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Biotechnology
1. The Origins of Biotechnology date back to at least 10 000 years ago
Describe the origins of biotechnology in early societies who collected seeds of wild
plants and domesticated some species of wild animals
 Biotechnology refers to using reactions that occur in living organisms by applying them for
human use
 Has a long history
 Origin can be traced to the beginning of agriculture
 Human society changed from nomadic hunter-gatherers to farmers
 Began when societies collected seeds in various places such as South-east asia and fertile
crescent of Middle East
 Wild species were domesticated and were bred for characteristics that were favourable for
human use (artificial selection)
 This was also applied to agriculture in which good seeds were cultivated for harvest, thus
improving crop
Explain why the collection of seeds and breeding of animals with desired characteristics could
be described as early biotechnology
 Can be considered early biotechnology
 Collection of seeds that produced varieties of better quality crop and yield were cultivated
 Humans replaced environment as the selection agent as they selected desired characteristics
by humans (artificial selection)
 People began to control the genetic make-up of the next generation of organisms
 Crops that matured quickly and animals with placid nature and for hides
 Human selection for desired characteristics uses organisms to make products for human use
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329 Describe the changes in one group of animals and one group of plants as a result of artificial
selection of characteristics suitable for agriculture stock
Use available evidence to describe the changes in a species of grain or animal as a result of
domestication and agricultural processes
Agricultural Crop – Wheat
 Part of the Genus Triticum
 Before 10 000, it was a wild small grass, wild Triticum
 Species Triticum monoconnon was harvested in the Middle east
 Fertile hybrid occurred when wild T was bred with T. monoconnon
 The offspring was polyploidy (Supernumerary chromosomes) as the parents had 2 sets of 7
(14) while a meiotic error occurred and the offspring had 4 sets of 7 (28)
 Called T. Turgidum – enmer wheat, bigger, more nutritious grain, ear attached so wind
could not blow away
 Cross between T.Turgidum and another strain with another meiotic error formed 6 pairs of 7
chromosomes (42)
 Modern wheat was T. astivum
 Had greater vitality due to elimination of harmful recessive genes
 Ears tightly attached to stalk
 High yielding, larger, nutritious, short stalk
 some are rust-resistant
Domesticated Animal – Aberdeen Angus
Requirements for domestication
 Diet: Herbivorous, more efficient to turn plant biomass into animal biomass rather than
plant to animal to animal
 Growth rate: reasonably fast
 Captive breeding: able to breed in domestic situations
 Disposition: Absence of aggression
 Social structure: herd, tolerance
Angus
 6000 BC humans domesticated ancient cows called Auroch
 18th century Scotland
 Farmer interbred two strains of polled (no horns) cattle
 decided they wanted a black herd
 Angus was bred so it was moderate in size
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329  Symmetrical shape and strength
 ability to accumulate muscle for good meat and high-quality beef
 fertility, docile, maternal instincts
 milk quality
Process information to outline an ancient Aboriginal use of Biotechnology
Aquaculture – Eel Farming
 Aboriginals constructed artificial streams (canals) to trap eels into constructed 'farms'
 These were a set of channeled streams that led from natural water bodies into traps
 The traps were specially made nets which allowed smaller eels to pass into yet larger eels
were unable to enter
 the smaller eels were taken out of the stream as they were undesirable and were smoked and
sold to other tribes (human use)
 The eel population thus improved in size for human consumption
2. Biotechnology was come to be recognised as the use of living organisms to make or
modify a product, to improve plants or animals or to utilise micro-organisms for
specific uses
Outline the key events that led to the use of biotechnological practices including:
− Yeast in the manufacture of bread
− Yeast and fermentation for alcohol production
− the use of other micro-organisms for the manufacture of yoghurt and cheeses
 Traditional fermentation is anaerobic fermentation
 Produces readily usable ATP from compounds such as sugars
 By-products depend on the reaction but generally include water, CO2 and alcohol
Yeast in the manufacture of bread
 The ancients discovered that heated grains were tastier and easier to digest than raw grains
 Hieroglyphics suggest Egyptians used yeast to leaven bread over 5000 years ago
 Ancient Egyptians had warm climates and wild yeasts in the air were attracted to grain
mixtures leading to leavened bread
 Small portions of dough were used to starts new bread, good batches were saved
 Ancient swiss 10 000 years ago discovered that leaving bread outside for a day before
baking improved taste and texture
