processing, as the more steps there are, the higher the
chance of losing some of the product (thus getting a poorer yield); and
also, the more steps, the more expensive the process.
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Extracting cells from liquid culture
An example of when cells are removed from liquid medium is during
alcohol production, such as in the making of beer. Removal of the yeast
causes the beer to become clearer. Alcohol is formed when yeast cells
undergo anaerobic respiration, a process known as fermentation.
One of the reasons why yeast can be removed from the liquid medium in
this fermentation process is because yeast cells naturally flocculate, or
clump together. The flocculated cells precipitate out of solution (by
becoming a more solid mass) and so they can be separated relatively
easily from the liquid culture medium. Yeast cells used to make lagers
flocculate and sink to the bottom of the fermentation vessel, whereas
yeast cells used to make ales rise to the top of the fermentation vessel
after flocculating. Yeast cells that are removed from the liquid medium
are used to start another fermentation. By using the same yeast, the
brewers are able to maintain the flavour of the beer.
In some fermentation processes, flocculating agents are added to the
yeast suspension to help them to flocculate.
Following flocculation, the yeast cells may be removed from the culture
medium by filtration. In this process, a filter retains the flocculated
cells, while allowing the liquid culture medium to flow through. The
yeast cells are retained by the filter because of their large size in relation
to the pores in the filter.
Some strains of yeast used in alcohol production do not flocculate and
they are centrifuged from the culture medium. In this process, the
culture medium and yeast cells are spun round in a centrifuge at very
high speed, thus causing high centrifugal forces. This causes the yeast
cells, which are more dense, to form a solid pellet. The liquid culture
medium is then easily separated from the pellet of yeast cells.
Centrifugation is not routinely used to separate yeast cells after alcohol
fermentation, due to the high initial cost of the centrifuge and the
associated energy and maintenance costs.
A method used to remove bacterial cells from liquid culture is to freezedry the cells. The bacterial cells are frozen, then the frozen liquid is
removed by sublimation (this is the removal of the liquid as a gas). This
leaves the cells completely dehydrated. They can then be stored or
transported without becoming denatured.
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When freeze-dried bacteria are required for use, they are re-hydrated by
the addition of a sterile aqueous solution.
Obtaining solvent and solute from liquid medium
The previous section described the methods used to obtain whole cells
from the fermentation process. However, at the end of a fermentation
process, it can be a solvent, or a solute, that has been made and secreted
by the microbial cells (such as antibiotics, proteins, organic acids or
amino acids), that is the desired end-product, rather than the cells.
In the extraction and purification of solvents and solutes, some of the
techniques employed are similar to those used to separate whole cells,
for example flocculation, precipitation, filtration and centrifugation.
Two organic acids that are produced by fermentation are citric acid and
lactic acid. Both are used in the food industry as acidifiers.
The extraction of both these organic acids involves the addition of chalk
or lime, which causes precipitates to form. The precipitates (which are
solid) are removed from the liquid solution by filtration. Both acids then
undergo further downstream processing.
Proteins and polysaccharides can be precipitated out of solution by the
addition of several different types of chemicals (such as acetone and
ammonium sulphate). The precipitate can be separated from the
remaining liquid by filtration or by centrifugation.
Ultrafiltration
This technique makes use of a semi-permeable membrane (this means
that the membrane contains pores). When a solution is passed through
the membrane, any molecules that are larger than the pore size are
retained by the membrane, while molecules of a smaller size than the
pores flow through the membrane.
Ultrafiltration is involved in the separation of molecules in the range of
0.001µm to 0.02µm (a µm is 10–6 of a metre).
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Distillation
This method is used in the production of consumable alcohol (such as
whisky), fuel alcohol (such as gasohol), acetone and acetic acid
(vinegar).
Different molecules have different boiling points, so they can be
separated from each other on this basis. The fermented liquid
containing the product is heated. Alcohol is a volatile substance and so
has a fairly low boiling point and turns to vapour at lower temperatures.
The vapour is collected in a condenser (which is cooled with cold
water), cools down, condenses back into a liquid, and is collected as a
pure substance.
The diagram below shows a distillation in process.
Figure 5: Distillation apparatus
Protein purification
The first stage in protein purification is often to precipitate the protein
from the liquid medium, as described on p18. The precipitate will
contain the protein, but it may also contain other contaminating
substances. Depending on the end-use of the protein, it may have to be
further purified.
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To do this, the precipitate is dissolved in a small volume of liquid. It is
then subjected to a technique known as column chromatography. In
this technique, a metal or glass column is packed with a resin that
separates proteins according to their size, charge or shape.
Figure 6: Separation of proteins according to their size
Figure 6 shows proteins being separated by size. The mixture of
proteins is loaded into the top of the column. The proteins are washed
through the column using a buffer and, as they pass down the column,
the large proteins move faster than the small proteins. The proteins in
the mixture are thus separated from each other. This separation is
followed by measuring the absorbance of the liquid as it elutes from
(comes off) the column. When a protein elutes from the column, it
causes an increase in absorbance.
