Biotechnology
Unit 1: Microbiology
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. The drawings on pages 16, 21 and 41 are based on
illustrations in Foundations in Microbiology, by Kathleen Park Talaro and Arthur Talaro
(WCB/McGraw-Hill, 1999).
First published 2004
© Learning and Teaching Scotland 2004
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 048 2
CONTENTS
Introduction
1
Section 1:
Structure of micro-organisms
3
Section 2:
Microbial metabolism
19
Section 3:
Patterns of growth
29
Section 4:
Copying and translating genes
39
Section 5:
Genetic engineering
55
Section 6:
Infection and immunity
67
Bibliography
75
Appendix:
79
Advice for problem-solving outcomes
UNIT 1: MICROBIOLOGY (HIGHER BIOTECHNOLOGY)
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UNIT 1: MICROBIOLOGY (HIGHER BIOTECHNOLOGY)
INTRODUCTION
This unit introduces you to the micro-organisms that are used in
biotechnology. A micro-organism is any small organism that cannot be
clearly seen without the help of a microscope. The study of microorganisms is known as microbiology. The micro-organisms that you will
study are bacteria, fungi and viruses.
Before starting the study of micro-organisms, you should be aware of the
system used to name micro-organisms as you will be introduced to
several micro-organisms in this unit. Most micro-organisms are given two
names and, when the name of the micro-organism appears in printed
text, it is written in italics, for example Eschericia coli and
Saccharomyces cerevisiae. If you are handwriting the name of a microorganism, the convention is to underline its name, for example
Eschericia coli.
You may have noted that the first name of the micro-organism is given a
capital, upper case letter whereas the second name is written using a
small, lower case letter.
Finally, once you have written the full name of a micro-organism you can
abbreviate the first name the next time you write it. Eschericia coli is
abbreviated to E. coli and Saccharomyces cerevisiae is shortened to S.
cerevisiae.
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SECTION 1
The purpose of this section is to introduce you to the following
concepts:
• the structure of bacteria, fungi and viruses
• the function of some of the structures found within these microorganisms
• the uses of bacteria, fungi and viruses in biotechnology.
Understanding how micro-organisms work allows you to understand
why micro-organisms are so important in the processes used by the
biotechnology industry.
Prokaryotes and eukaryotes
All living organisms, including most micro-organisms, can be divided
into two groups depending on their basic cellular structure. The two
groups are known as prokaryotes and eukaryotes.
A prokaryote is an organism whose cells have a genome that is not
contained within a nucleus. The genome is the genetic material or
information that controls the activities of the cell. All bacterial cells are
prokaryotes.
Fig. 1 shows a typical bacterial cell whose genome is organised into a
single circular chromosome.
Figure 1: A typical bacterial cell
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STRUCTURE OF MICRO-ORGANISMS
In eukaryotes the genetic material is organised into chromosomes and
stored within a membrane-bound structure called the nucleus, found
inside the cell. Eukaryotic cells also have other membrane-bound
structures known as organelles that are not found in prokaryotic cells.
The cells of animals, plants and fungi are examples of eukaryotic cells.
Figure 2: A typical eukaryotic cell
Lysosomes
Table 1 on the next page outlines the general functions of these
organelles.
While bacterial cells are classified as being prokaryotes and fungal cells
are eukaryotes, it is not possible to classify viruses in the same way. As
you will find out later, viruses do not have a cellular structure and so
they are neither prokaryotes nor eukaryotes.
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Table 1: The functions of organelles
Organelle
Function of the organelle
Mitochondrion
(plural Mitochondria)
Involved in the production of energy
within the cell through the process of
aerobic respiration.
Chloroplast
Used in the process of photosynthesis that
involves the making of sugar using light as
an energy source. Found only in plant cells
and some algae.
Endoplasmic reticulum
Rough endoplasmic reticulum is involved
in the production and transport of
proteins. Smooth endoplasmic reticulum is
involved in the making and transport of
lipids.
Golgi apparatus
Stores, modifies and packages proteins to
be transported out of the cell.
Lysosomes
These contain digestive enzymes which
help to breakdown materials taken into the
cell, e.g. bacteria. Found mainly in animal
cells.
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STRUCTURE OF MICRO-ORGANISMS
Test yourself on prokaryotes and eukaryotes
Before moving onto the next part of this unit, read over your notes on
prokaryotes and eukaryotes again and then answer the following
questions.
1.
Write down a definition of a prokaryote and a eukaryote.
2.
What is the function of the genome in a prokaryote?
3.
Give three examples of organisms composed of eukaryotic cells.
4.
What is the function of mitochondria?
5.
Name the organelle involved in the storage, modification and
packaging of proteins.
6.
Name an organelle found only in plant cells and some algae.
7.
What is the function of a lysosome in an animal cell?
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Bacteria
Bacteria are single-celled organisms. This means that each bacterial cell
is capable of surviving on its own. Individual bacterial cells can be seen
using a light microscope. Although bacteria are single-celled, they often
exist in a colony consisting of many thousands of bacterial cells. A
bacterial colony can be seen with the naked eye.
As mentioned previously, bacteria are prokaryotes. This means that the
genome (in the form of a circular chromosome) is not contained within
a nucleus. Also, prokaryotes do not have the membrane-bound
structures (organelles) found in eukaryotes.
Fig. 1 shows some of the main structures that are found in a typical
bacterial cell such as flagellum, gelatinous capsule, cell wall, ribosomes, a
circular chromosome and a plasmid. It should be noted that not all
bacteria have flagella, nor do they all have gelatinous capsules and
plasmids. However, the other structures are found in all bacteria.
Table 2 shows the functions of these structures within a bacterial cell.
Table 2: Bacterial cell structures and their functions
Structure
Function within in a bacterial cell
Flagellum
Has a rotating motion which enables the bacterial cell
to move. It is found on some motile (actively moving)
bacteria.
Gelatinous
capsule
Allows the bacterium to survive in dry areas. It can
trap other bacteria. It can help the bacterium evade the
immune system of a host.
Cell wall
It gives shape and support to the bacterial cell. It
protects the cell from physical damage and from
changes in the water content of its environment.
Ribosome
Involved in making protein for the bacterial cell.
Circular
chromosome
Contains all the genetic information (in the form of
genes) needed to control all the activities of the
bacterial cell.
Plasmid
A small circular piece of DNA in addition to the circular
chromosome. It gives the bacteria extra properties
such as the ability to resist certain antibiotics or to
produce toxins. It can be transferred from one
bacterial cell to another. It is not present in all bacteria.
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When viewed under a microscope, bacteria are observed to have a
definite shape. Three shapes are commonly seen – round, rods and
spirals. Microbiologists use the shape of bacteria to help identify and
categorise them.
Round bacteria are called cocci, rod-shaped bacteria are called bacilli
and spiral bacteria are called spirilla.
Figure 3: Shapes of bacteria
Another method that microbiologists use to identify and categorise
bacteria is to stain them using the Gram stain.
A sample of the bacterial cells to be identified is smeared onto a
microscope slide, soaked in a violet dye (crystal violet) and then treated
with iodine. The violet dye binds irreversibly to some types of bacteria
but not to others, depending on the composition of their cell walls. The
slide is washed with alcohol to remove the violet dye (if it has not bound
irreversibly to the bacteria), then counterstained with a red dye
(safranin). Bacterial cells that do not bind the violet dye become stained
with this red dye. At the end of the staining procedure, the bacterial
cells are either stained purple or red.
Bacterial cells that appear purple have retained the crystal violet dye
and are called Gram positive (G+).
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Bacterial cells that appear red have not retained the violet dye and are
called Gram negative (G-).
The different staining reactions are due to differences in the cell walls of
the different types of bacteria. Gram positive bacterial cell walls are thick
with over 40% peptidoglycans (a type of carbohydrate) in their
structure whereas Gram negative bacterial cell walls have significantly
less peptidoglycans.
Penicillin is an antibiotic that is effective against Gram positive bacteria
because it interferes with the cross linking of the peptidoglycan in the
cell wall. This causes gram positive bacteria to produce weak cell walls
which, in turn, results in the bacteria swelling as water enters the cell.
When treated with penicillin, Gram positive bacteria also divide less
frequently. Penicillin is less effective against infections caused by Gram
negative bacteria.
Bacteria are commonly used in biotechnology processes. The two main
areas that make use of bacteria are genetic engineering and
fermentation.
Plasmids are used in genetic engineering because they are easily
modified by the addition of new genes. The modified plasmids are
introduced into bacteria which then produce a useful new substance.
The genetically modified bacteria are grown in industrial-scale
fermenters to produce large quantities of the new product, which might
be a vitamin or a drug.
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STRUCTURE OF MICRO-ORGANISMS
Test yourself on bacteria
Before you move onto the next part of this unit, spend a little time
reviewing your notes on bacteria, then see if you can answer the
questions below.
1.
Name the structure that gives shape and support to the bacterial
cell.
2.
Give the function of a flagellum.
3.
Describe the composition of the cell wall of a bacterium that stains
purple with the gram stain.
4.
Describe how penicillin prevents the growth of gram positive
bacteria.
5.
Explain why plasmids are used in genetic engineering.
6.
The diagrams in Fig. 4 show the effect of using penicillin at
increasing concentrations (from 0 to 50%) on the growth of two
different bacteria:
10
(a)
Describe the effect the antibiotic has on the growth of
(i) E.coli and (ii) S. aureus.
(b)
What was the purpose of including 0% antibiotic?
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Figure 4: The effect of using penicillin at increasing concentrations on
the growth of two different bacteria
•••••••
= paper disc soaked in different % concentrations of antibiotic
•••••••
•••••••
•••••••
•••••••
•••••••
•••••••
•••••••
•••••••
•••••••
•••••••
•••••••
•••••••
Agar plate containing
E.coli
Agar plate containing
S.aureus
Fungi
Fungi are eukaryotes. This means that their genomes are stored in a
membrane-bound nucleus and that they have organelles within their cell
structure. (Look back at the section on prokaryotes and eukaryotes to
remind yourself of the structure and function of organelles.)