 In the 1860's, Louise pasture found that yeast was responsible for fermentation
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329  Sacchromyces Cerevisiae is used
 Yeast ferments sugars in bread to produce CO2 and ethanol, ethanol evaporates
 Sugar (and yeast) → Carbon dioxide + Ethanol (without the presence of oxygen)
Alcohol production
 Sumerians accidently brewed on Babylon before 6000 BC, left fruits outside in damp area
and it created a pleasant tasting beverage
 Ancient Egyptian papyrus includes directions for making beer from dates
 3000 BC, Egyptians were making Boozah beer from fermented dough and barley
 Barley was crushed and partly baked, soaked in water and beer was strained out
 Middle ages, people drank beer from the Hops flower and this was safer to drink than water
 James Watt during the late Industrial revolution discovered refrigeration which greatly
increased the production of beer
 Pastuer discovered that yeast living on the skin of grapes was responsible for fermentation
 Vat (wine fermentation tanks) began to be used
 Sacchromyces Cerevisiae
 Sugar (and yeast) → Carbon dioxide + Ethanol (without the presence of oxygen)
Cheese production
 Rock drawings 10 000 years ago show cattle being milked
 Domesticated cattle in Rome, Greece, Egypt
 Milks forms curds from stomach enzymes (renin), heat and natural bacteria
 Travelling arab merchant carried sheep stomach pouch with milk, milk had curdled
 Curdled milk could be salted
 Improvements occurred over the next 10 000 years
 Bacteria came from air while enzymes were deliberately added
 Bacteria made it sour
 Why was separated from the curd and the curd was dried
 Left to ripen in caves were it grew mould and flavour
 Streptococcus lactis
Yoghurt Production
 occurred in the Balkans and Middle-east
 Chinese were using Lactobacillus in the 4000 BC
 Added to heat treated milk
 Ancients preferred to make yoghurt than store milk
 Semifluid fermented milk with smooth texture and sour taste of lactic acid
 Milk is boiled in an uncovered pot and yoghurt from previous batch is added to introduce
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329 bacteria
 Lactobacillus Bulgaris forms lactic acid
Plan, choose equipment or resources, perform a first-hand investigation to demonstrate the use
of fermentation processes in bread or alcohol production
Ginger Beer Practical
Aim: to demonstrate that microorganisms can be used to make products for human use and to
determine that the gas produced in fermentation process is carbon dioxide
Hypothesis: Saccromycs Cerevisiae will convert the sugars into Carbon dioxide and Ethanol
(fermentation)
Method
1) Measure 2 x 50g of ginger, crush and place 50g into one bowl and the other into a separate
one.
2) Measure 150g of raw sugar and place it in one of the bowls, measure another 150g and place
it in the second
3) Pour 2L of boiling water into each bowl and mix the solution
4) Let both bowls cool in room temperature by waiting 2 hours
5) Measure 15g of yeast (7g of dried yeast) and place it in one of the bowls, labelling it
'YEAST'
6) Label the other bowl 'NO yeast'
7) Mix the solutions
8) Wrap the bowl in two tight layers of glad wrap and cover it with a cloth
9) Leave both bowls to set for 24 hours at room temperature
10) Dip two pH indicator paper into distilled water
11) Open one the 'YEAST' bowls slightly and place the indicator paper directly within the
gaseous part of the container, record any colour change. In a table.
12) Repeat step 12 on the 'NO yeast' bowl. Record any colour change in a table
13) Repeat experiment
Results
Experiment tested
Colour of indicator paper
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329 YEAST
Orange-red
NO yeast
Green
Note: acidic entails that CO2 is dissolved in the water as CO2 dissolves as hydrogen carbonate ions,
lowering pH
Variables:
Independent (change, only one)
Presence of Yeast (Sacchromyces cerevisiae)
Dependent (measure, only one)
Amount of CO2 released (acidity of water pH)
Controlled (at least three things that are kept the same)
− Temperature of Water
− Amount of water
− Size of container
− Amount of Sugar
CONTROL (without the independent variable)
Experiment with no yeast
Risk Assesment
Risk
Assessment
Management
Pathogen microbes may grow Low
unintentionally on the substrate
Ensure that the system is
anaerobis
Wear safety gloves
Ensure hands are washed before
and after practical
Sterilise all equipment before
and after use
Do not ingest ingredients
Glassware may break
Ensure that glassware is not
kept at the edge of the bench
Low
Link to Real life:
Pasteur was the first to show that microorganisms had a role in fermentation, discovering that yeast
present on the skin of grapes produced ethanol (alcohol) in wine. Before this, the ancients utilised
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329 fermentation without this knowledge to produce a variety of products for use including bread, wine,
yoghurt and cheese. 10 000 years ago, the Swiss discovered that leaving bread dough outside for a
few days before baking increased the taste and texture of the bread which rose. This was probably
due to wild yeast fermenting the dough.