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Figure 7 shows a typical graph of absorbance against volume of liquid
eluted. Each peak on the graph corresponds to a protein that has eluted
from the column shown in Figure 6. This graph shows two peaks, so the
two proteins have been separated according to their size. The peak on
the left corresponds to the larger protein as it eluted first; the peak on
the right represents the smaller protein.
Figure 7: Elution profile of proteins
large protein
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small protein
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Figure 8 shows the purification of lysozyme, an enzyme that is positively
charged at pH10. It is applied to a column filled with a negatively
charged resin. Lysozyme binds to the negatively charged resin, whereas
all other proteins flow through the column. Lysozyme is then eluted
from the column by changing the buffer running through the column to
one that breaks the bond between lysozyme and the resin.
Figure 8: Purification of lysozyme, a positively charged protein
Lysozyme is eluted
from the column with
buffer that breaks the
lysozyme–resin bond
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Figure 9 shows the purification of interferon (a protein molecule used
to fight viral infections). A mixture of substances, containing interferon,
is applied to a column filled with antibodies with a complementary
shape to interferon. Interferon binds specifically to the antibodies; other
substances do not. Interferon is then eluted by changing the buffer
flowing through the column to one that breaks the bond between
interferon and the antibody.
Figure 9: Purification of interferon
Interferon is eluted
from the column with
buffer that breaks the
interferon–antibody
bond
Solvent extraction
Penicillin (an antibiotic) is extracted from the fermentation medium by
solvent extraction. The fermentation medium is mixed with a solvent.
Penicillin is more soluble in the solvent than in the fermentation
medium, so it moves from the fermentation medium into the solvent.
The solvent and the fermentation medium do not mix (just as oil and
water do not mix), so they can be separated from each other.
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Drying
Spray dryers are used for drying large volumes of liquid. The liquid is
turned into an aerosol of very small droplets that are passed into a
stream of hot air. Water in the droplets evaporates, leaving behind a
solid product.
Chamber dryers are used to dry smaller volumes of liquid. The liquid is
placed on shelves inside the cabinet and water evaporates from it. A
solid product is formed.
Enzymes in production
The majority of industrial enzymes are produced from gram-positive
bacteria and from fungi grown in batch culture.
Generally, the enzymes are extracellular, which means that, during
fermentation, they are secreted from the micro-organisms into the
culture medium in large quantities. After fermentation, downstream
processing takes place. The enzymes are purified from the culture
medium by precipitation and filtration, which removes contaminants.
Extracellular enzymes are relatively easy to extract from the medium.
However, some enzymes are intracellular, which means that the
microbial cells must first be harvested, then broken open and the
enzyme released. Detergents or enzymes are often used to break down
cell walls and membranes in order to release the desired enzyme. After
the enzyme is released, downstream processing using precipitation and
filtration continues.
Both intracellular and extracellular enzymes may be dried to make them
more concentrated.
High-value medical and pharmaceutical enzymes that require a high
level of purity may be further purified by the use of column
chromatography. Enzymes such as those in biological washing powders
often have limited downstream processing, as their purity is not as
critical.
A flow diagram to show the extraction of an intracellular enzyme is
shown in Figure 10.
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Figure 10: Obtaining an intracellular enzyme
Uses of enzymes
Enzymes have a wide variety of uses, some of which are:
• in biological washing powders, to degrade protein, starch and lipidbased stains
• in the baking and brewing industries, to break down starch in flour
and barley
• in the dairy and confectionery industries, to produce sweeteners
• in the leather industry, to make the leather more pliable
• in the textile industry, to make cloth softer
• in the medical/pharmaceutical industry, in diagnostic kits.
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Some specific enzymes and their uses are as follows.
Pectinase is an enzyme that breaks down pectin, a component of the
cell walls of plants. Pectinase is used to clarify (make clear) fruit juice. It
is also used to release juices, oils and colour from fruit. These are then
added as flavourings and colourings to soft drinks.
Urokinase
Heart attacks and strokes can be caused by an obstruction in a blood
vessel, or by a blood clot. Normally, a protein called plasmin breaks
down blood clots in your body. The enzyme urokinase helps your body
to make plasmin. Urokinase can be given therapeutically to help remove
blood clots, so is useful in preventive medicine.
Cellulase
This is an enzyme that breaks down cellulose, the main component of
plant cell walls. Cellulase is used to convert plant waste material into
edible feedstock for animals. It is also used to treat denim to give the
stone-washed effect.
Lysozyme
This enzyme is used to disrupt the cell walls of bacteria and yeast cells. It
can be used in downstream processing to break open cells to release
intracellular enzymes.
Immobilisation of enzymes
As demand has increased for the use of enzymes in industrial and
pharmaceutical processes, a technique has been developed called
enzyme immobilisation. Briefly, this technique involves attaching an
enzyme to an insoluble support. The enzyme makes the product, the
product is removed, and the enzyme can be used again and again.
There are three main methods of immobilising enzymes.
Adsorption
The enzyme is attached to the solid support by non-covalent bonds
(see Figure 11 overleaf).
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Figure 11: Adsorption of an enzyme onto a solid support
Entrapment
The enzyme is trapped in a jelly-like matrix or capsule.
Figure 12: Entrapment of an enzyme
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Bonding
The enzyme is attached to the solid support by covalent bonding.
