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Title: BIOLOGY OCR F215
Description: OCR Board A2 Level Biology F215 SECTION 2: BIOTECHNOLOGY AND GENE TECHNOLOGIES
Description: OCR Board A2 Level Biology F215 SECTION 2: BIOTECHNOLOGY AND GENE TECHNOLOGIES
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BIOLOGY
F215: Control, Genomes and Environment
1) Biotechnology and Gene Technologies
a) Cloning in Plants and Animals
i) Outline the differences between reproductive and non-‐reproductive cloning
(1) Clone – a gene, cell or whole organism that carries identical genetic material because
they are derived from the same original DNA
(2) Eg
...
animal with the same genotype as the donor organism
(7) Asexual reproduction – mitosis in eukaryotes, binary fission in prokaryotes (all the
resulting bacteria are clones of the original bacterium)
(a) Advantages
(i) Quick – organisms can take advantage of resources in the environment
(ii) Can be completed if sexual reproduction fails eg
...
therapeutic cloning – using embryonic, undifferentiated
stem cells to create cloned cells (rather than a complete organism)
(a) Generate cells, tissues and organs to replace those damaged eg
...
Nerve cells could be grown to repair those damaged in an accident or
those destroyed by diseases such as multiple sclerosis
2
...
Spinal cord can be repaired of those paralysed by an accident that has
resulted in a broken back or neck
(e) Disadvantages of therapeutic cloning
(i) Risk of rejection – stem cells would be genetically different to host’s
(ii) Ethical objections – use of human embryonic material
(iii) Scientific concerns – lack of understanding of how cloned cells will behave
over time
ii) Describe the production of natural clones in plants using the example of vegetative
propagation in elm trees
(1) Vegetative propagation – production of structures in an organism that can grow into
new, individual clones as they contain the same genetic information as the parent,
asexual reproduction in plants without production of seeds or spores
(2) English elm (Ulmus procera) – adapted to reproduce asexually following damage to
the parent plant which allows it to survive catastrophes such as disease or burning
(a) Root suckers/basal sprouts – grow from meristem tissue in the trunk close to the
ground (least likely area to be damaged) within 2 months of main destruction
(b) Root suckers grow all around the original trunk – help the elm spread
(c) Clonal patch – circle of new elms the suckers grow into when the tree is stressed
or dies eg
...
potatoes
(5) Bulbs – condensed shoots containing nutrients with very short stems, fleshy leaf
bases and buds at the sides that develop into new buds eg
...
strawberries
iii) Describe the production of artificial clones of plants from tissue culture
(1) Grafting (for rosebushes or fruit trees)
(a) Shoot section of a woody plant is joined to an already growing root and stem
(b) Side-‐grafting – rootstock is cut to match the wedge-‐shaped stem to be grafted
(c) Scion – part of the plant taken which is a portion of the stem with many buds,
selected for the quality of its fruit
(d) Rootstock – stump to which the scion is attached, selected for qualities such as
disease resistance and hardiness
(e) Vascular tissue is linked up
(f) Bindings are wrapped around the graft area to hold it in place until growth
supports the grafted section
(g) Graft grows genetically identical to the parent plant, but the rootstock is
genetically different
(2) Cuttings
(a) Section of parent plant removed
(b) Plant it into a suitable growth medium
(c) Treat it with plant hormones eg
...
root/shoot tips
(c) Explant – small piece of meristem tissue
(i) Explant is taken from the parent plant eg
...
between leaf joints
(d) Explants are sterilised using sodium hypochlorite, bleach or an alcohol
(e) Explants are placed on a suitable, nutrient growth medium with optimal
concentrations of amino acids, proteins, glucose, nitrates and phosphate
(f) Callus – mass of undifferentiated but dividing cells from the explant tissue
(g) Single callus cells removed from mass and placed on another growing medium
(h) Sub-‐culturing – callus can be subdivided to grow on different media
(i) Medium has plant hormones (auxin and cytokinins) to encourage shoot growth
(j) Growing shoots are transferred onto a different growing medium containing
different hormone concentrations that encourage root growth
(k) Hormone concentration ratios can be changed eg
...