Some types of fungi are unicellular (single celled) whereas other types
are multinucleate (the fungus has more than one nucleus within each
compartment).
An example of a unicellular fungus is yeast, which is larger than a
bacterium and more complicated in structure.
One method by which yeast can increase its numbers is by the process of
asexual reproduction. In this process, which is called budding, each
new yeast cell that is produced is identical to the parent yeast cell from
which it is formed. Fig. 5 shows the process of budding in a yeast cell.
As you can see from this figure, the parent cell develops a bud or
swelling. The nucleus and other organelles of the parent yeast cell
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STRUCTURE OF MICRO-ORGANISMS
divide into two, and a nucleus and the new organelles move into the
bud. The bud continues to grow and eventually the bud separates from
the parent. At the end of budding, two yeast cells are present which are
identical to each other.
Figure 5: The process of budding in a yeast cell
Yeasts are important in biotechnology as they have been used for
thousands of years to make bread and to ferment alcoholic drinks such
as wine and beer. In more recent times, yeasts have been genetically
engineered to produce a variety of pharmaceutical proteins.
Mucor is an example of a multinucleate fungus. It consists of long, thin,
branched threads called hyphae that form a tangled mass called a
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mycelium, which looks like cotton wool. You may have seen evidence
of the growth of Mucor on mouldy bread! The hyphae are enclosed
within a cell wall and the cytoplasm passes through the hyphae. This is
shown in Fig. 6. As you can see from this diagram, there are several
nuclei within the cytoplasm and so it is referred to as multinucleate.
Remember, like yeast, Mucor is a eukaryote and so the cytoplasm also
contains all the organelles associated with a eukaryote.
Figure 6: Mucor is an example of a multinucleate fungus
Mucor can reproduce asexually (from a single parent) and the new
fungus produced is identical to the parent. In asexual reproduction,
Mucor produces lots of identical spores enclosed within structures
called sporangia as shown in Fig. 6. The spores are dispersed by means
of air currents. A new fungus will grow where a spore lands, assuming
the conditions are right for growth.
Mucor can also reproduce by sexual reproduction (from two parents)
which produces new fungi that are genetically different from the
parents. Fig. 7 shows the process of sexual reproduction in Mucor. This
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STRUCTURE OF MICRO-ORGANISMS
involves the fusion (joining) of two nuclei from different Mucor parents
(the parents are referred to as + and – hyphae). The fused nuclei form
a zygospore that eventually germinates to produce a new mycelium.
Mucor is one example of a multinucleate fungus but there are others –
some of which are very important in biotechnology. Multinucleate fungi
have been used for the large-scale production of a wide variety of
enzymes (for example, those used in washing powders) and for the
production of antibiotics, such as penicillin.
Figure 7: Sexual reproduction in Mucor
Developing
zygospore
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STRUCTURE OF MICRO-ORGANISMS
Test yourself on fungi
Before you move onto the next part of this unit, spend some time
reviewing your notes on fungi, then see if you can answer the questions
below.
1.
What do you understand by the following terms:
(a)
(b)
Multinucleate
Unicellular.
2.
Look at Fig. 5. State one feature in the diagram which shows that
yeast is a eukaryote and not a prokaryote.
3.
Describe the process of budding in yeast.
4.
Describe the process of sexual reproduction in Mucor.
5.
Give some uses of yeast and fungi in biotechnology.
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STRUCTURE OF MICRO-ORGANISMS
Viruses
Viruses do not have a cellular structure and so cannot be described as
either a prokaryote or a eukaryote. Instead, most viruses have a protein
coat, called a capsid, which encloses a central core of nucleic acid that
can either be DNA or RNA. Also, some viruses have an envelope that
surrounds the capsid. Fig. 8 shows the structures of some viruses.
Figure 8: The structure of viruses
Herpes virus
Bacteriophage
nucleic acid
protective
envelope
nucleic acid
capsid head
capsid
containing
nucleic acid
tail
fibres
Tobacco mosaic virus
helical RNA
capsid
Viruses can only reproduce inside living cells. Animal cells, plant cells
and bacterial cells are all attacked by viruses.
A virus that infects and reproduces itself inside a bacterial cell is known
as a bacteriophage. The process by which a bacteriophage replicates is
shown in Fig. 9 and is known as the bacteriophage lytic cycle.
The bacteriophage attaches to a specific site on the cell wall of the
bacteria and its DNA is injected into the bacterial cell. The viral DNA
prevents the bacterial cell from carrying out its normal metabolic
reactions and, instead, causes the bacterial cell to start replicating
(making new copies of) the viral DNA.
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The new copies of the viral DNA are used to produce the proteins
needed to form the capsid of the bacteriophage. These proteins then
arrange themselves around the copies of the viral DNA so that many new
bacteriophages are formed.
Finally, the bacterial cell wall is weakened which causes the bacterial cell
to burst (lyse) open, releasing the new bacteriophages. Each newly
released bacteriophage can now infect another bacterial cell and so the
cycle continues.
Sometimes, when a virus enters its host cell, the viral DNA transfers into
the host cell’s chromosomes, so that the viral DNA becomes part of the
host cell’s DNA. In this way, viral genes can become part of the host
cell’s genetic make up.
Viruses are important in biotechnology for several reasons. They are
cultured in large numbers for use in the production of vaccines against
viral diseases such as smallpox, polio, rubella and measles. Also, viruses
are used in genetic engineering to introduce new genes into animals
and plants where they are known as cloning vectors.
Figure 9: The bacteriophage lytic cycle
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STRUCTURE OF MICRO-ORGANISMS
Test yourself on viruses
Before you move onto the next part of this unit, spend a little time
reviewing your notes on viruses, then see if you can answer the
questions below
1.
What is the capsid of a virus?
2.
Put the following sentences into order to correctly describe the
bacteriophage lytic cycle:
(a)
(b)
(c)
(d)
(e)
(f)
New capsid proteins are produced.
A bacteriophage attaches to a bacterial cell wall.
New copies of bacteriophage DNA are made.
New bacteriophages are made.
Bacteriophage DNA is injected into the bacterium.
Bacterial cell lyses releasing new bacteriophages.
3.
What is the function of a cloning vector in a biotechnology
process?
4.
What type of micro-organism does a bacteriophage infect? Tick the
correct answer.
(a)
(b)
(c)
(d)
5.
Bacteria
Fungi
Viruses
All of the above
A bacteriophage is 0.2 µm in length. Given that 1 µm =1000
nanometres, calculate the length of the bacteriophage in
nanometres.
You have now completed the structure of micro-organisms. By now
you should be familiar with the differences between prokaryotes
and eukaryotes, the structures of bacteria, fungi and viruses and
have an appreciation of the uses of these micro-organisms in
biotechnology.
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MICROBIAL METABOLISM
SECTION 2
This section introduces you to the processes that occur within bacterial
and fungal cells to produce energy.
Energy release: the role of adenosine triphosphate (ATP)
The word metabolism refers to all the biochemical reactions that take
place inside any prokaryotic or eukaryotic cell. These biochemical
reactions can be split into two categories:
• those that are involved in the making of compounds inside the cell
• those that are involved in the breakdown of compounds in the cell.
Some of these biochemical reactions result in the production of energy,
others need energy to proceed.
In a cell the energy that is made or used up is in the form of a chemical
compound called adenosine triphosphate (ATP). As the name implies,
ATP is made up of an adenosine (A) unit linked to three phosphate (P)
groups, as shown below:
Figure 10: The structure of ATP
A
P
P
P
When the last phosphate is removed from ATP, energy is released. A
molecule of adenosine diphosphate (ADP) and a single phosphate
(known as inorganic phosphate or Pi) is also produced. This is shown
below:
ATP → ADP + Pi + Energy
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MICROBIAL METABOLISM
When energy becomes available to the cell, ATP can be regenerated by
reversing this process. ADP combines with Pi to form ATP as shown
below:
ADP + Pi + Energy → ATP
The cell uses the released energy to carry out a number of cellular
processes. For example, in a micro-organism, one of the processes that
energy is used for is reproduction, which increases the numbers in a
microbial population. When the supply of ATP is used up, microorganisms usually stop growing and die.
Although micro-organisms use ATP as a readily available source of
energy, it is not a suitable molecule for storing energy. Instead microorganisms use the energy released from ATP to make nutrient molecules
for energy storage. These can then be broken down to release energy to
produce ATP for the cell to use when required. Thus for microorganisms to grow in culture, they must be provided with the correct
nutrients that they can break down to release the ATP necessary for
their continued reproduction and growth.
A nutrient that is used to produce energy is glucose. It is broken down
by micro-organisms in a series of stages known as:
• glycolysis
• Krebs cycle (also known as the citric acid cycle or the tricarboxylic
acid (TCA) cycle)
• Cytochrome system (also known as the hydrogen carrier system or
the electron transport chain).
Collectively the three stages are referred to as respiration. When
oxygen is present, it is known as aerobic respiration and when oxygen
is absent from the cell, it is referred to as anaerobic respiration.
Glycolysis takes place in the cytoplasm of all cells.
The Krebs cycle and the cytochrome system occur inside the
mitochondria of eukaryotes. Fig. 11 shows the internal structure of a
single mitochondrion.
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Figure 11: Internal structure of a mitochondrion
Glycolysis
This process takes place in both eukaryotic and prokaryotic cells. The
following points summarise the main events of glycolysis:
• It occurs in the cytoplasm of the cell
• Glucose, which contains 6 carbon atoms, is broken down into pyruvic
acid, a 3-carbon molecule (2 molecules of pyruvic acid are produced)
• There is a net production of 2 ATP molecules
• Hydrogen is released which immediately binds to a coenzyme. When
hydrogen binds to this coenzyme, it is called a reduced coenzyme.