Similar occurrences led to the ancient production of wine in Sumeria and 'boozah' in Egypt in which
dates and other fruits were fermented. During the middle ages, bear from the fermentation of hop
seeds was integral to society as it was often more safe to drink than water. As the science behind
fermentation was understood, the products of the reaction were commercialised. Bread yeast
(Saccharomyces cerevusiae) was proliferated in bioreactors on mass aerobic reactions to make yeast
biomass for the baking industry. Products including yoghurt which relies on lactic acid fermentation
became recognised as beneficial to health and also became mass produced.
Commercially available products of fermentation are indicative of its significance. Today, different
products of the process are widely available for use including glycerol, citric acid and acetic acid.
3. Classical Biotechnology exploited knowledge of cell biochemistry to produce
industrial fermentation Procedures
Describe the expansion of fermentation since the early 18th century to include the production
of several compounds, including glycerol, lactic acid, citric acid and yeast biomass for bakers
yeast
New Knowledge
 Pastuer demonstrated that yeast was responsible for fermentation
 Observed soured beer and identified that lactobacillus was responsible for souring (lactic
acid)
 Demonstrated the pastuerisation process, heating milk below boiling point to kill pathogens
 Hansen set up a pure culture of Yeast
 Industrial revolution led to massive changes in Western society
 Discovery of steam engines allows for empowerment of newly discovered bioreactors
 Gaint steel tanks with subtrate and temperature and parameters could be controlled
 Discovery of refirgeration
 improvements in aseptic techniques and sterlisation practices
Knowledge on Cell biochemistry
 Knowledge increased on how microbes grew and metabolised
 6 distinct phases of microbial growth and reproduction
 Small cultures placed in large nutrient cultures
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329  1. Lag Phase: No growth, high activity for adjusment
 2. Acceleration phase: growth begins, cells divide
 3. Exponential phase: maximum growth, favourable conditions
 4. Deceleration phase: slow down, nutrients finish
 5. Stationary phase: no growth
 6. Death phase: cells are broken down, no more output, toxic for living cells
 Occurs in the fermentation tank
 Batch culture are utilised to start this process
Glycerol
 Trihydric alcohol
 Sacchromyces Cerevisiae produces alcohol
 Neuberg realised that if sodium bisulfite was added, yeast formed glycerol instead of Carbon
dioxide and alcohol
 Glyrcerol was used to make explosives
 USA made it from by=product of soap yet Germany lacked the ingredients for soap
 Germans had to use fermentation to make glycerol
Lactic Acid
 Lactobacilli used
 Manufactured for either food or industrial processes
 Glucose (Lactobacillus) → Lactic acid
 preservative and acidifies food, probiotic, bone health, anti-cancer chemicals
 production of synthetic fibres and polymers including PolyLacticAcid (PLA)
 Fermentation can proceed at higher than usual temperatures
Citric Acid
 Several species of moulds that can convert sugars into citric acid, Aspergillus niger
 Many sugars can serve as the substrate
 Molasses commonly used
 Aspergillus is submerged fermentation of molasses aerobically
 Important chemical in foods, flavourings, sweets, manufacture of ink, dyeing and engraving
Yeast Biomass
 Past bakers obtained yeast from brewers
 Varied quality meant that resulting bread was of varied quality
 Strains of Saccromycs cerevisiae best used for brewing is different from that that is used for
baking
 Became possible from the 1800s to grow yeast in fermentation tanks to make Baker's yeast
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329  'Stock strain' to be reproduced is added to a food medium
 pH 4-5 is kept
 Aerobic conditions so it is continuly aerated
 Yeast is harvested by washing the cells and separating through a centrifuge
Fermentation Tanks
 Commonly used in biotechnology
 Supply of air may be bubbled or paddled
 pH and temperature is controlled
 Large steel tanks
Describe strain isolation methods developed in the 1940s
 Almost all microbes used in fermentation have undergone a process of artificial selection
 Selected to find those with greater yields
 until late 1800s, these strains were found accidently rather than planned
 Robert Koch developed the agar plates, strain isolation could be pursued
 Methods of causing mutations have helped
Pennecillin
 Alexander Flemming accidently discovered Peniccilin
 Colonies that soroounded a mould appeared to be broken down
 Mould was releasing a chemical that was killing the bacteria
 determined that it was a species of Peniccilin
 Acts on growing bacteria by interfering with cell wall development
 Howard Florey and Chain read his dicovery, increased its output
 tested on mice by 1940
 Major industrial production began 1944
Methods
 Screening is used to test microbes on pathogens
Method 1
 Antibiotic producing microde is streaked on an agar plate using aseptic techniques
 allowed to grow
 various other microbes are streaked at right angles
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329  If antibiotic has been produced, one or more will fail to grow
Method 2