Figure 13: Bonding of an enzyme
There are many advantages associated with the use of immobilised
enzymes:
• immobilised enzymes can be recycled and used again
• immobilised enzymes are more stable
• there is easier and cheaper separation of enzyme and product, so
reducing costs
• they are ideal for use in a continuous flow process.
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Production of transgenic organisms
A transgenic organism is one with DNA (a gene) inserted into it from a
different (foreign) organism. A common laboratory exercise to
demonstrate this is where bacteria have a fluorescent gene from a
jellyfish inserted into them, so that the transgenic bacteria are
fluorescent too!
Micro-organisms, animals and plants can be made transgenic. The
techniques associated with introducing foreign DNA into microorganisms is covered in Unit 1 (Microbiology (Higher)). You may find it
useful to revise these techniques before proceeding with this section.
Any organism that has been genetically modified by the insertion of
foreign DNA is known as a genetically modified organism (GMO).
Transgenic animals
Animals can have foreign DNA inserted into them by microinjection or
by viral infection.
Microinjection involves injecting foreign DNA into a newly fertilised egg
cell using a small glass-needled syringe.
Viral infection involves introducing the foreign DNA into a virus, then
using the virus to infect a fertilised egg cell.
In many cases, the inserted foreign DNA codes for a drug that the animal
then makes and secretes. The use of transgenic animals is an alternative
to the production of drugs using cell culture and fermenters.
Transgenic plants
Foreign DNA can be inserted into plants using a plasmid from the
bacterium Agrobacterium tumefaciens. The plasmid is genetically
modified with the foreign DNA.
The following procedure outlines how a transgenic plant can be
produced using A. tumefaciens and its plasmid.
1.
30
The plasmid (containing an antibiotic resistance gene) is obtained
from A. tumefaciens.
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2.
The plasmid is cut open with a restriction enzyme endonuclease
and the foreign DNA is inserted into the plasmid to produce a
genetically modified plasmid.
3.
A. tumefaciens is transformed with the genetically modified
plasmid.
4.
Protoplasts are made from plant cells. (A protoplast is a plant cell
that has had the cell wall removed, using the enzyme cellulase).
5.
Protoplasts are incubated with transformed A. tumefaciens.
6.
Protoplasts are plated out onto nutrient medium containing the
same antibiotic as the antibiotic-resistant gene in the plasmid.
7.
Only those protoplasts that are infected with A. tumefaciens grow
in the selective media.
8.
Growing plant cells are isolated and grown on to produce
transgenic plants.
There are several reasons as to why biotechnologists want to produce
transgenic plants:
•
•
•
•
•
to
to
to
to
to
improve crop yields
protect against pests and diseases
protect against herbicides (weedkillers)
protect against harsh environments
increase the variety of available plants.
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New breeding techniques
Gardeners have been cloning their favourite plants for many years, using
plant tissue culture techniques. (These were discussed in Unit 2
Microbiological Techniques (Higher)). These techniques can be used on
transgenic plants too, so that large numbers of a particular transgenic
plant can be obtained. Until recently, cloning was restricted mainly to
plants, but now it has been extended to animals.
The reason why animal cloning has been developed is to produce a
number of identical animals with desired characteristics. For example, if
a transgenic sheep has been produced that can secrete a drug in its
milk, then it makes sense to clone this animal so that a flock of identical
sheep are produced, all making the same drug in their milk.
Before moving on to the different types of new breeding techniques,
let’s find out a bit about embryo developmental biology, because some
of that subject’s terms are used when describing the new breeding
techniques.
When an egg cell and a sperm cell fuse, a new cell is produced (called a
fertilised egg cell, or a zygote) that contains all the necessary genetic
information in its nucleus to produce a new individual. The fertilised
egg cell divides into two (this is known as the two-cell stage) and each
new cell is identical to the fertilised egg cell, with the same genetic
information inside their nucleus. The two cells are clones of each other.
Both of these cells now divide into two and the four cells produced also
contain identical genetic information.
Cell division continues until a ball of cells is produced, which is called a
blastocyst. Again, each cell is identical genetically to the original
fertilised egg cell. The cells in the blastocyst are said to be
undifferentiated, because they have the capability of becoming any type
of cell in the organism and have not yet followed a developmental
pathway to become a particular type of cell.
After many, many further cell divisions, a multicellular organism is
produced. The majority of the cells of this organism (the somatic cells)
have the same genetic information in their nucleus as the original
fertilised egg cell. However, these cells are now called differentiated
cells, because they have followed a developmental pathway and become
a particular type of cell (such as a muscle or nerve cell) and cannot
become any other type of cell.
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Embryo manipulation
This technique has been used by sheep and cattle breeders to increase
the numbers of animals in a herd.
An egg is fertilised and grown to the two-cell stage. At this point, the
two cells are separated and each cell is transplanted into the uterus of
the mother. Each transplanted cell now behaves like a fertilised egg cell
and continues to divide to produce a new individual. This results in
twins being born which are genetically identical (or clones). This
process mimics the naturally occurring process where identical twins are
produced.
The advantage of this technique is that it doubles the reproductive rate
of the animal.