(In biology, the word ‘reduced’ refers to the binding of a hydrogen
atom to a compound, not to a decrease in the size of the compound!)
A coenzyme is an extra part of an enzyme that is needed for the
enzyme to function correctly.
• The reduced coenzyme is used by the cytochrome system
• It occurs whether oxygen is present or not.
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Figure 12: A simplified flow diagram of glycolysis
Glucose
(6-carbon molecule)
2 ATP produced
reduced coenzyme produced
Pyruvic acid
+
Pyruvic acid
(3-carbon molecule)
Krebs cycle
The Krebs cycle takes place only when oxygen is present in the cell, so it
is involved only in aerobic respiration.
In eukaryotes, the Krebs cycle takes place in the matrix of the
mitochondria.
Figure 13: A simplified flow diagram of the Krebs cycle
Pyruvic acid
V
CO2
V
Acetyl coA
V
4-carbon
molecule
V
Krebs
cycle
V
V
V
Reduced
coenzyme
Tricarboxylic acid
CO2
ATP
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The main points of the Krebs cycle are as follows:
• Pyruvic acid (formed from glycolysis) diffuses into the mitochondria
where it loses a carbon atom to become a 2-carbon molecule called
acetyl coenzyme A (acetyl Co A). The carbon that is removed diffuses
out of the mitochondria as carbon dioxide
• Acetyl Co A (with 2 carbons) reacts with a 4-carbon compound to
form a 6-carbon compound called tricarboxylic acid (also known as
citric acid).
• Tricarboxylic acid is gradually converted back, step by step, to the 4carbon compound. This is why this series of reactions is known as a
cycle, as the original 4-carbon compound is regenerated
• 2 ATP molecules are produced
• Carbon dioxide is released
• Hydrogen is released that immediately binds to a coenzyme which
becomes a reduced coenzyme
• The reduced coenzyme is used by the cytochrome system.
Cytochrome system
The cytochrome system is found in the inner folds, the cristae, of the
mitochondria of eukaryotes. It occurs only when oxygen is present in
the cell, so it is involved in aerobic respiration. Its function is to
produce ATP molecules in large quantities.
The reduced coenzymes formed during glycolysis and the Krebs cycle
are said to be energy-rich molecules because they contain a pair of
electrons that are passed to other electron carriers. At the same time
that the electrons are transferred to another carrier, the hydrogen that
the reduced coenzyme was carrying passes into the cytoplasm. Each
time a pair of electrons passes from one carrier to the next, an ATP
molecule is produced.
Fig. 14 below shows how the cytochrome system works:
Figure 14: The cytochrome system
ADP + Pi
ADP + Pi
ADP + Pi
Cytochrome
V
V
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V
MICROBIAL METABOLISM
Reduced coenzyme (NADH) gives its pair of electrons to coenzyme 2
(FAD). Reduced coenzyme (NADH) becomes coenzyme again (NAD),
and so it can pick up another hydrogen from glycolysis or the Krebs
cycle.
When coenzyme 2 (FAD) accepts the pair of electrons from reduced
coenzyme (NADH), coenzyme 2 (FAD) now becomes reduced coenzyme
2 (FADH 2). The energy released from this electron transfer is used to
form ATP from ADP and Pi.
Reduced coenzyme 2 (FADH2) now passes the pair of electrons to
cytochrome and so becomes coenzyme 2 (FAD) again. It is now able to
accept another pair of electrons from reduced coenzyme.
Cytochrome, in accepting the pair of electrons, now becomes reduced
cytochrome. Again, when the pair of electrons pass from reduced
coenzyme 2 to cytochrome, the released energy is used to make another
molecule of ATP.
A third ATP molecule is produced when the pair of electrons from
reduced cytochrome is passed to molecular oxygen. When oxygen
accepts the pair of electrons, along with hydrogen from the cytoplasm,
water is formed as a by-product. Because oxygen is the final electron
acceptor, the cytochrome system functions only when oxygen is present
in the cell.
In total 34 ATP molecules are formed from the cytochrome system.
Anaerobic respiration
As mentioned, the Krebs cycle and the cytochrome system work only
when oxygen is present in the cell.
However, if oxygen is absent, glycolysis still takes place. Pyruvic acid is
made and two molecules of ATP are produced. When glycolysis occurs in
the absence of oxygen, it is called anaerobic respiration and sometimes
it is referred to as fermentation.
Some bacteria convert their pyruvic acid into lactic acid and this is
known as lactate fermentation. Streptococcus lactis is a bacterium that
produces lactic acid and it is used by the dairy industry in the
production of buttermilk.
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Other bacteria, such as Acetobacter species, produce acetic acid
(vinegar) from pyruvic acid.
Yeasts convert pyruvic acid into ethanol and carbon dioxide when they
are grown in the absence of oxygen. This is known as alcohol
fermentation. Saccharomyces cerevisae is an example of a yeast that is
used to produce alcohol for the brewing industry.
Comparison of aerobic and anaerobic respiration
Table 3 gives a brief comparison of aerobic and anaerobic respiration.
Table 3
Feature of respiration
Type of respiration
Anaerobic
Location within the cell
Number of ATP
Aerobic
Cytoplasm
2
Mitochondria (in eukaryotes)
38
Lactic acid
Carbon dioxide and water
molecules produced
Products formed
Acetic acid
Ethanol
The 38 molecules of ATP formed as a result of aerobic respiration come
from glycolysis (2), Krebs cycle (2) and the cytochrome system (34).
Industrial fermentation
The large-scale industrial growth of micro-organisms is referred to as
fermentation, regardless as to whether the micro-organisms are grown
in the presence or absence of oxygen.
As to whether a fermentation process is carried out in the presence or
in the absence of oxygen depends on the micro-organism that is being
used in the fermentation and the product being formed.
The following table summarises the different types of micro-organisms
depending on their need for oxygen for growth:
UNIT 1: MICROBIOLOGY (HIGHER BIOTECHNOLOGY)
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MICROBIAL METABOLISM
Table 4
Name given to the microorganism Oxygen requirement for growth
Obligate aerobes
These micro-organisms grow only
in the presence of oxygen as it is
the final electron acceptor in
their cytochrome system
Obligate anaerobes
These micro-organisms grow only
when there is no oxygen present.
Oxygen is toxic to these
microorganisms
Facultative anaerobes
These micro-organisms can grow
in the presence or absence of
oxygen
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MICROBIAL METABOLISM
Test yourself on energy release
Before you move onto the next part of this unit, spend a little time
reviewing your notes on aerobic respiration, anaerobic respiration and
industrial fermentation, then see if you can answer the questions below
1.
How many ATP molecules are produced when one molecule of
glucose is broken down in the presence of oxygen?
2.
Compare the products produced when glucose is broken down by
aerobic respiration and by anaerobic respiration.
3.
Fig. 15 shows some of the steps of cellular respiration in yeast.
(a)
Name compounds X and Y.
(b)
Name process Z and cycle W.
(c)
What happens to hydrogen atoms when they are released
from cycle W?
(d)
Name the organelle in which aerobic respiration takes place.
Figure 15
ATP
ATP
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MICROBIAL METABOLISM
4.
Outline the role of the electron transport chain in the production
of ATP.
5.
Yeast is able to respire in the presence and absence of oxygen.
28
(a)
To which group (obligate aerobe, obligate anaerobe or
facultative anaerobe) does yeast belong?
(b)
What products would you expect if yeast were grown in a
fermenter under anaerobic conditions?
(c)
When grown anaerobically, yeast produces energy in the form
of heat. How could you physically measure this energy
production in a fermenter?
UNIT 1: MICROBIOLOGY (HIGHER BIOTECHNOLOGY)
PATTERNS OF GROWTH
SECTION 3
The purpose of this section in the unit is to introduce you to the factors
that influence the growth of a micro-organism. This is important if you
want to grow micro-organisms in culture successfully or if you wish to
prevent their growth. This section also looks at the different phases that
a bacterial culture goes through as it is growing in a culture vessel.
For most micro-organisms, growth involves an increase in the size of the
cell, followed by cell division. Therefore, growth of a micro-organism is
an increase in the number of cells of the micro-organism.
Micro-organisms grow at their optimum rate only if all the external
factors are suitable.
Factors affecting growth
There are many factors that affect the growth of a culture. It is important
to have knowledge of these factors so that you understand why cultures
must be grown under certain conditions to achieve maximum growth.
For example, in an industrial situation it is important to have optimum
growth conditions so that the maximum product is formed.
Knowledge of factors that affect growth is not just important for
understanding how to grow micro-organisms to their maximum. This
knowledge can be applied also to prevent the growth of microorganisms. For example, in food preservation, the environment is
altered so that the growth of micro-organisms is slower and spoilage of
food prevented.
Temperature
Temperature is one factor that affects microbial growth. Micro-organisms
grow fastest in their optimum temperature ranges. Some microorganisms grow over a narrow range of temperature; for example, the
micro-organisms that cause disease grow between 30 oC and 38 oC.
Other micro-organisms grow over a broad range of temperature. Those
isolated from soil can grow from about 5 oC to about 40 oC or higher.
There are even some micro-organisms, such as those found in compost
heaps, which can grow at very high temperatures (above 45 oC).
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PATTERNS OF GROWTH
However, as temperature decreases below, or increases above the
optimum, growth of the micro-organism slows down. At temperatures
above the optimum, enzymes within the micro-organism become
denatured and so stop working. This prevents the growth of the microorganism.
pH
Another factor that affects growth is pH. Different micro-organisms grow
at different optimum pH values. In general, bacteria prefer to grow in
neutral conditions (pH 6.5 to pH 7.5) whereas fungi prefer acidic
conditions (pH 4.0 to pH 6.0). Most micro-organisms do not grow at
very low pH values and this knowledge is used in food preservation.