 determiens yield
 Grown in culture
 Discs of filter paper are dipped into liquid medium where microbes have grown
 discs placed on agar plates inoculated with bacteriaIf there is antibiotic on the dicsc, bacteria
will not grow on the region
Describe using a specific example, the benefits of strain isolation methods used in
biotechnology in the 20th century
 During WWII, more soldiers were dying of infection than of bullets
 Moved to USA as wartime Britian could not produce it
 Improvements in the chemical composition of nutrient medium led to yield
 Better penicillin strains were discovered P. chyrisegenum
 Development of submerged fermentation methods to replace surface fermentation
 better methods to remove penicillin
 Before penicillin, 30% of people with pneumonia sufferers died
 now down to 6%
 large epidemics of Diphtheria In Europe during that time
Identify that developments in the 1950s led to biotransformation technologies that could
produce required organic compounds such as cortisone and sex hormones
 Biotransformation is using microbes to convert one compound into another with very few or
one step
 Increased demand during the 1950s led to development of such techniques
 Thereputic role of steroids was discovered
 particularly cortisone and sex hormones
 Cortisone could be used for anti-inflammatory treatments to help relieve the pain of arthiritis
 Oestrogen and progoestrogen could be used in contraception
 Testorone for hormone therapy
 Large-scale production was problematic as it took far too long to be made in the lab
 Labour intensive, slow, a lot of material required, low yield = uneconomical
 If extracted from plants, most work was already done
 simply needed to add hydroxyl group into molecule
 Microbes transformed them including Rhizopius which converts progesterone into hydroxyElite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329 progesterone
 Fermentation tanks with molasses, glucose, yeast and light aeration
 Quick process = economical with good yield
Gather and process information from secondary sources to:
− Identify and describe a named industrial fermentation process
− identify the microorganism used in the fermentation and the products of the fermentation
− outline the use of the product of fermentation process
− use availble evidence to assess the impact of the use of the fermentation product on
society at the time of its introduction
Secondary Sources
 HSC credited textbooks including Biology in Context (Kennedy) and Spotlight HSC biology
(Heffernan) as well as peer-reviewed scientific journals and educational/government
websites
 First they were analysed for relevance to ensure that the topics and information that was
provided could be applied to the investigation of fermentation and its impact
 Reliability was ensured due to the wide variety of sources used in which information was
consistent
 The integrity of the sources were analysed for validity
 Dates were ensured to be recent, the publishers were credible (HSC endorsed), domain of
the website was checked (edu, gov, non-for-profit organisation, NGO), author was
researched to ensure credibility, publishing location, bibliography included, if experiments
were present then the methodology was analysed
Yeast biomass
 Sacchromyces cerevisiae strains that are ideal for baking instead of brewing
 Yeast biomass could make consistent bread that was of high quality instead of the variable
quality of brewers yeast
 No longer had to rely on the brewers industry for yeast
 Baking became a wide and commercial industry, bacth cultures
Process and analyse information from secondary sources to demonstrate how changes in
technology and scientific knowledge have modified traditional uses of biotechnology such as
fermentation
Yeast
 Original fermentation practices to produce bread utilised leaving dough and water out a few
days before baking
 Good batches were saved
 Louise Pastuer discovered yeast was responsible for fermentation, thus yeast began to be
used
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329  Brewers yeast initially used
 Steam engine and sterlisation techniques allowed for the development and powering of large
fermentation tanks
 this is coupled increased knowledge on cell biochemistry and cellular aerobic respiration
and division
 Development of bioreactors
 Bakers yeast was created which separeted the bakers and brewers industry
 Yeast Biomass
Biotransformation
 Original techniques were slow, non-profitable and labour intensive creating it in the lab
 enzymes would catalyse reactions
 New knowledge on microbe activity and how they could change compounds
 therepeutic value of steroids
 Allowed it to be more cheap, quicker and greater yield
Penecillin
 Initially low-yielding varieties
 New strain isolation techniques
 Aseptic sterilisation, improoved glassware
 Koch invented agar plates
 changes include that high yielding varieties were discovered including P.chrysogenum
4. Cell chemistry is utilised in Biotechnology
Outline, simply, the steps in the synthesis of protein in the cell, including:
 Difference between DNA and RNA
 Production of messenger RNA
 The role of transfer RNA
 The formation of the polypeptide chain
 The formation of the protein from the polypeptide chain
Difference between DNA and RNA
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329 DNA
RNA