Embryo cloning
The main purpose of this technique is to conserve desired features
within an animal for future generations.
Embryo cloning has been used to produce many genetically identical
mice. These mice are useful in experiments – fewer mice are required
and the results are more reliable.
In addition to this, the embryos of mice have been frozen, so they can
be stored for long periods of time. Thus scientists will be able to repeat
the experiments in the future.
The stages used in embryo cloning in mice are as follows:
1.
An egg is taken from a donor mouse and fertilised.
2.
The fertilised egg is grown to the blastocyst stage.
3.
The undifferentiated cells from the blastocyst are separated from
each other.
4.
More egg cells are taken from the donor mouse and the nuclei are
removed from these cells and discarded. The cells are now said to
be enucleated.
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5.
The nuclei are removed from the separated blastocyst cells and
each nucleus is transferred into an enucleated egg cell.
This has the effect of creating lots of new fertilised egg cells, each
one genetically identical and each capable of dividing into a new
individual mouse.
6.
Each newly formed cell is grown to the blastocyst stage.
7.
Blastocysts are transferred into surrogate mother mice.
8.
The surrogate mice give birth to many genetically identical mice (a
clone of mice).
Somatic cell cloning
This new breeding technique is used to create clones from an animal,
using a nucleus from one of its mature differentiated cells. The most
famous animal associated with somatic cell cloning was Dolly the Sheep,
who was born in July 1996 at the Roslin Institute outside Edinburgh. She
was the first animal to be cloned successfully from a cell taken from an
adult animal.
Since Dolly, many animals have been cloned in this way, including cattle,
goats, pigs and mice. However, cloning animals in this way is not
problem-free, for example many cloned offspring die during pregnancy
or shortly after birth. Some have health problems, such as respiratory
and cardiovascular dysfunction. However, as the technology advances,
these problems may be overcome.
The procedure for somatic cell cloning is very similar to that for embryo
cloning. A nucleus is removed from a differentiated cell from an adult
(in the case of Dolly, the nucleus was removed from a cell from the
udder of a female sheep). The nucleus is transferred into an enucleated
egg cell. The new nucleated egg cell is grown for five or six days, and
assuming it appears to be developing normally, it is transplanted into a
surrogate mother.
The animal that is born is identical genetically to the animal from which
the nucleus came. If the original animal was a transgenic animal
producing a drug in its milk, then the cloned animal will also produce
the drug in its milk. In this way, a flock of identical animals producing
the desired product can be produced.
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SECTION 2
Biotechnology applications
You have now completed the first section on biotechnological
processing and should now have an understanding of how many of the
products of biotechnology are made by using micro-organisms, plants
and animals.
In this section, you will be introduced to the applications (or uses) of
some of these products. You will look at the following areas:
• agriculture and horticulture
• clinical and forensic medicine
• environment.
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Agriculture and horticulture
Crop protection
Resistance to pests
Insects are pests that cause billions of pounds of damage to crops every
year. One way to remove insects is to introduce micro-organisms that
kill them.
Bacillus thuringiensis is a bacterium that infects the caterpillars of the
Gypsy Moth. When this bacterium produces an endospore (a resistant
form of the bacteria), it makes a crystalline protein toxin called Bt
toxin. Gypsy Moth caterpillars are specifically killed by Bt toxin. It has
no harmful effect on humans or other vertebrates.
B. thuringiensis and Bt toxin have been sprayed over crops that have
been infected with Gypsy Moth and the numbers of the insect have
decreased.
As an alternative to spraying the crops with B. thuringiensis and Bt
toxin, transgenic tobacco and tomato plants have been produced with
the gene for Bt toxin inserted into them (using a technique similar to
that described on p30). The transgenic tobacco and tomato plants
produce the crystalline protein toxin themselves (without spraying),
which kills the Gypsy Moth caterpillars.
Resistance to herbicides
Weeds are a major problem in cultivated crops, as they compete with
the crop plants for available light, water and nutrients. When a farmer
sprays his crops with herbicide (such as glyphosate) to kill the weeds,
the crops sometimes die too.
However, transgenic wheat and maize plants have been produced that
have had a herbicide-resistant gene inserted into them. The gene codes
for a protein that degrades and detoxifies glyphosate. These transgenic
wheat and maize plants can then be sprayed with glyphosate to kill the
weeds growing among the crops, leaving the crop plants unaffected.
Plant production
Tissue culture is a technique used to grow a large number of identical
plants with desired characteristics, such as pest or herbicide resistance.
An additional benefit of producing large numbers of plants using tissue
culture is that it is relatively cheap.
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The procedures used to produce plants in this way are described in
detail in Unit 2 (Microbiological Techniques (Higher)).
Clinical and forensic medicine
Producing vaccines by genetic engineering
Vaccination is used to give immunity against specific diseases. In
developed countries, such as the UK, there are vaccination programmes
in place that have managed to completely eradicate some diseases, such
as smallpox.