Vinegar, citric acid and lactic acid are widely used as food preservatives
as they stop the growth of micro-organisms. Now you know why onions
are pickled in vinegar!
When growing micro-organisms in culture, the medium is often
buffered to prevent changes in the pH of the culture medium.
Oxygen
Oxygen concentration is another factor affecting the growth of microorganisms. Look back at the previous section (Table 4) to remind
yourself of the names given to different micro-organisms depending on
their requirement for oxygen for growth.
• What micro-organisms grow only in the presence of oxygen?
• What micro-organisms grow only in the absence of oxygen?
• What micro-organisms can grow in the presence or absence of
oxygen?
Many micro-organisms that spoil meat and fish are obligate aerobes. This
is why meat and fish are sometimes vacuum packed in airtight wrapping
to prevent these micro-organisms from growing and so spoiling the
food.
Water
The concentration of solutes and water in the growth medium also
affects the growth of micro-organisms.
Water is essential for microbial growth as all the substances required for
growth are dissolved or suspended in water within the micro-organism.
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PATTERNS OF GROWTH
All micro-organisms have a natural internal concentration of solutes,
such as salts and sugar. If a micro-organism is placed in culture medium
that has a greater concentration of solutes than that inside the microorganism, solutes may enter the micro-organism by diffusion and water
may leave by osmosis. This upsets the balance within the micro-organism
and its growth slows down.
[Diffusion is the movement of solutes from an area of high
concentration to an area of lower concentration. Osmosis is the
movement of water from where it is in high concentration (for example
a dilute solution) to an area of lower concentration (a more
concentrated solution).]
Similarly, if a micro-organism is cultured in a medium with a lower
concentration of solutes than that inside the micro-organism, then
solutes will leave the micro-organism by diffusion and water will enter by
osmosis. Again, this upsets the natural internal balance and the
microorganism’s growth slows down or stops.
Pressure
Pressure is another factor to affect the growth of micro-organisms. Most
micro-organisms grow at atmospheric pressure, although small increases
in pressure do not generally affect their growth. Some micro-organisms
that live deep in the oceans have adapted to survive pressures higher
than atmospheric pressure while micro-organisms that live in high
mountains survive in pressures slightly lower than atmospheric
pressure. If micro-organisms that normally grow at atmospheric pressure
are placed in too high or too low a pressure, then they are unable to
grow in these extremes of pressure.
Nutrients
The last factor to be considered which affects the growth of microorganisms is nutrient availability. A nutrient is said to be available if it is
in a form that the micro-organism can take up directly. Available
nutrients include simple sugars (such as glucose) and amino acids.
Starch is a large complex molecule made up of many glucose units
bonded together. It is too large to be taken up by micro-organisms and
so the glucose within this molecule is unavailable to the micro-organism.
However, some micro-organisms secrete enzymes that can digest starch
to glucose, so making this nutrient available to them.
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PATTERNS OF GROWTH
Similarly, protein is made up of many amino acids joined together and
some micro-organisms secrete an enzyme that breaks down protein into
amino acids. Again this makes the amino acids available to the microorganism.
When micro-organisms are cultured, the growth medium generally
contains available nutrients for the micro-organism to use directly for its
growth.
Also, growth media contain mineral nutrients such as nitrate and
phosphate. Nitrate is needed by micro-organisms for making protein
and nucleic acids while phosphate is needed for making nucleic acids
and phospholipids.
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PATTERNS OF GROWTH
Test yourself on factors that affect growth of micro-organisms
Before you move onto the next part of this unit, spend a little time
reviewing your notes on factors affecting growth, then see if you can
answer the questions below.
1.
Why is it important for culture medium to contain readily available
glucose?
2.
Fig. 16 shows the effect of temperature on the growth of bacteria.
(a)
Over which range of temperature is there optimum growth of
bacteria?
(b)
Explain why at 50oC, there is no growth of bacteria.
Figure 16
5
3.
10
15
20
25
30
35
40
45
50
What are the meanings of the following terms:
(a)
obligate aerobe
(b)
facultative anaerobe?
4.
Explain why the growth of a micro-organism slows down if it is
placed in culture medium with a higher concentration of solutes
than the intracellular concentration of the micro-organism.
5.
Why do you think it is important to monitor pH in a fermenter
being used to grow micro-organisms?
UNIT 1: MICROBIOLOGY (HIGHER BIOTECHNOLOGY)
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PATTERNS OF GROWTH
The bacterial growth curve in liquid medium
Now that you have an understanding of some of the factors that affect
the growth of micro-organisms, we shall look at the growth of bacteria in
a culture medium with the correct oxygen concentration, containing all
the nutrients needed by the bacteria and at the bacteria’s optimum pH
and temperature.
Will the number of living (viable) bacterial cells increase and continue to
increase indefinitely? (Remember that the number of viable bacterial
cells is a measure of the growth of a micro-organism.)
Look at Fig. 17. This shows a typical bacterial growth curve of the
number of viable bacteria in the culture medium in relation to time. You
can see that the growth of the bacterial cells follows a number of phases.
These phases are called the lag (or latent or initial) phase,
exponential (or log) phase, stationary phase and final (or death or
senescent) phase.
In answer to the question above, the graph clearly shows that viable
bacterial cells do not continue to grow indefinitely despite being placed
initially in medium containing all the factors needed for growth.
Figure 17: A bacterial growth graph
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PATTERNS OF GROWTH
What happens to the bacteria during each of these growth phases?
The lag phase begins with the bacterial cells being introduced
(inoculated) into the new culture medium. During the lag phase there is
little or no increase in bacterial cell numbers, although the cells may
increase in size. During this phase, the bacterial cells are adapting to
their new growth conditions, for example by producing enzymes to
process the nutrients present in the growth medium.
During the exponential phase the bacterial cells double at a constant
rate. The actual time that the bacteria take to double depends on the
culture medium and the temperature. The time taken for the numbers
of bacterial cells to double is called the doubling rate.
It is the exponential phase that is the most suitable phase for carrying
out experiments to find out growth rates and to investigate the factors
that affect growth.
In the stationary phase there is no increase in the number of viable
bacterial cells. The number of new cells being produced is equivalent to
the number of bacterial cells that are dying. During this phase there is
no further increase in bacterial cell growth because the available
nutrients are starting to be used up. Also, conditions such as pH may
have altered to such an extent that they are now inhibiting the growth
of the bacteria.
During the death phase the bacterial cells die due to starvation and/or
the adverse environmental conditions.
UNIT 1: MICROBIOLOGY (HIGHER BIOTECHNOLOGY)
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PATTERNS OF GROWTH
Test yourself on the bacterial growth curve
Before you move onto the next part of this unit, spend a little time
reviewing your notes on the bacterial growth curve then see if you can
answer the questions below.
1.
(a)
Sketch a graph to show the growth curve of bacteria.
(b)
Label the following phases on the graph:
lag phase
log phase
stationary phase
2.
Describe the events that occur during lag phase and stationary
phase.
3.
A fungus produces an antibiotic. The fungus is grown in a
fermenter and the antibiotic, released into the growth medium, is
measured over a period of time. The results are shown in Table 5.
Table 5
36
Time (hours)
Antibiotic concentration (mg/ml)
0
0
15
8
30
40
45
72
60
100
UNIT 1: MICROBIOLOGY (HIGHER BIOTECHNOLOGY)
PATTERNS OF GROWTH
(a)
Draw a graph to show the increase in antibiotic concentration
with time.
Antibiotic
concentration
(mg/ml)
(b)
4.
From your graph work out the time taken to produce 55 mg/
ml of antibiotic.
A bacterium was grown in a fermenter. The mass of the bacterium
at the beginning (0 hours) was 2 g/l. After 30 minutes, the mass of
bacteria had risen to 62 g/l. Calculate the increase in mass of
bacteria per hour.
You have now completed this section on the growth of microorganisms. You should now be able to carry out the following
tasks:
•
•
•
•
•
Name the factors that affect growth of micro-organisms;
Explain why these factors affect growth in the way that they do;
Draw the general shape of a bacterial growth curve;
Name the phases observed in the growth curve;
Describe the events that occur in each phase of the growth curve.
UNIT 1: MICROBIOLOGY (HIGHER BIOTECHNOLOGY)
37
38
UNIT 1: MICROBIOLOGY (HIGHER BIOTECHNOLOGY)
COPYING AND TRANSLATING GENES
SECTION 4
In Section 1 of this unit (Structure of Micro-organisms), you were
introduced to the concept that all cells have a genome organised into
chromosomes, which control all the activities of the cell.
The genome itself consists of a series of genes, many of which code for
proteins. These genes are made of a nucleic acid called
deoxyribonucleic acid (DNA).
In this section you will find out about the following:
• the structure of deoxyribonucleic acid;
• how genes control the cell by directing the making of proteins within
the cell;
• how genes in prokaryotes are regulated.
The structure of DNA
The genes that make up a chromosome are made of a nucleic acid called
deoxyribonucleic acid (shortened to DNA). This is a long, threadlike
molecule consisting of two strands twisted into a helical (spiral)
molecule.
The building blocks of each strand of the DNA molecule are called
nucleotides that are joined together to form a long chain. Each
nucleotide consists of three components: a phosphate group, a sugar
molecule called deoxyribose and an organic base.
These three components are arranged in the following way:
5′
4′
1′
3′
2′
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COPYING AND TRANSLATING GENES
The deoxyribose sugar is given a special numbering system in that each
carbon atom (of which there are 5) is given a number. The carbon atoms
of the sugar that are known as 3′ (said 3 prime) and 5′ (said 5 prime) are
shown in the above diagram.