sodium hypochlorite, bleach or an
alcohol, before being transferred to the agar medium
(n) Advantages of micropropagation
(i) Larger-‐scale cloning than cutting and grafting
(ii) Plants reproduce better than in cutting and grafting
(iii) Only a very small amount of plant material is needed
(iv) Easy to do
(v) Fast process
(vi) Genetically-‐identical – so can be disease-‐free
iv) Discuss the advantages and disadvantages of plant cloning in agriculture (HSW6a, 6b,
7c)
(1) Advantages of tissue culture – a ‘refinement’ of selective breeding
(a) Clones generated with the best qualities eg
...
pests, disease or environmental change (1845-‐51 potato famine)
(b) Attempts to minimise the risks are limited eg
...
mammary cell from cow’s udder
(ii) Remove the nucleus of an ovum (egg cell)
(iii) Place the differentiated cell’s nucleus in the enucleated egg cell
(iv) Fuse the cell to stimulate growth
(v) Allow the egg to go through the stages of development using the genetic
information inserted into the nucleus
(vi) Implant reconstructed cell ‘culture’ into a tied oviduct of a surrogate mother
(vii) Recover the early embryo and check for successful development
(viii) Implant the embryo into a surrogate mother’s uterus with exactly the correct
balance of hormones
(ix) Offspring is a clone of the adult whose differentiated cell was donated
(x) Cow has been genetically engineered to produce a human growth hormone in
milk and two more have been produced via cloning (only 15 are needed to
supply the world’s requirement for this hormone)
(xi) Fusion cell was not transplanted directly into the surrogate mother
1
...
Oviduct was used a culture medium
(xii) Only one out of 277 attempts were successful in Dolly the Sheep’s case
1
...
Some failed to develop into early embryos – perhaps due to difficulty in
reprogramming the embryo to behave as a zygote
3
...
skin cells into
pluripotent cells and almost identical to embryonic stem cells
(a) Uses four essential regulator genes
(b) Advantages – could replace the more controversial nuclear transfer method used
by scientist working on therapeutic cloning (see disadvantages above)
(c) Disadvantages – increased risk of developing cancers eg
...
sheep that produce pharmaceutical chemicals in their milk
(b) Large numbers of high-‐value animals can be produced quickly
(c) Preservation of endangered species
(d) Used for animals with low reproductive rates eg
...
Aspergillus
(d) Healthcare and medical processes – production of drugs by microorganisms, gene
therapy to treat some genetic disorders
(i) Penicillin antibiotic – fungus Penicillium grown in culture produces the
antibiotic as a by-‐product of its normal metabolism
(ii) Insulin hormone – bacteria E
...
niger can be grown in
certain conditions to produce and secrete pectinase enzyme
(ii) Calcium citrate used in detergents – fungus A
...
waste water treatment – bacteria and fungi
use organic waste in their water as nutrients and make the waste harmless
(g) Bacterium Clostridium acetobutylicum – produced acetone for WWI explosives
(4) Blue biotechnology – biotechnology applied to marine and aquatic environments
ii) Explain why microorganisms eg
...
proteins or chemicals that are given out into the
surrounding medium and can be harvested
(4) Can be genetically engineered to produce specific products
(5) Can often be grown using nutrient materials that would otherwise be useless, or
even toxic, to humans
(6) Grow well at relatively low temperature (more economic) – much lower than those
required in chemical engineering of similar processes
(7) Tend to generate products that are in a purer form than chemical engineering
(8) Cells themselves can be harvested and processed
iii) Describe, with the aid of diagrams, and explain the standard growth curve of a
microorganism in a closed culture
(1) Key definitions
(a) Culture – growth of microorganisms
(b) Pure broth culture – single of species of microorganism grown in nutrient broth
(c) Mixed culture – more than one species of microorganism grown in nutrient broth
(d) Closed culture – growth of microorganisms in an environment where all
conditions are fixed
(i) No new materials added
(ii) No waste products or organisms are removed
(e) Petri dish of nutrient agar – nutrient broth in a solid surface of agar
(2) Growth rate of microbes (see ecosystems and sustainability for graph) – only in a
fresh and ‘closed’ culture
(a) Lag/latent/initial phase
(i) Little or no increase in cell number – little or no reproduction
(ii) Acclimatisation – cells are adjusting to the surrounding conditions
(iii) Cell shows intense metabolic activity – exploiting culture medium
1
...
Cell expansion
3
...
Cells induce enzymes
5
...