The principles of vaccination are outlined in Unit 1 (Microbiology
(Higher)). Briefly, an antigen (a micro-organism or part of a microorganism) is artificially introduced into your body. Your immune system
makes antibodies against the antigen to destroy and remove it. After the
antigen has been removed from your body, memory cells (B
lymphocytes) are produced that help you to fight the micro-organism,
should it enter your body again. Thus you have acquired immunity
against that particular disease.
There are a number of ways in which conventional vaccines are made.
Vaccines normally contain inactivated (killed) micro-organism or live,
attenuated micro-organism (this means that the micro-organism has
been artificially mutated so that it does not cause disease) or it can
contain part of a micro-organism, such as surface proteins or toxins.
There are a number of problems associated with conventional vaccines:
• Use of inactivated micro-organisms can lead to disease in recipients of
the vaccine, as the inactivation process is not always 100% successful.
Any surviving micro-organisms can potentially cause disease.
• Some attenuated micro-organisms become pathogenic again, so can
cause disease in recipients of the vaccine. Attenuated vaccines must
be monitored carefully.
• Many vaccines rely on tissue culture, which is expensive and can have
low production rates.
• Some micro-organisms cannot be grown in tissue culture, for
example Hepatitis B virus.
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Producing vaccines using genetic engineering techniques allows some of
these problems to be overcome, for example:
• Genes in a micro-organism that are known to cause disease have been
deleted by genetic engineering. This type of attenuated microorganism cannot become pathogenic again.
• For micro-organisms that cannot be grown in tissue culture, the
genes coding for antigenic proteins have been isolated and
introduced into E.coli, or other eukaryotic cell systems. These cells
are then grown in large scale in a fermenter, the antigenic proteins
produced, isolated and used in vaccines.
• Large quantities of vaccine can be produced.
Production of Hepatitis B vaccine
Hepatitis B is caused by a virus that infects the liver and may cause liver
cancer.
Figure 14: Hepatitis B virus
Patients who have been infected with Hepatitis B have a surface protein
from the virus present in their blood, which acts as an antigen. This
surface protein is called Hepatitis B surface antigen (HBsAg).
The gene coding for HBsAg was isolated and cloned by inserting the
gene into the nucleus of a yeast cell. The genetically modified yeast cell
produces HBsAg protein, which is secreted into the culture medium.
The secreted HBsAg protein is extracted from the culture medium,
purified and used as a vaccine.
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Use of plants to produce vaccines
Some research has been carried out, whereby plants have been
genetically engineered to produce a vaccine. Instead of injecting the
vaccine into the patient, it is intended that the plant is eaten and the
patient will acquire immunity in this way. An added advantage is that it is
cheaper to mass produce plants using tissue-culture techniques than it is
to run fermenters, so producing vaccines in plants may be cheaper. Also,
plants can be dried and stored, which overcomes the problem of
refrigeration that is needed to store many conventional vaccines.
Research has been carried out on producing Hepatitis B vaccine in
bananas and tomatoes.
Monoclonal antibodies
Antibodies are proteins that are secreted by B lymphocytes in response
to an antigen. Although different antibodies are produced by different B
lymphocytes, all antibodies produced by a single B lymphocyte are
identical, and all bind to the same part of the antigen. Antibodies
produced by a single type of B lymphocyte are known as monoclonal
antibodies. Biotechnologists want to produce monoclonal antibodies,
because they can be used as a reliable analytical reagent to identify and
quantify specific antigens.
In order to produce monoclonal antibodies, individual B lymphocytes
must be isolated and each individual B lymphocyte grown in culture, so
that all the antibodies produced are identical. However, a problem in
culturing B lymphocytes is that they last only for a few days before
dying. This problem has been overcome by fusing (joining) the B
lymphocytes with cancer cells that live indefinitely (they are said to be
immortal, assuming they are looked after properly!). The fused B
lymphocyte and cancer cell are called hybrid cells and can live for a
long time and produce identical antibody molecules. After the hybrid
cells have been produced, the ones that are making and secreting the
desired antibody are selected and these hybrid cells are further cultured
to produce hybrid cell clones.
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Uses of monoclonal antibodies
There are many uses of monoclonal antibodies in scientific research,
biotechnology and medicine, some of which are outlined below.
• Research tool
There are times when a researcher may want to know whether an
antigen is present in a body tissue. To find this out, a monoclonal
antibody complementary to the antigen is labelled with radioactivity
or fluorescence so that it can be tracked. The monoclonal antibody is
incubated with the tissue and, if the antigen is present, the
monoclonal antibody binds to it. The tissue will become radioactive
or fluorescent at the exact location where the monoclonal antibody
has bound.
• Tissue typing before an organ transplant
Monoclonal antibodies have been used to ensure that an organ that is
to be transplanted into a patient will not be rejected. This is known as
tissue typing. The better the match between the surface proteins on
the organ to be transplanted and those found in the patient, the less
chance there is of rejection. Monoclonal antibodies are used to check
the compatibility of these surface proteins.
• Pregnancy testing
The urine of a pregnant woman contains the hormone human
chorionic gonadotrophin (hCG). One type of home pregnancy kit
consists of a dipstick that contains coloured monoclonal antibodies
that bind to hCG. The dipstick is dipped into the urine of the person
doing the test.