There are 4 organic bases found in a DNA molecule, namely ADENINE
(A), GUANINE (G), CYTOSINE (G) and THYMINE (T), and so there are 4
different types of nucleotides found in DNA, each containing a different
organic base.
As mentioned, the nucleotides join together to make a long strand of
DNA. The phosphate group linked to the 5’ end of one nucleotide joins
to the 3’ of the sugar of the neighbouring nucleotide, thus forming a
phosphate–sugar backbone. In this way the nucleotides form a long
single strand of DNA, one end with a 5’ phosphate and the other end
with a free 3’ group on the sugar.
Two strands of nucleotides link together with weak hydrogen bonds
between their organic bases. The two strands of nucleotides run in
opposite directions to each other, so they are said to be antiparallel.
One strand starts with a 5′ end and finishes with a 3′ end, while the
other strand starts with a 3′ end and finishes with a 5′ end:
5′
3′
3′
5′
Each organic base in a nucleotide from one strand can form a hydrogen
bond with only one other type of organic base in a nucleotide in the
other strand:
• A bonds only with T
• C bonds only with G
• A-T and C-G are known as base pairs.
A fragment of double stranded DNA showing only the sequence of
organic bases in each strand is represented below:
5′ AGCTTGCATTAACGTCGC 3′
3′ TCGAACGTAATTGCAGCG 5′
One strand is known as the sense strand, while the other is called the
antisense strand. You will come across these terms again when the
40
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COPYING AND TRANSLATING GENES
making of another type of nucleic acid (called messenger ribonucleic
acid) is discussed.
The double strand of DNA is twisted into a structure called a double
helix. This resembles a spiral staircase with the phosphate-sugar
backbone forming the uprights and the base pairs forming the rungs
(Fig. 18).
Figure 18
V
Base pairs
V
Phosphate-sugar
backbone
V
V
Sugar
Phosphate
Chromosomes and genes in eukaryotes
In eukaryotes chromosomes are found in the nucleus. Under a very high
powered microscope the chromosomes appear to be striped. Each
stripe represents one single gene, so eukaryotic chromosomes are made
up of lots of genes.
Many of the genes in a chromosome contain the genetic code (DNA)
needed to make proteins. When a protein is made from a gene (DNA),
the gene is said to be ‘expressed’. It has been found that only some
parts of a gene (DNA) are expressed. These parts are known as exons or
coding regions of the gene. The parts of the gene that do not code for
protein are called introns or intervening, non-coding regions.
Chromosomes and genes in prokaryotes
Prokaryotes have a single circular chromosome. It has been found that
genes which have a related function are grouped together in
prokaryotes. This group of genes is called an operon. You will find out
more about operons in prokaryotes later.
Not all genes needed by a prokaryote are found on the circular
chromosome. Some bacterial genes are found on a plasmid. This is a
small circular piece of double stranded DNA. Plasmids are found
naturally in bacteria and generally carry genes that are advantageous to
the bacteria, but are not essential for their survival. For example, some
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COPYING AND TRANSLATING GENES
plasmids have genes that allow the bacteria to grow on certain
antibiotics. Plasmids can be transferred from one bacterium to the next,
so the advantageous genes can be passed on.
Plasmids have been isolated and manipulated by biotechnologists for use
in genetic engineering. It is now possible to use plasmids as cloning
vectors to introduce new genes into bacteria, to grow the bacteria and
for the bacteria to express the new genes and so produce new proteins.
DNA replication
When a cell divides into two, the two new cells are called daughter
cells. The daughter cells have exact copies of the chromosomes that
were present in the original parent cell. Before the cell can divide, the
DNA molecules must be duplicated exactly. The duplication of the DNA
molecules is known as DNA replication.
Several factors are required for the replication of a DNA molecule:
•
•
•
•
Double stranded DNA (to act as a template for the new DNA);
An enzyme called DNA polymerase;
Each of the four nucleotides (A, T, C and G bases);
Energy in the form of ATP.
The steps involved in the replication of DNA are as follows:
1.
The parental double stranded DNA to be replicated (or copied)
begins to untwist from its helical shape.
2.
Hydrogen bonds between complementary bases (A-T and G-C) are
broken. This causes the two strands to separate, forming two single
strands of DNA.
3.
A free DNA nucleotide finds its complementary base on the single
strand of DNA. For example, if there is a T on the single strand of
DNA, a free A nucleotide lines up with it, similarly if there is a G on
the single strand, then a C nucleotide lines up with it.
4.
A hydrogen bond forms between the free DNA nucleotide and its
complement.
5.
The 5′ phosphate group of this new nucleotide joins to the free 3′
of the adjacent nucleotide, thus continuing the formation of the
new DNA strand. The enzyme which joins (polymerises) one
nucleotide to the next is called DNA polymerase.
42
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COPYING AND TRANSLATING GENES
6.
The newly formed daughter DNA molecule rewinds into a double
helix.
Figure 19: The replication of DNA
At the end of DNA replication, two new strands of DNA are formed that
are identical to each other and the parental DNA molecule.
DNA mutations
Sometimes when DNA is being replicated, mistakes happen and the
wrong nucleotide is inserted into or a nucleotide is missed out of the
new DNA. This is known as a mutation in the DNA. Table 6 shows some
of the mutations that can occur in a DNA molecule:
UNIT 1: MICROBIOLOGY (HIGHER BIOTECHNOLOGY)
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COPYING AND TRANSLATING GENES
Table 6
Mutation
Description of the mutation
Substitution
This is when a nucleotide is substituted for another
nucleotide. For example, in the DNA sequence
TTGCTAAGCCGT, the 5th T may be substituted for a G.
The new sequence would be TTGCGAAGCCGT.
Insertion
This is when an extra nucleotide is introduced into a
DNA molecule. Taking the above sequence as an
example, the mutated sequence may be
TTGCTAAGACCGT where an extra A nucleotide has
been inserted.
Deletion
This is when a nucleotide is removed from the original
sequence. For example, TTGCTAAGCCGT may become
TTGCTAGCCGT where the 6th A has been deleted from
the sequence.
Inversion
This is when two nucleotides are inverted. For
example, in the sequence TTGCTAAGCCGT the 3rd and
4th nucleotide may change place so that the mutated
sequence becomes TTCGTAAGCCGT.
The structure of protein
As mentioned previously, many genes in the chromosomes of
eukaryotes and prokaryotes code for proteins.
Proteins are large, complex molecules that carry out many functions in
the cell as described below.
• Some proteins have a structural role in the cell.
• Some proteins are enzymes and carry out biochemical reactions in
the cell such as those involved in respiration.
• There are proteins that are involved in preventing infection in the
body. These proteins are known as antibodies.
• Other proteins are involved in the transport of substance around the
body. For example, haemoglobin is involved in the transport of
oxygen in red blood cells.
Proteins are made of building blocks called amino acids that join
together by strong peptide bonds to make large polypeptide (protein)
molecules.
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COPYING AND TRANSLATING GENES
A polypeptide molecule is shown in Fig. 20.
Figure 20: A polypeptide chain
The amino acids that join together to form a polypeptide chain are
known as the primary structure of a protein. In a cell this polypeptide
chain folds again into a secondary, then again into a tertiary structure (a
three dimensional (3D) shape). The 3D shape is the most compact,
stable structure that the protein can form. This 3D shape is held in place
by weak hydrogen bonds. A protein must be in its correct 3D shape for it
to work (function) properly in the cell. Anything that causes a change in
the 3D shape of the protein (such as a change in temperature or a
change in pH) can affect the function of the protein.
Before proteins can be made by a cell, another type of nucleic acid is
needed, called RNA.
The structure of RNA
Ribonucleic acid (RNA) is the second type of nucleic acid found in the
cell. It consists of nucleotides polymerised together, although the
structure of an RNA nucleotide is slightly different to a DNA nucleotide.
An RNA nucleotide consists of the following:
• a phosphate group
• a ribose sugar group
• an organic base
In RNA, the organic bases are Adenine (A), Cytosine (C), Guanine (G)
and Uracil (U).
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COPYING AND TRANSLATING GENES
The ribose sugar group from one RNA nucleotide joins to the phosphate
group of a second RNA nucleotide, forming a single polymerised chain.
RNA does not exist as a double stranded molecule, instead it is single
stranded.
The differences between the structures of DNA and RNA are shown in
Table 7.
Table 7
Feature
DNA
RNA
Number of nucleotide strands
present in one molecule
Two
One
Bases found in the nucleotides
A, G, C, T
A, G, C, U
Sugar present in the nucleotides
Deoxyribose
Ribose
The synthesis of RNA
RNA is made in the nucleus of the cell, using one of the strands of DNA
as a template. The strand that is used as the template is known as the
sense strand. Thus, the information that is coded for in the DNA
molecule is transferred to the RNA molecule which is then exported
from the nucleus to the cytoplasm. The synthesis of RNA is called
transcription.
There are several types of RNA transcribed from DNA. One type of RNA
is called messenger RNA (mRNA) and another type of RNA is called
transfer RNA (tRNA). Both types of RNA are involved in the synthesis of
protein.
The synthesis of mRNA
In eukaryotes, the genes from the DNA strand that are used to
synthesise mRNA are not continuous. This means that the DNA contains
nucleotide sequences that do not appear in the mature mRNA. These
intervening sequences are called introns and they are cut out of newly
formed mRNA molecules in a process known as splicing.
This is shown in Fig. 21.
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COPYING AND TRANSLATING GENES
Figure 21
GENE (DNA)
Exon 1
Intron 1
Exon 2
Intron 2
Exon 3
RNA synthesis
V
RNA
Exon 1
Intron 1
Exon 2
Intron 2
Exon 3
Introns are removed
V
Mature mRNA
Exon 1
Exon 2
Exon 3
The mature mRNA molecule is exported from the nucleus consisting
only of exon sequences and is then used to synthesise protein.