Every 20-‐30 minutes, dependent on the species
(v) Length of the phase is variable – dependent on how quickly the organisms
reproduce and take up the available nutrients and space
(vi) Cannot be maintained indefinitely (unless system isn’t closed)
(c) Stationary phase
(i) Birth rate = death rate – individual organisms die at the same rate at which
new individuals are being produced
(ii) Slight fluctuations when birth rate is greater than death rate at some points
and death rate is greater than birth rate at others
(iii) Nutrient levels decrease
(iv) Metabolites (including waste products) build up – may have a toxic effect eg
...
porous carbon, glass beads, collagen, resins
(e) Covalent Bonding – binding enzymes to a support
(i) Formation of a covalent bond between the enzyme and the support medium
(ii) Enzyme molecules are covalently bonded to each other and to a support
(insoluble material)
(iii) Can use a cross-‐linking agent to cross-‐link enzymes molecules to each other
eg
...
Carboxymethylcellulose, clay particles
(f) Entrapment – hold enzymes in place without binding
(i) Enzymes trapped in a network, eg
...
not bound to another molecule
(ii) Enzyme molecules are free in solution
(iii) Enzyme molecules are restricted in movement by the lattice structure of the
support medium
(iv) Porosity of the lattice/semi-‐permeable membrane is controlled so that
enzyme leakage is prevented, but substrate and product can move freely
(v) Accessibility of active sites is reduced – substrate molecules need to get
through the trapping barrier so reaction rates are reduced
(vi) Eg
...
Nylon, cellulose nitrate, erythrocytes (red blood cell ‘ghosts’) or liposomes
v) Explain why immobilised enzymes are used in large-‐scale production
(1) Features of enzyme molecules that make them advantageous to use
(a) Specificity
(i) Higher rates of reaction – can catalyse reactions between specific chemicals
(even in mixtures)
(ii) Higher yields
(iii) Fewer by-‐products are formed
(iv) Less purification of products is necessary
(v) Can be reused as it is not used up in the reaction itself
(b) Temperature of enzyme reaction
(i) Lower temperatures and pressures can be used than for industrial chemical
processes – more economic as you save money on fuel costs
(ii) Thermophilic bacteria – thrive at high temperatures and can be used in
reactions that need a high temperature
(c) More efficient – product of only a single chemical reaction is needed so it is more
efficient to use isolated enzymes rather than growing a whole organism/using an
inorganic catalyst
(2) Advantages of immobilised enzymes
(a) Purification/Downstreaming process costs reduced – to separate soluble enzymes
from the product at the end of an industrial fermentation process is expensive
(b) Enzymes are immediately available for reuse – particularly useful in allowing for
continuous culture/processes
(c) More stable because the immobilising matrix protects the enzyme molecules
(3) Disadvantages of immobilised enzymes (usually offset by considerable advantage)
(a) Time (and therefore money) spent immobilising enzyme
(b) Expensive equipment and materials required for the immobilisation procedure
(c) Any contamination is costly to deal with – whole system would need to stopped
(d) Could decrease the effectiveness of the enzyme (rate of reaction) –
immobilisation may affect the shape and properties of the enzyme molecule
(i) Especially true in covalent bonding attachment which is near the active site
(e) Immobilised enzymes less active because they do not mix freely with substrate –
transport of substrates to enzyme slows down the reaction
(f) NB: Impossible to predict effect immobilisation will have on a particular enzyme
(4) Producing new antibiotics
(a) Increase in the number of antibiotic-‐resistant strains of pathogenic bacteria
(b) Focus on changing the structure of available antibiotics – form new antibiotics
(c) Target microorganisms will no longer be resistant
(d) Eg
...
cheese and yoghurts
(c) Disadvantages
(i) Growth rate is slower – nutrient levels decline with time
(ii) Less efficient – fermenter is not in operation all of the time
(iii) Tedious – sterilization process needs to be carried out after each run
(iv) Catalyst for an enzyme is lost at the end of each run
(2) Continuous culture – human hormones such as insulin are produced from continuous
culture of genetically modified Escherichia coli bacteria
(a) Process
(i) Organisms are grown continuously in a particular phase (usually log phase)
(ii) Products are removed from the fermentation tank at regular intervals
(iii) Nutrients are added to the fermentation tank to exactly balance the product
being removed
(b) Advantages
(i) Growth rate is higher – nutrients levels are continuously maintained as
nutrients are added to the fermentation tank
(ii) More efficient – fermenter automated to run continuously day and night
(iii) Smaller vessels can be used due to higher productivity – cost-‐effective
(iv) Quicker as no need to sterilise machinery
(v) Suitable for producing primary metabolites
(c) Disadvantages
(i) Set up is more difficult – maintenance of required growing conditions can be
difficult to achieve and system can very easily become imbalanced
(ii) Considerable wastage should the system be contaminated – whole plant
would have to be shut down
(iii) Foaming and clumping of cells can occur, blocking the inlets and outlets
vii) Describe the differences between primary and secondary metabolites;
(1) Metabolism – the sum total of all of the chemical reactions that go on in an organism
(a) New cells and cellular components
(b) Chemicals eg
...