If hCG is present in the urine, it binds to the coloured monoclonal
antibodies and together they move up the dipstick. They are stopped
about one third of the way up by a row of immobilised monoclonal
antibodies that also bind to hCG. A coloured band appearing here
indicates that the woman is pregnant.
If hCG is not present in the urine, the coloured monoclonal
antibodies continue to move further up the dipstick until they are
stopped by a second set of immobilised monoclonal antibodies that
bind to the coloured monoclonal antibodies. The appearance of a
coloured band further up the dipstick indicates the woman is not
pregnant.
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• Identifying infective agents
Monoclonal antibodies are routinely used in clinical labs to find out if
a patient has been exposed to infectious micro-organisms, such as
human immunodeficiency virus (HIV), or if they are suffering from
infectious diseases such as glandular fever. A sample of the patient’s
blood is mixed with a labelled monoclonal antibody – if the sample
changes colour, then the patient has been exposed to the microorganism.
• Anti-cancer medicines
Monoclonal antibodies are used as therapeutic agents in the fight
against cancer. Anti-cancer drugs are attached to monoclonal
antibodies that bind specifically to cancer cells. The patient is given
the monoclonal antibody–drug complex and the monoclonal
antibody finds and binds only to cancer cells in the patient. The drug
is delivered straight to the cancer cells and not to healthy cells.
Transgenic animals
As mentioned in the Section 1 of this pack, a transgenic animal is one
that has had its genome altered by the insertion of a gene using
recombinant DNA technology (another name for genetic engineering).
In many cases, the gene codes for a drug that the animal then makes and
secretes in its milk. Successful transgenic animals have included sheep
and cattle.
Drugs that have been produced using transgenic animals include
interferon (used to fight viral infections), blood clotting factors and
alpha-1-antitrypsin (AAT) (used to treat emphysema and cystic
fibrosis).
Production of medical products by transgenic animals
Firstly, the gene coding for the medical product, such as AAT, is
isolated. This gene is then attached to another gene that is involved in
milk production. In this way, the inserted gene becomes part of the
sequence of genes involved in milk production. These are inserted into
a vector and the vector is introduced into a fertilised egg cell of the
animal, such as a sheep. The vector, containing the gene for AAT and the
milk-producing gene, becomes incorporated into the sheep’s DNA. The
egg is placed in the uterus of a surrogate mother sheep. The lamb that is
born contains the DNA needed to produce AAT. However, as the AAT
gene was attached to the milk-producing gene, AAT is produced and
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secreted only when the lamb becomes an adult and starts to produce
milk. The AAT protein is present in the sheep’s milk.
Advantages of using transgenic animals
• It is considered to be cost effective because:
– there is no need for expensive large-scale fermentation vessels
– there is no need to continuously monitor or maintain equipment.
• Animals modify proteins after they have been made, for instance sugar
groups are added to proteins as they pass through the Golgi
apparatus before being secreted from a cell. Such modifications are
sometimes needed to activate a protein.
• The ability to make and secrete the drug is passed from one
generation to the next.
• The animal does not need to be sacrificed to harvest the drug.
Disadvantages of using transgenic animals
• It is relatively difficult to make a transgenic animal (it took 276
attempts before Dolly was created) and the gene does not always
insert into the sheep DNA.
• The transgenic animal may die before it reaches adulthood.
• The transgenic animal may die before it produces offspring.
• Only female transgenic animals produce milk.
• There are ethical, social and moral issues. Is it right to artificially
change the genetic make-up of an animal?
Stem cell culture
Stem cells are cells that are produced early in embryo development
(generally between the two-cell stage and the blastocyst stage – see p32
to remind you of these terms). Stem cells obtained during this time are
undifferentiated cells – they have the potential of developing into any of
about 200 different types of cells found in the adult organism. Stem cells
can also be obtained from the blood in the umbilical cord, which can be
obtained after a baby is born.
Stem cell culture is still in the very early days of research and
development, but it is an area that has generated a lot of interest, both
among the scientific community and the general public.
Stem cell research could eventually lead to cells and tissues being
formed that may be used in the treatment of diabetes, Parkinson’s
disease and many other disorders and diseases. It is thought that it may
even lead to whole organs being cultured to be used in transplants.
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Stem cell culture may also be used to test new drugs without using
animal or humans.
However, there are a lot of social, moral and ethical issues surrounding
stem cell research. Some of these issues surround the source of the
stem cells. Human embryonic stem cells can be obtained from in vitro
fertilisation programmes where more embryos are created than are
needed. They can also be obtained from an aborted foetus. Both sources
are highly emotive and have given rise to much debate. Some people
believe that it is better for embryos to be used in stem cell research than
to be destroyed. Others believe that using human cells is totally
unacceptable, as the cells constitute a ‘person’ and have the right to life.
Many people fear that there will be a ‘black market’ in the sale of human
embryos. What are your views on stem cell culture?
DNA profiling
Everyone, except identical twins, has a different genetic make-up. These
differences in DNA can be used to uniquely identify an individual using a
technique known as DNA profiling.