Double-stranded molecule of nucleotides in the
shape of a spiral helix
Single stranded molecule of necleotides
Deoxyribose sugar
Ribose sugar
Thymine
Uracil
One Type
Two: Messenger and transfer
Production of Messenger RNA
 Transcription
 Occurs in the nucleus
 A section of DNA (gene) is copied
 Nuclear information is synthesised into complimentary strand of mRNA
 Thymine is replaced by Uracil
 Double helix is unwound at the site of transcription then rewound when it is finished
Role of tRNA
 Clover-shaped RNA molecules
 float in the cytoplasm
 carry an amino acid on one end which is determined by an anti-codon on the other
 in the ribosome, the codon and the anti-codon are complimentary
Formation of the Polypeptide sequence
 mRNA sttaches to ribosome, tRNA bring amino acid to the site of synthesis
 Anti-codons on tRNA recognise codons on mRNA
 Amino acids then form peptide bonds to form a polypeptide
Formation of Protein from polypeptide chain
 Polypeptide is sent to the endoplasmic reticulum
 folded in a particular way to form a protein with active site
 structural or functional protein
Plan and perform a first-hand investigation to test the conditions that influence the rate of
enzyme activity
Temperature
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329 Aim: To investigate the impact of temperature on the activity of renin
Method:
1. Prepare 20 test tubes and divide them into two groups of six placed in separate racks
2. label one of the groups 'enzyme' and the other 'control'
3. label each test tube in both groups either 10, 20, 30, 40, 50, 60
4. prepare an electronic heater
5. heat several beakers of water until they reach 30, 40, 50 and 60 using a thermometer to
measure them
6. Pour these into styrofoam cups and place the test tubes labelled with the appropriate
temperature into each and wait for the temperature to adjust, continue to monitor using a
thermometer
7. Use ice to cool two styrofoam cups with water to 10 and 20 and place the respective test
tubes into each and monitor, settle for 5 minutes
8. Pour milk into each test tube
9. Crush junket tablets (renin) with a mortar and pestle
10. place equal amounts of renin into each of the test tubes labelled enzyme and time
11. Time the time it takes for the milk to clot
12. Record results graphically and on a table
Acidity
Aim: To investigate the impact of acidity on the activity of catalase
Method:
1. Prepare 8 test tubes and divide them into two equal groups and two distinct test tube racks
2. Label one group 'Enzyme' and the other group 'control'
3. Pour 10ml of distilled water into each test tube
4. Label a test tube from each group consecutively pH 3,5,7,9
5. adjust the pH to the level labelled on the test tube by either adding dilute drops of sodium
hydroxide (increase pH) and dilute drops of hydrochloric acid (decrease pH) and monitor
using pH paper indicator
6. Add 5ml of hydrogen peroxide (this is toxic) to each test tube and place equal protions of
cut potato into the test tubes labelled 'enzyme'
7. Wait ten minutes
8. Measure the length of froth that forms on the surface
9. Tabulate and graph results
Substrate Concentration
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329 Method:
Same as above but don't tamper with pH, basically changing the concentration of hydrogen
peroxide used. Use 5 test tubes (2 groups of them, one being control without catalase). Test tube 1
will have 10 ml distilled water, 2 will have 2.5 HP and 7.5 water, 3 will have 5 water and 5 HP, 4
will have 2.5 water and 7.5 HP and 5 will have 10ml HP
5. Modern biotechnology includes recombinant DNA technology
Describe the three essentials of gene manipulation as:
− Cutting and joining DNA
− Monitoring the cutting and joining
− Transforming hosts, such as bacteria, with the recombinant DNA
Cutting and joining DNA
 The DNA (gene) that is required from the chromosome is cut using restriction enzymes
 ECOR1, HIND3
 The same restriction enzymes as each are specific to a specific site on both genes so that
they are cut at sticky ends so they join
 A circular piece called a plasmid is taken from the bacteria
 Sticky ends are joined by the enzyme ligase, they match
Monitoring the cutting and joining
 This has to be done in order to make sure that the correct gene is spliced into the plasmid
 Unwanted DNA fragments must be removed by electrophoresis
 Bacteria must be isolated with host gene
 Anti-biotic resistance is placed on the bacteria
 Colonies are grown in antibiotic medium, those with the gene will survive
Transforming Host
 Replacing plasmid that contain gene into E.coli and make more of the generating
 Calcium chloride, Ca ions weaken the cell membrane
 They can be placed on ice and heat shocked
Describe the following recombinant DNA technique used in biotechnology including:
− Gene splicing using restriction enzymes and ligases to produce recombinant DNA
− Polymerase chain reaction to amplify or modify DNA sequences
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329 − Use of DNA vectors and microinjections for carrying genes into nuclear DNA in the
production of transgenic multicellular organisms
Gene splicing
 Recombinant E.coli to produce insulin
 Restriction enzyme ECOR 1
 Ligase stick together at sticky ends
 Monitoring with anti-biotic resistance
 transforming hosts
Polymerase Chain Reaction