Protein synthesis
Each protein in the cell of an organism is coded for by a gene found in
the chromosomes of that organism. The gene (DNA) is used to
synthesise a mRNA molecule which, in turn, is used to direct the
synthesis of the protein molecule.
The information on the DNA is known as the genetic code. The
sequence of bases along a DNA strand represents a code for making
proteins.
DNA contains 4 bases (ACGT) yet proteins contain about 20 amino acids.
The relationship cannot be that 1 base represents (codes) for 1 amino
acid as this would allow only 4 amino acids to be coded. Even 2 bases
coding for 1 amino acid is insufficient as this allows for only 16 amino
acids.
It has been found that 3 bases in the DNA code for 1 amino acid. The
triplet of 3 bases is known as a codon. There are 64 codons and some
amino acids have more than one codon. The codons are arranged in a
specific order to code for a specific protein.
Remember that the DNA is transcribed into mRNA. The mRNA that is
produced contains the complementary sequence of codons to the sense
strand of DNA. Remember also that RNA has uracil (U) instead of
thymine (T).
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COPYING AND TRANSLATING GENES
For example:
{
DNA
TTTCTTTAGGGT
AAAGAAATCCCA (sense strand)
The sense strand is used as the template to make mRNA
V
mRNA
UUUCUUUAGGGU (this sequence is complementary to the sense
strand of DNA)
Table 8 shows some of the codons that specify different amino acids.
Table 8
Codon
Amino acid
Codon
Amino acid
UAG
Tyrosine
GUG
Valine
UUU
Phenylalanine
CCA
Proline
AGU
Serine
UGG
Tryptophan
CUU
Leucine
AGA
Arginine
GGU
Glycine
UCA
Serine
Using this table we can work out the sequence of amino acids that would
be produced using the mRNA (UUUCUUUAGGGU) from the above
example:
Phenylalanine-Leucine-Tyrosine-Glycine
This table can also be used to show that a mutation in the gene can
cause a change in the sequence of the protein. If there was a mutation
such that the first U in the sequence was replaced by a C (look back to
the previous section to find out the name of such a mutation), then the
sequence of amino acids would change to:
Leucine-Leucine-Tyrosine-Glycine
Sometimes a change in the amino acid sequence has no effect on the
function of the protein but in some cases, the protein may become
inactive.
mRNA is not the only RNA molecule involved in making protein, tRNA is
needed too. tRNA is a small molecule that attaches to an amino acid in
48
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COPYING AND TRANSLATING GENES
the cytoplasm of the cell. There is a different tRNA molecule for each
amino acid. At the opposite end to where the amino acid is attached to
tRNA, there is a triplet of bases called the anticodon. The anticodon
corresponds to a particular amino acid. The tRNA carries the amino acid
to the ribosome, where proteins are made.
Figure 22: The making of a protein
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COPYING AND TRANSLATING GENES
The interaction between mRNA, tRNA and the ribosome is shown in Fig.
22. When two tRNA molecules are present within a ribosome, a peptide
bond forms between the amino acids.
Ribosomes are small spherical structures found in the cytoplasm of the
cell. They are the site of protein synthesis. Each ribosome contains all
the components (proteins and RNA) required to make new proteins in
the cell. In prokaryotes, ribosomes are free in the cytoplasm whereas in
eukaryotes they are often found attached to internal membranes,
forming the organelle known as the rough endoplasmic reticulum.
The rough endoplasmic reticulum is involved in transporting the newly
made protein to another organelle, the Golgi apparatus. Proteins are
modified, then packaged by this organelle before being secreted out of
the cell.
Control of gene action
Some proteins are required by a cell only under certain conditions, e.g.
E.coli require the enzyme β-galactosidase only where the bacteria are
growing on lactose. When E.coli are growing on a different medium,
such as glucose, the genes that code for β-galactosidase are switched off.
The advantage of this control is that resources within the bacterial cell
are not wasted.
Three areas of bacterial DNA are involved in the control of βgalactosidase activity:
• The structural gene codes for the enzyme.
• The regulator gene codes for a protein known as the repressor.
• The operator is where the repressor binds.
These three areas are found together on the DNA in an area known as
the lac operon as shown in Fig. 23.
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Figure 23: The lac operon
β-galactosidase
Repressor
Control of the lac operon
In the absence of lactose
When no lactose is present in the culture medium, E.coli does not need
the β-galactosidase enzyme. Therefore the gene coding for this enzyme
is switched off.
The gene is switched off due to the presence of the repressor protein
(coded for by the regulator gene). The repressor protein binds to the
operator and switches off the structural gene. This is shown below:
Figure 24: The lac operon in the absence of lactose
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COPYING AND TRANSLATING GENES
In the presence of lactose
When E.coli is grown in culture medium containing lactose, βgalactosidase is produced. The enzyme breaks down lactose into
glucose and galactose and the bacteria use the glucose for growth:
V
Lactose
glucose + galactose
Lactose is called an inducer as it switches the structural gene on, so
producing the enzyme. This is shown in Fig. 25.
Figure 25: The lac operon in the presence of lactose
Lactose binds to the repressor molecule, which prevents the repressor
from binding to the operator. Therefore RNA is produced from the
structural gene. If RNA is made, then it can be used to synthesise the
enzyme.
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Test yourself on DNA structure and protein synthesis
Before you move onto the next part of this unit, spend time reviewing
your notes on the above section. It contains a lot of information.
1.
Give two differences between the structure of DNA and RNA.
2.
The sense strand of a piece of DNA has the following sequence:
5′ AGTGGTACCGAACAC 3′
(a)
Write down the sequence of the corresponding antisense
strand.
(b)
Write down the sequence of mRNA that would be produced if
the sense strand was transcribed.
(c)
Use Table 8 to find out the sequence of amino acids that
would be produced using the mRNA from (b).
3.
A DNA molecule consists of 24% cytosine bases. Calculate the
percentage number of thymine bases that would be present in this
DNA molecule.
4.
Describe the steps involved in the replication of a DNA molecule.
5.
Complete Table 9, which is about mutations:
Table 9
Type of mutation
Description of mutation
Substitution
When an extra nucleotide is inserted
into a DNA molecule
Inversion
When a nucleotide is removed from a
DNA sequence
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COPYING AND TRANSLATING GENES
6.
Describe the process of splicing in the synthesis of mRNA.
7.
What is the role of the following types of RNA in the synthesis of
protein:
(a)
(b)
8.
Describe the functions of the following organelles in the cell in
protein synthesis:
(a)
(b)
(c)
9.
mRNA
tRNA.
ribosome
rough endoplasmic reticulum
Golgi apparatus.
Fig. 26 shows the lac operon found in bacteria such as E.coli.
(a)
Name protein Y and protein Z.
(b)
State whether protein Y and protein Z are produced:
(i)
(ii)
in the absence of lactose
in the presence of lactose.
Figure 26
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GENETIC ENGINEERING
SECTION 5
Genetic engineering may be defined as the deliberate change of the
genetic makeup of an organism. This can be achieved by the
introduction of genes from another organism. In this way, organisms
with new characteristics are produced in a way that is not possible using
conventional breeding methods.
Genetic engineering is a rapidly growing technology and it is thought
that it will have profound effects on our everyday lives. Some examples
of how it may affect us are given below.
• In the field of medicine it may improve the diagnosis and cure of
hereditary defects and disease.
• It is being used for the development of new drugs and vaccines for
use by humans and animals.
• In agriculture it is being used to improve food production.
• It is being used to monitor and reduce environmental pollution.
In Scotland, one of the fastest growing industries is biotechnology.
Numerous biotechnology companies have been set up, many using the
techniques of genetic engineering.
In this section of the unit you will be introduced to some of the
techniques used in genetic engineering. The most basic technique
associated with genetic engineering is gene cloning.
Gene cloning itself involves several techniques including:
•
•
•
•
the isolation and purification of DNA
cutting DNA into smaller fragments with enzymes
separating fragments of DNA using electrophoresis
introducing fragments of DNA into organisms using cloning vectors.
The end result of gene cloning is the production of an organism that is
able to make many copies of the newly introduced DNA.
Purification of DNA
The first step in many genetic engineering processes is the isolation of
DNA from cells. There are several steps involved in DNA purification and
these are outlined below.
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GENETIC ENGINEERING
Firstly, the cells must be disrupted to release the soluble intracellular
components, including the DNA. This can be done mechanically by
putting the cells into a liquidiser/blender – similar to the one in your
kitchen!
Alternatively, cells can be disrupted using enzymes. The soluble
intracellular components are separated from insoluble cellular debris by
centrifugation, a technique that separates components using high speed
centrifugal forces.
The second step in DNA purification involves separating the DNA from
proteins. This is achieved by extracting the proteins into an organic
solvent and/or using enzymes that degrade the proteins, leaving purified
DNA.
Finally, the DNA is precipitated using alcohol and then resuspended in a
suitable buffer.
Restriction endonucleases
After DNA has been purified, it is cut into smaller fragments using
restriction endonucleases. These are enzymes that are found naturally
in bacteria. These enzymes recognise and cut short specific sequences
(between 4 and 8 base pairs) within DNA. Biotechnologists have
isolated many of these enzymes and they are now routinely used in
genetic engineering for cutting DNA.
One of the most commonly used restriction enzymes is called EcoR1. It
recognises the following 6-base pair DNA sequence:
5′ GAATTC 3′
3′ CTTAAG 5′
EcoR1 then cuts the DNA sequence as follows:
5′ G
3′ CTTAA
AATTC 3′
G 5′
When EcoR1 cuts DNA it produces two double stranded fragments, but
the cuts do not occur at the same position. Instead the cut is staggered
by four nucleotides, so that the DNA fragments have single stranded
overhangs (known as sticky ends). If another piece of DNA is cut with
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GENETIC ENGINEERING
the same enzyme and so has the same sticky ends, the pieces of DNA can
be joined together by base pairing between the sticky ends.