Firstly, DNA from an individual is isolated (generally from a blood
sample), then digested into smaller fragments using specific restriction
endonucleases. The sizes of the fragments from this digestion are
unique to each individual. These fragments are then separated
according to their size by gel electrophoresis. The DNA fragments are
then transferred onto a membrane filter (which is much more robust
than a gel). A special labelled probe that binds to certain regions of the
DNA is incubated with the membrane. After an appropriate length of
time, the membrane is washed and the probe that has bound to the DNA
demonstrates a pattern of bands, rather similar to a bar code. Each
individual has their own unique ‘bar code’, or DNA profile. (The
techniques used in DNA profiling are described in more detail in Unit 1:
Microbiology (Higher).)
Figure 15 shows simplified DNA profiles of a mother, father and one of
their children.
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Figure 15: DNA profiles
1
Mother
2
Child
3
Father
Half of our DNA is inherited from our mother; the other half from our
father.
If you look at the child’s profile in Figure 15, you will see which bands
were inherited from the mother and which from the father. Some bands,
you will observe, are common to all three individuals, so they could
have been inherited from either parent. These are non-informative
bands.
DNA profiling can be used to work out the parentage of a child in
paternity disputes. It can also be used to compare, for example, the DNA
profile from a sample of blood, semen or tissue at the scene of a crime,
to a sample from a suspect.
DNA profiling is also used to detect genetic disorders. In this case, a
probe is used that binds to a particular band in the DNA profile and it is
used as a marker for that particular genetic disorder.
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Environment
Biosensors as detectors of pollution
Biosensors have been developed that allow the monitoring and
measurement of pollution in the field (as opposed to collecting samples
and taking them back to lab for analysis).
A biosensor consists of a transducer that is linked to an enzyme,
antibody or cell which specifically recognises a particular contaminant,
such as a heavy metal. When the biosensor is exposed to this
contaminant, the enzyme, antibody or cell cause a change in the
transducer so that a signal is produced. This signal can be electrical, or a
coloured dye, or light can be emitted as luminescence. The higher the
level of contamination, the greater the signal emitted.
Bioremediation
Bioremediation is the use of micro-organisms to clean up pollution from
the environment. The micro-organisms can degrade the pollutant,
detoxify it to a less harmful substance, or remove it by accumulation, so
reducing the levels of the pollutant. The most common pollutants are
hydrocarbons, heavy metals, polychlorobiphenyls (PCBs) and
chlorinated solvents.
Bioremediation of contaminated soil involves one of two processes:
• naturally occurring micro-organisms in the soil can be activated so
that they increase in number and remove the contamination
• where the soil is contaminated with a specialised compound,
genetically engineered micro-organisms can be released into the soil
to specifically target the contamination.
Oil spillages are treated in a similar manner to soil contamination. For
example, during the Exxon Valdez oil spill in Alaska in 1989, naturally
occurring micro-organisms were stimulated by the addition of nutrients.
Genetically modified micro-organisms have also been used to clean up
oil spills.
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Some suggested reading materials for teachers/lecturers
The following is a commentary on some published reading materials that
may be useful when delivering Higher Biotechnology. This list is in no
way exhaustive and is meant only as a starting point for any tutor
delivering the units for Higher Biotechnology for the first time.
Foundations in Microbiology (3rd edition)
by Kathleen Park Talaro and Arthur Talaro
Published by WCB/McGraw-Hill
ISBN: 0-697-35452-0
This is a general introductory microbiology book that is a good teacher’s
resource, especially if you do not have a microbiology background. The
book is aimed at undergraduates, so it is too detailed and advanced to
be used as a student resource, but it is easy to read and has lots of good
illustrations and diagrams. There is an interactive CD-ROM that can be
purchased to accompany the book. It provides lots of detailed
background knowledge on many of the topics in all of the three units
that comprise Higher Biotechnology.
Fundamentals of Microbiology (5th edition)
by I Edward Alcamo
Published by Benjamin/Cummings Publishing Company
ISBN: 0-8053-0532-7
This is another general microbiology book that is a good teacher’s
resource. Again, it is easy to read with lots of diagrams and anecdotes
(although they are all American). This book is a good source of graphs
that could be the basis for problem-solving questions. It also provides
lots of detailed background information for all three units of Higher
Biotechnology.
Micro-organisms and Biotechnology (1st and 2nd editions)
by Jane Taylor
Published by Nelson Thornes
ISBN: 0-17-448255-8 (second edition)
This book is now into its second edition and may be used as a teacher
and student resource. Both the first and second edition provide
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background knowledge for all three units comprising Higher
Biotechnology and the book is especially good for the Enumerating
Micro-organisms section in Unit 2 (Microbiological Techniques). The
second edition also covers some ethical issues surrounding some
biotechnology processes.
Basic Biotechnology (2nd edition)
Edited by Colin Ratledge and Bjorn Kristiansen
Published by Cambridge University Press
ISBN: 0-521-77917-0
This is a book for teachers who are enthusiasts and want to have a
detailed knowledge of biotechnology. It provides all the background
knowledge (and more!) required for delivering Unit 3 (Biotechnology).