 TAQ polymerase used (from thermal aquatic organisms to resist heat)
 Amplify DNA
 Denaturation, annealing and extending
 Primers and free nucleotides
 Used for paternal tests and crime scenes to amplify evidence to provide a non-destructive
method
 (explained further)
Microinjection
 Used mainly in animals but also in plants
 Plant cells: Cell wall is removed leaving protoplasm, electric current forms small holes,
desired gene is shot through
 Mammals: Genes can be directly injected into cells through a glass pipette to punctured
holes in the cell
 Human gene has been placed gene into sheep called antitrysin which can produce proteins
which help people with bad lungs
DNA vectors
 Virus or plasmid vector
 Viral vectors are modified so that they will not replicate or cause disease
 gene is incorporated into the virus through recombinant DNA technologies
 binds with embryo cell
 advantage is that only a simple copy of transgene is integrated, if planted into eggs and all
cells will have it
 Disadvantage is that it is time and labour-intensive to prepare viral vector → changed vector
may revert to original state or recombine with pathogenic
Perform a first-hand investigation to identify and extract DNA from a suitable source
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329 DNA extraction
Aim:
To extract DNA from Barley
Materials:
 Marley solution prepared by CSIRO
 SdS detergent
 Ethanol
 Blue plastic loop
 centrifuge
 magnetic stirrer
 Transfer pipette
 Centrifuge tubes
 Triton X buffer and MgCl2 salt solution
Methods:
1. Barley was mixed using a magnetic stirrer with a solution of MgCl2, NaCl buffer and triton
X for 20 minutes, then strained
2. Place tubes in centrifuge, ensure it is balanced and spin for 3 minutes
3. pour off the supernatant into sink quickly
4. Add SDS up to the 5ml on the side of the centrifuge tube
5. Stir gently with the blue loop (blunt end) for 1 minute, ensuring the pellet is resuspended
6. Return to the centrifuge and spin for 5 mins
7. Holding the tubes on the side, add 2ml of the tube. You should get two distinct layers of
liquid
8. Using the blue loop, dip into the SDS layer and drag up into the ethanol
9. Continue until DNA precipitates a white fibrous and stringy solid
Process information to produce a flow chart on the sequence of events that result in the
formation of recombinant DNA
E.Coli diagram
Gather and analyse information to outline the purpose of a current application of transgenic
technology, naming the organism and gene transfer technique involved
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329  E.Coli
 DNA splicing
 Human insulin gene
Process and analyse secondary information to identify that complimentary DNA is produced
by Polymerase Chain Reaction
Denaturation
 Long strands of DNA are heated
 Specific gene is then denatured
 Heated to about 95-98
Annealing (Sticking Together)
 Primers are added and they stick onto the single strands of DNA at complimentary locations
(either side)
 Stuck outside of the desired gene so that the desired gene is definitely copied
Extension
 Extension of DNA by adding TAQ polymerase and free nucleotides
 Results in the synthesis of more copies of the required DNA molecule from the starting
sequence formed by primers
 strands are reheated and process is repeated 5 mins can copy 100 pairs
 fast
 even a trace from fingerprint or nail can be amplified
6. There are many applications and areas of research in biotechnology
Outline one way that forensic scientists use DNA analysis to help solve cases
Electrophoresis
 Basis of DNA fingerprinting
 DNA is cut into fragments
 Placed in an agar gel layer on a glass plate submerged in a buffer solution
 Buffer solution is required as buffers contain ions which conduct electricty
 DNA is negatively charged and placed in the negative area
 Voltage is put through, larger fragments travel less than smaller fragments
 Different lengths separate
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Tandem Repeats
 Every person has unique length of Tandem repeats
 Within introns
 Called TAG units (base pairs) that repeat, everyone has different repeat
Producing DNA fingerprints
 Restriction enzyme cut up DNA into many fragments
 Can be amplified via PCR if sample too small
 fragments are sorted by electrophoresis
 A radioactive probe is used containing the base sequence that is complimentary to the
tandem repeat being examined (our example is TAG)
 DNA band pattern from the electrophoresis gel is firstly moved onto a nylon membrane
 the probe runs through and binds with the complimentary repeat units
 X-ray is placed next to the membrane to determine pattern
 Developed X-ray film show DNA fragments
Describe one example of biotechnology in medicine
− Production of a synthetic hormone, such as insulin
 Production of insulin by recombinant E.Coli
 recombinant DNA technology
 Allows production of insulin for diabetics
Describe one example from the the following application of animal or plant biotechnology
− Recombinant vaccines to combat virulent animal disease
 Tickgaurd
 Affects cattle and makes them weaker producing bad meat
 Protein from tick BM-86 from the Tick is used as antigen
 Recombinant technique to take the gene that creates BM – 86 is placed in E.coli
Describe one example from the following applications of aquaculture
− The forming of a marine