CO2 and O2 to soluble urea, ammonia and nitrates
(2) Primary metabolites – substances produced as part of organism’s normal growth
(a) Eg
...
Antibiotic chemicals
(b) Production usually begins after the organism’s main growth phase
(c) Production does not mirror the population growth
(d) Only a relatively small number of microorganisms produce secondary metabolites
in comparison to primary metabolites
viii) Explain the importance of manipulating the growing conditions in a fermentation vessel
in order to maximise the yield of product required
(1) Bioreactor – vessel in which the activity of cells is optimised which permits efficient
conversion of relatively inexpensive raw materials to products of greater value
(2) Industrial applications – microorganisms must be grown in a bioreactor/fermenter
(3) NB: Fermenter is misleading – large number of processes carried out are not
fermentation reactions (originally applied only to the use of anaerobic respiration to
produce substances, in particular, the production of ethanol from yeast)
(4) Fermentation – now also refers to the culturing of microorganisms, both aerobically
and anaerobically, in fermentation tanks to produce a final, useful product
(5) Advantages of using a bioreactor
(a) Enables conditions to be monitored and controlled eg
...
for a cell count
(6) Scaling up – manipulating laboratory procedures so that they can be used on an
industrial scale to grow particular microorganisms on an enormous scale
(a) As conditions for small and larger scale production may not be the same
(b) Normally done through intermediate models
(c) Need a larger ‘starter’ population of microorganisms – obtained by taking a pure
culture and growing it in sterile nutrient broth
(d) Cooling water jacket
(i) Too hot – enzymes will be denatured
(ii) Too cool – growth will be slowed
(iii) Excess heat produced
(iv) Microorganisms produce heat as they grow
(v) Motor that drives the mixer produces a lot of heat
(e) Use sterilisable probes to monitor conditions more carefully and periodically
(i) Measurements can be recorded electronically
(ii) Electronic control systems and automatic valves can be used
(f) Inlet for the addition of nutrients
(i) Growth of microorganisms requires a nutrient supply, including sources of
carbon, nitrogen and any essential vitamins and minerals
(ii) The time of nutrient addition can be manipulated depending on whether the
process is designed to produce a primary or secondary metabolite
(g) Add a sparager and air inlet
(i) Aerobically respiring microorganisms rapidly consume dissolved oxygen
(ii) Sufficient oxygen in sterile air must be made available (pumped in)
(iii) In large volumes, diffusion is too slow
(iv) Lack of oxygen – unwanted products of anaerobic respiration that could lead
to a reduction in growth rate eg
...
Heat in flame, by UV light, steam-‐sterilised in an autoclave for 15 minutes
(b) Carry work out in a fume cupboard where air circulation carries away airborne
contaminants
(c) Cultures of microorganisms kept closed where possible
(d) Cultures of microorganisms kept away from bench surface when open and in use
(5) Aseptic techniques at large-‐scale culture level
(a) Fermenter, input and output pipes sterilised, disinfected and steam-‐cleaned
(b) Sterilising all nutrient media before adding to the fermenter
(c) Probes to monitor conditions such as temperature and pH must be sterilised
(d) Fine filters on inlet and outlet pipes
(i) Incoming air must be filtered to remove microorganisms
(ii) Outgoing air must be filtered if the organisms within it are particularly harmful
(e) Seal all entry, exit, observation and sampling ports when not in use
(i) Desired microorganisms must be contained
(ii) Other microorganisms must be excluded
(f) Polished, stainless steel fermenter surfaces
(i) Corrosion resistant containers – prevent trace metal contamination of the
culture
(ii) Prevent microbes sticking to surfaces
(iii) Capable of withstanding repeated sterilisation
(g) Pipework arranged so separate parts can be isolated and sterilised – avoids the
whole plant from being shut down
(h) Transparent materials wherever possible to enable visual inspection of culture
c) Genomes and Gene Technologies
i) Outline the steps involved in sequencing the genome of an organism
(1) Key definitions
(a) Genomics – study of genomes
(b) Genomes – the whole set of genetic information in the form of DNA base
sequences within the cells of organisms of a particular species
(i) Sequenced genomes are placed on public access databases
(c) Coding DNA – code for the production of polypeptides and proteins, only 1
...
