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Biotechnology

Unit 3: Biotechnology

Student Materials

[HIGHER]

Margot McKerrell

abc


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Acknowledgement

Learning and Teaching Scotland gratefully acknowledge this contribution to the National

Qualifications support programme for Biotechnology. The advice of Jim Stafford isacknowledged 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

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.

Learning and Teaching Scotland


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UNIT 3: BIOTECHNOLOGY (HIGHER, BIOTECHNOLOGY) 3

CONTENTS

Introduction 5

Section 1: Biotechnological processing 7

Large-scale cell and tissue culture production 9

Comparison of batch and continuous flow processes 14

Downstream processing 16

Enzymes in production 25

Production of transgenic organisms 30

New breeding techniques 32

Section 2: Biotechnology applications 35

Agriculture and horticulture 36

Clinical and forensic medicine 37

Environment 45

Bibliography 47

Appendix: Advice for problem-solving outcomes 51



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UNIT 3: BIOTECHNOLOGY (HIGHER, BIOTECHNOLOGY)4



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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, geneticallyengineered 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

Sterilisation

Micro-organisms

Fermentation in a

fermenter vessel

Separation of liquid

and solid waste

Extract liquid

Concentrate

product

Purify product

Solid waste

Disposal



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UNIT 3: BIOTECHNOLOGY (HIGHER, BIOTECHNOLOGY)8

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 pH required for maximum growth of the micro-organism

the range of temperature needed for maximum growth of the micro-

organism

the quantity of oxygen needed by the micro-organism

the nutrient supply needed by the micro-organism.

Laboratory models can also be used to investigate:

the range of substrates that can be used

the rate at which nutrients are used up

the stage when useful products are produced

the 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 micro-

organism. 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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UNIT 3: BIOTECHNOLOGY (HIGHER, BIOTECHNOLOGY)1 0

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 theculture 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 processknown as downstreamprocessing).

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 fermenteris 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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UNIT 3: BIOTECHNOLOGY (HIGHER, BIOTECHNOLOGY) 1 1

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 aerobicconditions

(where oxygen is added) or anaerobicconditions (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 andharvesting 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 valveis 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 micro-

organism 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 ratesmay 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 spargercan 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 anychanges 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 monitoringthe 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 cultureor 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 ofnutrients 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 notcritical 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 continuousremoval 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, suchas 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

Advantages of continuous culture include:

increased productivity

continuous supply of product.

Downstream processing

After fermentation, the desired product must be extracted from theculture medium and purified. The extraction and purification of a

product is known as downstream processingbecause, 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.

Product (1 metabolite)



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Figure 4: Steps involved in downstream processing

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 inliquid 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.

Liquid

product

Fermentation

Disruption

of cells

Separation of cells

from liquid medium

Cells

Freeze drying

Dried cells

Liquid medium

Extraction of

product

Purification of

product

Drying

Solid

product



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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 vesselafter 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. Theyeast 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 centrifugedfrom 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 theassociated energy and maintenance costs.

A method used to remove bacterial cells from liquid culture is to freeze-

drythe 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 theaddition 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

0.001m to 0.02m (a m is 106of 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 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

lysozymeresin 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

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.

Interferon is eluted

from the column withbuffer that breaks the

interferonantibody

bond



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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 highlevel 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 lipid-

based stains

in the baking and brewing industries, to break down starch in flourand 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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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 organismis one with DNA (a gene) inserted into it from adifferent (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 micro-

organisms 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 offoreign 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 geneticallymodified with the foreign DNA.

The following procedure outlines how a transgenic plant can be

produced using A. tumefaciens and its plasmid.

1. 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. Protoplastsare 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 improve crop yields

to protect against pests and diseases

to protect against herbicides (weedkillers)

to protect against harsh environments

to increase the variety of available plants.



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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 rateof 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, andassuming 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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BIOTECHNOLOGY APPLICATIONS


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 transgenicwheat 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 micro-

organism) 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 mustbe 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 micro-

organism 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. Thissurface 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 cellsthat live indefinitely (they are said to be

immortal, assuming they are looked after properly!). The fused B

lymphocyte and cancer cell are called hybrid cellsand 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 thedesired 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 testingThe 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 immunodeficiencyvirus (HIV), or if they are suffering from

infectious diseases such as glandularfever. A sample of the patients

blood is mixed with a labelled monoclonal antibody if the sample

changes colour, then the patient has been exposed to the micro-

organism.

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 antibodydrug complex and the monoclonalantibody 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 sheepand cattle.

Drugs that have been produced using transgenic animals include

interferon(used to fight viral infections), blood clotting factorsand

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 inmilk 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 sheeps 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 sheeps 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, Parkinsons

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 areunique 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

Half of our DNA is inherited from our mother; the other half from our

father.

If you look at the childs 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 inpaternity 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.

1

Mother

2

Child

3

Father



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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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BIBLIOGRAPHY

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-HillISBN: 0-697-35452-0

This is a general introductory microbiology book that is a good teachers

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 teachers

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 HigherBiotechnology.

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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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.

http://www.ncbe.reading.ac.uk/http://localhost/var/www/apps/conversion/tmp/scratch_2/www-saps.plantsci.cam.ac.ukhttp://www.scottishbiotech.org/http://www.sserc.org.uk/http://www.scottish-enterprise.com/http://www.scottish-enterprise.com/http://www.sserc.org.uk/http://www.scottishbiotech.org/http://localhost/var/www/apps/conversion/tmp/scratch_2/www-saps.plantsci.cam.ac.ukhttp://www.ncbe.reading.ac.uk/


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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 thecandidate.

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) The problem to be solved is identified.

(b) Resources required to solve the problem are identified and

obtained.(c) Procedures appropriate to solving the problem are planned

and designed.

(d) The planned procedures are carried out.

(e) 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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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 problemFollowing 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.

Documents

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