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329  Yabbie, Cherax destructor
 Farmed extensively
 Market demand
 Restaurant quality
 Selection for more breeding seasons (twice), size
Identify data sources, gather, analyse and process information to present one case study on the
application of biotechnology in each of the following:
− medicine
− animal biotechnology
− aquaculture
− Give details of the process used
− Identify the organisms or tissue involved
− Describe the outcomes of the biotechnological processes
− Evaluate the efficiency of the process and discuss advantages and disadvantages
associated with either the product or the process
E.Coli Insulin - Medicine
 Human gene for insulin from pancreatic cells is spliced into a vector plasmid and then
transferred into E.coli bacteria
 Gene for production of insulin is cut from DNA using restriction enzyme ECOR1
 Creates a very specific sticky ends on the sequence as the same restriction enzyme is used
 Spliced into plasmid, sticky ends match up and ligase is then used to join the ends together
 Transformed into the E.coli bacteria, reproducing via cloning, production of insulin
production escalated
Process outcome
• Process aims to produce a recombinant E.coli with a plasmid containing the human gene for
insulin production
• Ideally both recombination and transformation will occur
• A new gene has been recombined with plasmid
• Plasmid has been transformed into bacteria
Advantages
• Some bacteria reproduce asexually, all of the offspring will have the identical gene for
human insulin production
• Bacteria reproduce at an escalated rate so mass production can be made
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• Outcomes are always unknown and it is not guaranteed that the process will be successful,
despite this many methods of monitoring have been put in place to ensure that minimal
discrepancies occur
•
DNA promoter is needed to remove introns from eukaryotic DNA as Bacteria do not have
introns
• Plasmid may not recombine
• Transformation may not occur
Evaluation
• More productive and efficient than from removing from bovine pancreas
• Loses efficiency dues to the difficulty in purification of insulin
• Loss of efficiency due to the difficulty in purification of the insulin
• Loss of efficiency in the transformation rate of successful recombinant plasmids
• However, these issues can be monitored and minimised and certainly outweigh many of the
negative issues at using bone insulin
Tick GUARD – Recombinant Vaccine
• Antigen Bm 86 is made from intestinal cells of Bophilus micrplus
• Genes for making this antigen are spliced into E.Coli plasmid
Process
• Purpose of a vaccine is to expose the body to the antigen as a weakened form to stimulate
the immune response to the disease under controlled conditions
• The antigen must be identified in the pathogen, Bm 86
• The gene for producing the antigen is extracted from the gut cells using restriction enzymes
and spliced into the DNA from another species, commonly an E.coli plasmid
• Plasmid is transformed into the E.coli bacteria
• As the bacteria grow exponentially, a large quantity of the antigen is produced
• A weakened form of the purified antigen is then injected into the animal
• A primary immune response is stimulated in the vaccinated animal and thus it produces
antibodies against the antigen, it already has the necessary means of defence if it encounters
the antigen again
•
Antigen is the protein that the tick makes in the stomach
• When it injects saliva into the beef or sheep, it injects the protein in the animal and that is
what causes the disease in the animal
• Therefore, a vaccine will make sure that the animal has already developed antibodies against
the protein so if infected, a secondary response will occur and the animals will not get too
sick
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329 Outcome of process
• Process produces recombinant E.coli that can produce a protein called BM-86
• This protein can be purified and made into a vaccine which, when injected into an animal’s
blood are sucked into the tick’s gut and destroy the tick from the inside
• Tick can’t reproduce and vaccinated herd can decimate tick populations quickly
Advantages
• Ecologically and environmentally safe alternative for controlling ticks in beef and dairy
cattle because no pesticides are sprayed into the environment
• Can be produced cheaply and consistently in large quantities using bacterial cells
• Recombinant batches do not vary in response against specific parasite strains
Disadvantages
• There has been difficulty in ensuring purification of the product so no other bacterial
components are still present in the final product
• Parasite populations may undergo mutations
• Cost more to immunise entire herd than use pesticide
• Every 6 month requires a booster
Evaluation
• More efficient production of bm 86 than to take it from ticks
• E.coli produce lots of antigen quickly difficulty of purification
• Loss of efficiency in transformation rate of successful recombinant plasmids
• Monitored
Yabby farming – Aquaculture
• Common yabby
• Cherax destructor
• Size, breeding speed, hardiness
Process
• Obtained samples of Yabbie populations that showed diversity
• Population is artificially selected in a selective breeding program