Other restriction endonucleases cut in the middle of their recognition
sequence so producing blunt ends.
Agarose gel electrophoresis
This is a technique used to separate fragments of DNA according to their
size. It is often used to separate fragments of DNA after digestion with
restriction endonucleases.
A solution of warm agarose is poured into a casting tray. A comb is
inserted in one end of the tray and the gel is allowed to cool causing the
agarose to set. After it has set, the comb is removed forming a number of
wells.
Different concentrations of agarose can be used, the higher the
concentration of agarose, the slower the rate of movement of the DNA
fragments.
The agarose gel has very small pores that act as a molecular sieve and
causes DNA of different sizes to separate from each other as follows:
• Small fragments of DNA move fastest through the gel.
• Large DNA fragments move slowly through the gel.
The DNA fragments to be separated are mixed with a tracking dye and
loaded into the wells. DNA is negatively charged and, when a voltage is
applied to the gel, the DNA migrates towards the positively charged
anode. The power supply is switched off when the tracking dye reaches
the end of the gel.
After electrophoresis the DNA fragments can be visualised by staining
the gel with a dye that binds to the DNA.
Fig. 27 shows an agarose gel with DNA fragments that have been stained
and so can be easily seen.
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GENETIC ENGINEERING
Figure 27: DNA fragments separated by electrophoresis
Lanes 1 and 7 contain DNA fragments of known size.
Lanes 2–6 contain plasmid DNA of different sizes.
Locating a fragment of DNA separated by electrophoresis
After separating DNA fragments on an agarose gel, one particular DNA
fragment may need to be located. For example, if chromosomal DNA is
cut up into smaller fragments, one of the smaller fragments may contain
a gene that a biotechnologist is interested in. How is this fragment
located?
Firstly, the DNA is transferred from the agarose gel to a membrane filter.
This step is needed because the double stranded DNA must be
denatured into single strands. This is almost impossible to do while the
DNA is in agarose. The DNA is transferred to the membrane by a
process known as blotting. Then the DNA is denatured.
The membrane containing the single stranded DNA is incubated with
either single stranded DNA or RNA (known as a probe) that contains
some bases complementary to the fragment of DNA to be located. The
complementary bases in the probe and the desired fragment of DNA join
together, forming double stranded DNA.
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The fragment of DNA located by the probe is visualised because the
probe is labelled either with radioactivity or with a chemiluminescent
label, making it easy to see.
The production of complementary DNA from RNA
Sometimes biotechnologists do not want to work with genes because
they contain introns that are not used to make protein. (Look back at
Section 4 to remind yourself about introns and exons.)
Instead, some biotechnologists work with messenger RNA (mRNA)
which is the expressed form of the gene. However, working with RNA is
difficult because it is single stranded and so it cannot easily be inserted
into a cloning vector such as a plasmid. Also, RNA is degraded very easily
and so can be difficult to use.
However, these problems working with RNA can be overcome by
converting RNA into DNA (known as complementary DNA or cDNA)
using an enzyme called reverse transcriptase.
cDNA is a direct copy of the mRNA but, unlike the original gene, it does
not contain introns. cDNA can be inserted easily into a cloning vector
and cloned in the usual manner.
Fig. 28 shows the steps taken to make cDNA from a mRNA template.
Figure 28: The synthesis of cDNA
mRNA
Make a DNA copy of the mRNA
using reverse transcripase
mRNA
DNA
Remove RNA by
treating with alkali
Single strand
DNA
Make a double stranded DNA
using DNA polymerase
Double stranded
DNA
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GENETIC ENGINEERING
Firstly the mRNA is used by reverse transcriptase as a template to
synthesise the first strand of DNA. A DNA-RNA hybrid is formed.
The RNA in the DNA-RNA hybrid is removed using alkali.
The remaining single stranded DNA is used as a template by the enzyme
DNA polymerase to make a second complementary strand of DNA.
The cDNA can now be inserted into a cloning vector, such as a plasmid
and cloned to produce many identical copies of the cDNA.
Transformation and cloning
Transformation is the name used to describe the process when a foreign
sequence of DNA (such as a gene or cDNA) is introduced into microorganisms such as bacteria and yeast.
Two micro-organisms that are commonly used in transformations are the
bacterium E.coli and the yeast, S. cerevisiae. Both micro-organisms are
single celled (unicellular) organisms that have fast reproduction rates
and thus are quick growing. This makes them ideal for large scale
production in industrial fermenters (bioreactors).
E.coli
This is a prokaryote that is often used as a recipient for foreign DNA.
Large sequences of foreign DNA can be inserted into E.coli using a
plasmid. The DNA is transcribed and translated and it is possible for the
protein coded for by the foreign DNA to account for 60% of the total
protein produced by the bacterial cell. E.coli are relatively easy to
transform.
While there are many advantages of using E.coli, there are some
disadvantages – mainly due to the fact that it is a prokaryote and the
foreign protein produced may originally have come from a eukaryote.
The disadvantages are outlined below.
The foreign protein produced is not always secreted easily from E.coli.
This may be due to E.coli not being able to carry out modifications to
the protein after it is made, for example addition of sugar groups. If the
protein is not secreted by the bacterium, it causes problems for the
biotechnologist as E.coli must be harvested, the bacterial cells broken
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GENETIC ENGINEERING
open (lysed), and the protein purified. This increases the production
costs.
E.coli does not always fold the foreign protein into its natural 3D shape.
This causes the protein to be inactive.
S. cerevisiae
This is a eukaryote (it is a yeast) that can be used instead of E.coli as the
recipient for foreign DNA.
Since it is eukaryotic, it can fold proteins into their 3D shape which
allows the proteins to be active. Foreign proteins made by S. cerevisiae
are secreted from the cell as S. cerevisiae can carry out post-translational
modifications (e.g. it can add sugar groups to proteins) which allows the
proteins to cross the cell wall. Thus proteins secreted by S. cerevisiae
can be extracted from the culture medium.
The disadvantages of using yeast include the following:
• It can be difficult to transform, this means that it can be difficult to
introduce the foreign DNA into the yeast.
• It produces less protein, so yields of the foreign protein are smaller.
• Plasmid vectors may be lost from yeast if there is no advantage to the
yeast in having the plasmid.
Cloning vectors
Cloning vectors are used to introduce foreign DNA into microorganisms such as E.coli and S. cerevisiae. Cloning vectors must be able
to replicate within these host cells.
Two types of cloning vectors used to introduce foreign DNA sequences
into micro-organisms are plasmids and bacteriophages. Both of these
cloning vectors have been mentioned previously. Plasmids are discussed
in the section on bacteria and bacteriophages are mentioned in the
section on viruses. You might find it helpful to read these sections again
before continuing.
Both occur naturally in bacteria but biotechnologists have genetically
engineered them so that they can be used as cloning vectors.
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GENETIC ENGINEERING
Cloning vectors have been manipulated so that they have the following
characteristics:
1.
They can be cut with restriction enzymes and foreign DNA
sequences (cut with the same restriction enzymes) can be inserted
into them using an enzyme called DNA ligase.
2.
Antibiotic resistance marker genes have been added to them.
These genes code for proteins that breakdown antibiotics. If a
cloning vector is inserted into a micro-organism, the microorganism gains the antibiotic resistance gene and so is able to grow
in the presence of this antibiotic. The micro-organism becomes
resistant to the antibiotic.
3.
Some cloning vectors contain part of the lac operon. This is used
to control the expression of the foreign DNA sequences. The
foreign DNA is transcribed and translated only when the lac
operon is switched on.
After a foreign sequence of DNA has been inserted into a cloning vector
using DNA ligase, the cloning vector is mixed with the micro-organism
into which it is to be transformed. Some of the micro-organisms will take
up the cloning vector, some will not. To separate the transformed
micro-organism from those that are not, the micro-organism is grown in
media containing the antibiotic to which the transformed microorganism has acquired resistance. The transformed micro-organism has
the cloning vector that has the antibiotic resistance gene, so it is able to
grow in the presence of the antibiotic.
Any micro-organism that does not possess the cloning vector is unable
to grow in this medium.
The transformed micro-organism is isolated from the medium and
transferred to another medium where it is allowed to reproduce and
grow in large quantities. Each new micro-organism that is produced is
genetically identical to the original transformed micro-organism. Each
genetically identical micro-organism is called a clone. The process of
producing lots of genetically identical micro-organisms is known as
cloning. This is shown in Fig. 29.
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GENETIC ENGINEERING
Figure 29: Transformation of a bacterial cell with a plasmid
Plasmid containing the
gene is introduced into
a bacterial cell
Circular chromosome
of the bacteria
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GENETIC ENGINEERING
Test yourself on genetic engineering
Before you move onto the next part of this unit, spend a little time
reviewing your notes on genetic engineering, then see if you can answer
the questions below.
1.
Fig. 30 represents a human chromosome showing the possible
position of the human insulin gene.
Figure 30
(a)
Name the type of enzyme that can be used to break the
chromosome into smaller fragments.
(b)
The above chromosome is broken into smaller fragments with
the following sizes:
Table 10
Fragment
Size of fragment(base pairs)
W
250
X
345
Y
400
Z
750
Fragments were separated by agarose gel electrophoresis.
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GENETIC ENGINEERING
Complete the diagram to identify which band corresponds to
which fragment.
(c)
One of the bands is known to contain the gene for insulin.
Describe how you might use a probe to find out which band
contains the gene.
2.
Given the following components, describe how you could obtain
clones of an insulin gene:
Components available:
Insulin gene
plasmid vector with ampicillin resistant gene
Bacterial cells
restriction enzymes
Ligase
nutrient medium containing ampicillin
(Note: ampicillin is an antibiotic)
3.