Some suggested websites
Remember that web addresses are constantly being upgraded and
changed. The following web information is correct at the time of
publication (February 2005).
www.Biotechinstitute.org
This is an American website that has lots of biotechnology information. It
has links to biotechnology-related news stories from a range of sources,
e.g. Nature, Yahoo and the BBC. There are teachers’ resources and links
to other websites. Also, you can download back copies of the magazine
Your World; this is aimed at post-16 students. Each issue covers one
particular biotechnology topic and so can be used as a classroom
resource.
www.biowise.org.uk
This website provides downloadable case studies on industrial
biotechnology which may be useful for Unit 3 (Biotechnology). The case
studies highlight companies in the UK that actively use biotechnology;
they are a good way to show students the practical relevance of what
they are studying.
www.sgm.ac.uk
This is the Society for General Microbiology website, which has links to
current ‘hot’ topics and news items, so it is a good way of keeping up to
date with issues in microbiology. It also has educational resources and
links to online microbiology resources.
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BIBLIOGRAPHY
www.ncbe.reading.ac.uk
This website provides downloadable protocols for practical exercises, as
well as online learning materials. It has a good section on safety issues to
be taken into consideration when carrying out biotechnology practical
exercises. It also provides information about the Scottish Centre for
Biotechnology Education.
www-saps.plantsci.cam.ac.uk
This website has protocol information, details on how to purchase kits
that can be used as learning activities, and details of biotechnology
workshops for teachers and the annual biotechnology summer school.
www.scottishbiotech.org
This is the website of the Scottish Colleges Biotechnology Consortium,
who deliver technical training to industry and schools. Online courses
are available.
www.sserc.org.uk
This website provides information about the Scottish Institute of
Biotechnology Education (SIBE), which runs workshops for teachers
and pupils. Members can access an interactive manual on
microbiological techniques for schools and colleges. It includes a code
of practice on safety in microbiology and notes on micro-organisms for
investigations.
www.scottish-enterprise.com
This is the website of Scottish Enterprise. Information about the
Scottish biotechnology industry can be obtained here by clicking on
‘services to industry group’, then clicking on ‘life sciences’. It is very
useful for keeping up to date with the activities of biotechnology
companies in Scotland.
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ADVICE FOR PROBLEM-SOLVING OUTCOMES
APPENDIX
Advice for problem-solving outcomes
Unit 1: Microbiology, Outcome 3
Unit 3: Biotechnology, Outcome 2
Candidates are required to produce one report on a problem-solving
activity as part of the evidence for the achievement of Higher
Biotechnology. The report can be used as evidence for Outcome 3 to
achieve the Unit ‘Microbiology’ and for Outcome 2 in the Unit
‘Biotechnology’. The report must be the individual work of the
candidate.
One way that a problem can be solved is to carry out a practical
investigation, either as an individual or as part of a group. This enables
candidates to fulfil the five required performance criteria (PC):
(a)
(b)
(c)
(d)
(e)
The problem to be solved is identified.
Resources required to solve the problem are identified and
obtained.
Procedures appropriate to solving the problem are planned
and designed.
The planned procedures are carried out.
The problem-solving procedure is evaluated.
Alternatively, candidates can undertake a paper-based investigation by
identifying a particular problem, obtaining data from other sources (for
example, biotechnology journals or the internet), then analysing,
presenting and evaluating this data.
Whichever method is used to solve the problem, it is essential to ensure
that candidates produce sufficient evidence to fulfil all the required
performance criteria. Suggestions to aid professional judgement in
ensuring that performance criteria are covered are given in the support
notes of both unit specifications.
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ADVICE FOR PROBLEM-SOLVING OUTCOMES
A case study of a practical investigation that was used by candidates in a
presenting centre to solve problems is described below.
Title
‘Immobilisation of enzymes’
Introduction
As a learning activity to demonstrate immobilisation, candidates
entrapped yeast invertase within alginate beads, then assayed the
immobilised enzyme by quantitatively measuring product formed using a
standard curve. (Many experiments used as learning activities can form
the basis of problem-solving exercises.)
The problem
Following this activity, several candidates started to identify potential
problems associated with immobilisation. Some wanted to know if
immobilisation changed the pH and temperature optima of the enzyme;
others wanted to know how often the immobilised enzyme could be
used before it stopped making product. Both groups realised that these
problems may be genuine in the biotechnology industry if an enzyme is
to be immobilised for commercial purposes. (Note that these problems
have a real practical application that can help in the evaluation of the
exercise.)
The procedure
These candidates used the knowledge and practical skills they had
previously gained from immobilising enzymes to identify the resources
and to plan and design their problem-solving activities.
The evaluation
The candidates found out that the pH optimum changed, the
temperature optimum stayed the same and the immobilised enzyme
could be used three times before the quantity of product decreased.
Other learning activities that can be used as the basis of problem-solving
activities are given in the support notes of each unit specification. They
are as outlined below.
• Set up a small-scale laboratory fermenter and monitor and control
various conditions, such as pH and temperature.
• Autolyse yeast and test viability at different stages in a downstream
process.
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• Investigate the effect of pectinase, amylase, cellulose and RGase on
the production and clarity of fruit juice.
• Investigate the action of cellulase on cellulose.
• Investigate methods of removing immobilised enzyme beads from the
substrate.
• Analyse data on DNA profiling.
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