• Yabbies that possess the favourable market characteristics including size, breeding speed
and hardiness were selected
• These individuals are allowed to breed in controlled conditions so that the characteristics are
passed onto offspring
• Humans act as the selection pressure
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329 Outcome of process
• Yabbies that breed more quick and have 2 breeding cycles per year
• Increased size for more meat
• Reached market size more quick
• Hardiness, could survive in diverse conditions
Advantages
• Less labour intensive than altering environment
• Permanent
• Increased breeding, size, hardiness
Disadvantages
• Reduces genetic variability in captive populations as only favourable individuals are bred
• Disease
7. Ethical issues are relevant to the use of biotechnology are important and need to be
considered
Explain why different groups in society may have different views about the use of DNA
technology
 Different groups in society have different viewpoints because of their cultural background
and experience
 Issues that are contentious include environmental concerns, animal welfare, human safety
and confidentiality
 Scientists believe research and judicious decision-making is essential, they have a high code
of ethics and generally consider the technology as practical but have no involvement with
technology after it leaves the lab
 Biotechnology companies exist because of DNA technology, so the employment of many
people depends on a positive viewpoint, research on funding depends on stock market so
companies are aware of high ethical standards, but profit is significant and some companies
keep trade secrets of technology which may or may not be fair towards society
 Patients suffering disease are often desperate for a treatment, recombinant DNA technology
(humuline production), gene therapy (cystic fibrosis) and trials of monoclonal antibodies
(cancer) have show great benefits
 Farmers can be undecided as products such as tickGAURD improve survival and growth
but GM crops are debated and the question of profit for smaller farmers who can't afford this
technology
 Consumers are also undecided due to the unknown long term consequences of altering DNA
in viruses. The need for medicine by DNA technology has much greater acceptance than
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 Groups have different views as they are affected in different ways, profit, health,
employment, livelihood
 Animal liberationists are against the use of animals in biotechnology without considering
the benefits that come from such research, welfare of animal is paramount
Identify and evaluate ethical concerns related to one of the following:
− Development of genetically modified organisms
 A set of moral principles that determine an individual's or a group's behavious
 Necessary system to protect rights and fairness
 GMO's have had their DNA artificially altered by human, e.g. B-T Maize, soy bean
(recombinant)
 Safety of GMO's used in food production
 Transfer of genes from forbidden animal tissue may offend certain religions e.g. Jew and
Muslims do not eat pork
 Transfer of animal genes into plants may be an issue to vegetarians e.g. vegans
 may effect a large number of the population so GMO's should be discouraged if there is an
alternative
 Appropriate labelling must be mandated
Use available evidence to identify and discuss ethical and social issues associated with the use of
biotechnology
 Great potential for good, but due to progress, possible consequences, unforeseen events or
misuse
 ANZFA (Aus and NZ food authority) carry out safety assessments of GM foods such as
Round UP ready soy which resists Round Up herbicide
 Round Up Soy can have herbicide residue however this is inspected
 Genetic screening of human embryos for genetic defects may lead to termination which is
against religious views
 Eugenics could be applied, genetic discrimination
 Embryonic stem cell research, misuse of human beings? Life saving for disease? Adult stem
cells?
 GMF for developing nations with hunger issues, drought-resistant wheat, cold strawberries
Economic
 Increases employment
 Profit from previously unavailable food and food – insulin
 increased income from greater yields of agricultural goods Bt-cotton
Elite Education | The experts in private tuition www.eliteedu.com.au | 1300 914 329  Profits for small number of big businesses which can afford but smaller farmers may be outcompeted
 Ethical issues can be omitted from the public or manipulated such as 'the body shop'
incident
Scientific
 Research is essential for progress as it is practical
 high code of ethics
 enhance medicine and create new vaccines e.g. gardasil
 no involvement with technologies as it leaves lab
Cultural
 Can help save lives and therefore support families and quality of life for patients
 medical technology accepted
 desperate for medication
 religious beliefs are often discarded
 pork
Environmental
 Less harmful insecticides, pesticides used BT cotton
 Greater production of food
 More herbicides are used Round Up ready soybeans
 Possible cross pollination of transgenic crops with other varieties and native weeds with
unknown long term results
 May be allergic to GMO, Brazilian nut gene in soy beans, detected by rigorous tests
 All food must be labelled
 Less biodiversity
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