State 2 advantages of using yeast rather than bacteria in producing
clones of a gene.
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4.
66
The following questions refer to the making of cDNA.
(a)
Name the enzyme used to convert RNA into DNA.
(b)
What is the purpose of incubating the RNA/DNA hybrid with
alkali?
(c)
Name the enzyme that is used to make the second
complementary strand of DNA.
UNIT 1: MICROBIOLOGY (HIGHER BIOTECHNOLOGY)
INFECTION AND IMMUNITY
SECTION 6
Micro-organisms as pathogens
Micro-organisms such as bacteria and fungi can be advantageous to man
in that they can be used to produce useful substances such as yoghurt,
cheese, beer, wine and antibiotics, to name but a few.
However, it must be remembered that not all micro-organisms are
beneficial, some are harmful and cause disease. Micro-organisms that
cause disease are known as pathogens. Many species of bacteria, fungi
and viruses are pathogenic.
However, your body has developed an immune system that removes
pathogens and provides you with natural immunity if the pathogen
should enter your body again.
Production of antibodies and the role of blood cells
When a pathogen enters your body, your immune system responds by
producing antibodies. Any substance that causes your immune system to
produce antibodies is known as an antigen. So a pathogen is also an
antigen. An antigen is generally anything that is foreign to (or not
normally part of) your body.
Antibodies are protein molecules that have the following basic structure:
Figure 31
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INFECTION AND IMMUNITY
There are generally two sites on each antibody molecule that bind
specifically to a particular antigen.
The production of antibody molecules is part of your natural immunity.
You are constantly being exposed to pathogens (and other antigens)
and so you produce antibodies to build up a natural immunity to them.
There are two main cells that are involved in natural immunity: Blymphocytes and T-lymphocytes. Each of these different cell types is
discussed below:
B-lymphocytes and the humoral response
When a pathogen enters your body, a group of cells known as Blymphocytes bind to the pathogen. This causes the B-lymphocytes to
multiply into two different types of B-lymphocytes.
The first type of B-lymphocyte produces antibodies that then bind to the
pathogen and help to remove it from your body. The production of
antibodies by this type of B-lymphocyte is known as the humoral
response. It takes about two weeks for antibodies to be produced and a
pathogen cleared from your body.
The second type of B-lymphocyte circulates in your blood for many
years after the pathogen has first entered your body and been
destroyed. If the pathogen enters your body again at a later date, these
B-lymphocytes produce and secrete many antibodies very quickly and
these help to destroy the pathogen before it can do harm to your body
and before any symptoms of the disease appear.
T-lymphocytes and the cell-mediated response
When a T-lymphocyte is involved in immunity, it is known as the cellmediated response.
There are several different types of T-lymphocytes.
The first type of T-lymphocyte is one of the most important cells in the
immune system because it has a regulatory role. It activates and controls
B-lymphocytes, other T-lymphocytes and other cells of the immune
system.
The second type of T-lymphocyte destroys any body cell that has been
infected by a pathogen.
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The function of macrophage
B- and T-lymphocytes are not the only cells involved in pathogen
removal.
The antibodies produced by B-lymphocytes bind to the pathogen but
the antibody does not directly remove the pathogen. Instead, the
antibody acts as a chemical tag informing other cells in the immune
system that the pathogen is foreign and must be removed from your
body.
One of the cells of the immune system that is involved in removing the
pathogen is called a macrophage and the process by which it removes
the pathogen from your body is known as phagocytosis. This process
uses the organelle called the lysosome. Lysosomes are sacs that contain
digestive enzymes.
The process of phagocytosis is shown in Fig. 32 and the steps are
outlined below:
• Firstly the macrophage recognises and binds to the pathogen
• A vacuole then forms around the pathogen and it is engulfed within
the macrophage
• Lysosomes within the macrophage move towards the engulfed
pathogen and fuse with the vacuole surrounding the pathogen
• Enzymes are released into the vacuole from the lysosomes and the
pathogen is digested.
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Figure 32: Phagocytosis
Lysosomes fuse with
the vacuole and
digestive enzymes are
released into the
vacuole
Digestive enzymes
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INFECTION AND IMMUNITY
Immunity
The action of macrophage is considered to be part of the innate
immune response.
Innate immunity is a non-specific response to a pathogen. This means
that a macrophage will digest any pathogen that it encounters.
Other examples of innate immunity include:
• Skin which acts as a physical barrier against infection
• Acid in the stomach and in sweat. Pathogens are less likely to grow in
these acidic environments
• Lysozyme which is an enzyme found in tears that kills bacteria
• Interferon which is a molecule that stops viruses from replicating in
your body cells.
Naturally acquired immunity
When a pathogen enters your body naturally (for example, if you sit
beside someone who has chickenpox and is coughing and you breathe
in their chickenpox virus) your B-lymphocytes produce antibodies that
help you to remove this virus from your body. Unfortunately, this takes
about two weeks, so you get the symptoms of chickenpox too!
However, remember when the humoral response was discussed
previously, a second type of B-lymphocyte was mentioned. This other Blymphocyte circulates in your blood for many years after you have first
had chickenpox and if the chickenpox virus enters your body again, this
other type of B-lymphocyte quickly produces many antibodies and the
virus is removed before you get the symptoms of chickenpox again.
It is because of this natural acquired immunity that someone who has
had chickenpox as a child rarely gets chickenpox again. As you get
older, your naturally acquired immunity to many pathogens increases.
Artificially acquired immunity
Immunity can also be acquired artificially by the process of vaccination.
In the case of a vaccine, the pathogen (which has been weakened or
killed in some way) is injected into a person. This means that the person
has been artificially exposed to the pathogen. When the weakened or
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INFECTION AND IMMUNITY
killed pathogen enters their body, the immune system sets to work.
Antibodies are produced which help to remove the pathogen from their
body. (Remember that the pathogen has been weakened or killed and so
does not cause any symptoms in their body). Also, B-lymphocytes are
produced that circulate in the blood and will produce many antibodies
quickly if the natural pathogen enters their body at a later date. Thus the
person has artificially acquired immunity to the pathogen.
An example of a vaccine is the tetanus vaccine. Tetanus is the
uncontrolled contraction of muscles and can cause death in an
individual.
Tetanus is caused by a toxin produced by a bacterium. The tetanus
vaccine is made by purifying the toxin and then inactivating it to
produce a toxoid.
The toxoid is injected into an individual who then makes antibodies
against the toxoid to remove it from their body. The individual also
produces B-lymphocytes that circulate in the blood and which will
secrete antibodies if the naturally occurring toxin enters their body. The
antibodies that are produced are called antitoxins. These antibodies are
able to bind to and neutralise the naturally occurring toxin produced by
the bacterium.
Thus, if the individual is infected by the bacterium that causes tetanus,
they can quickly produce antitoxins that prevent the effects of the toxin.
Active immunity
This refers to the production of antibodies by an individual. The
antibodies can be made by the individual in response to a naturally
occurring infection or to the artificial injection (vaccination) of a
pathogen or toxoid.
Passive immunity
This refers to an individual receiving ready-made antibodies. These
ready-made antibodies can be gained either by natural or by artificial
means.
Natural passive immunity
This refers to someone receiving ready-made antibodies naturally. A
baby receives antibodies from its mother through the placenta and
through breast milk.
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INFECTION AND IMMUNITY
Artificial passive immunity
This refers to someone receiving ready-made antibodies through a
vaccine. For example, if someone cuts themselves badly and if they do
not have any natural antitoxins against tetanus in their blood (they may
not have kept up to date with their tetanus vaccines), then they can be
given ready-made antitoxins in a vaccine that allows them to fight the
bacteria that causes tetanus, if it has entered their body through the cut.
Generally, natural and artificial passive immunity do not last long as the
ready-made antibodies are removed from the body within a few months.
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Test yourself on infection and immunity
Spend time reviewing your notes on infection and immunity, then see if
you can answer the questions below.
1.
What do the following terms mean?
(a)
(b)
(c)
(d)
(e)
2.
pathogen
antigen
antibody
humoral response
cell-mediated response
White blood cells (wbc) are involved in the immune response.
Some of these wbc are listed below:
B-lymphocytes
T-lymphocytes
macrophage
Use the list to complete the following sentences:
3.
(a)
The wbc involved in humoral immunity is
(b)
The wbc involved in regulating the immune response is
(c)
The wbc involved in phagocytosis is
Put the following statements into the correct order to describe
phagocytosis:
(a)
(b)
(c)
(d)
(e)
(f)
Digestive enzymes are released from the lysosomes into the
vacuole.
The pathogen is digested.
The phagocyte recognises and binds to the pathogen.
Lysosomes within the phagocyte move towards the engulfed
pathogen.
A vacuole forms around the pathogen and it is engulfed
within the phagocyte.
Lysosomes fuse with the vacuole surrounding the pathogen.
4.
Describe what is meant by the terms ‘active’ and ‘passive’ with
reference to immunity.
5.
Describe two ways that a person may acquire natural passive
immunity.
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BIBLIOGRAPHY
Some suggested staff reading materials
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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BIBLIOGRAPHY
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
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 that may be useful for Unit 3 (Biotechnology). The case
studies highlight companies in the UK that actively use biotechnology;
so they are a good introduction to students to show 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.
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
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exercises. It also provides information about the Scottish Centre for
Biotechnology Education.
http://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) who run workshops for teachers and
pupils.
www.sebiotech.org.uk
This is the website of Scottish Enterprise that is dedicated to the
Scottish biotechnology industry. It is very useful for keeping up to date
with the biotechnology companies in Scotland.
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APPENDIX
Advice for problem-solving outcomes
Unit 1: Microbiology, Outcome 3 and
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 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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A case study of a practical investigation that was used to solve problems
by candidates in a presenting centre 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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