coli (bacterial) cells
(f) Clone libraries – cells are grown in culture so clones of the sections are produced
(5) BAC section sequencing
(a) Cells containing specific BACs are taken and cultured
(b) DNA is extracted from the cells
(c) Restriction enzymes are used to cut the DNA into smaller fragments because
sequencing can only operate on a length of DNA of about 750 base pairs
(d) Fragments are separated using electrophoresis in order of size
(e) Each fragment is sequenced
(f) To ensure accuracy of completed code, sequencing carried out many times, on
overlapping fragments
(g) Use of different restriction enzymes on a number of samples gives different
fragment types
(h) Computer programmes compare overlapping regions from the cuts made by
different restriction enzymes
(i) Whole BAC segment sequences reassembled
(6) Automated DNA sequencing – fragments of varying length are produced, with a
fluorescent marker as the last added base, then the sequence is shown by the order
of the colours as electrophoresis separates the fragments by length
(a) Sanger/dideoxy method – based on the use of dideoxyribonucleotides (ddNTP)
and a chain termination technique
(b) ddNTP – hydroxyl group on 3rd carbon of dNTP is replaced by a hydrogen
(c) Compose four different sequencing reaction mixtures
(i) Many copies of the single-‐stranded template DNA fragment to be copied
(ii) Four normal free-‐floating DNA nucleotides (A, T, G, C)
(iii) Small amount of one type (A, T, G, C) of dideoxynucleotide – each type has a
different coloured fluorescent markers
(iv) Primer for the nucleotides to join onto
(v) DNA polymerase
(d) DNA fragment to be sequenced is copied many times – process similar to PCR
(i) Primer anneals at the 3’ end of the template strand
(ii) DNA polymerase is now allowed to attach to the double-‐stranded section of
the template strand
(iii) DNA polymerase can add complementary nucleotides to the strand according
to base-‐pairing rules
(iv) Double stranded length of DNA grows
(v) DNA polymerase comes across a ddNTP and is thrown off
(vi) Further nucleotides cannot be added
(vii) ddNTP acts as a terminator – the reaction stops on that template strand
(e) Thousands of DNA fragments are produced in each reaction mixture
(i) Each DNA fragment is of a different length
(ii) Length dependent on when the ddNTP was added and when the DNA
polymerase was thrown off
(iii) In some, the template strand is completed
(iv) All have a final added nucleotide tagged with a specific colour
(f) DNA strands are run through a DNA sequencing machine
(i) Transfer fragments from each reaction mixture to tiny wells in a gel plate
(ii) Load gel plate into a DNA sequencing machine
(iii) Pass an electric current, via electrodes on either side, through the gel
(iv) As the fragments run down the gel to the positive electrode, a laser reads the
colour sequence, detecting the colour each fragment fluoresces
(v) Smaller fragments are read first as they move more quickly through the gel
and reach the end of the gel first
(vi) Data is recorded by the machine as a coloured peak
(vii) Colour of each peak identifies the ddNTP (final base of each strand) that
terminated the fragment, ordered from shortest to longest
(viii) Sequence of colours, and so the sequence of bases, can then be displayed
(g) Sequencing requires sections of DNA to be sequenced between 6 and 10 times to
be confident that the base sequencing information is accurate
ii) Outline how gene sequencing allows for genome-‐wide comparisons between individuals
and between species (HSW7b)
(1) Comparative genome mapping
(a) Sequences of bases in a gene of one organism is already known
(b) Comparing genes for the same/similar proteins across a wide range of organisms
(2) Gives clues to the relative importance of such genes to life – finding the same genes
coding in all/many living organisms shows it plays a great role in living
(3) Shows evolutionary relationships between different species – the more DNA
sequences organisms share, the more closely related they are likely to be
(4) Modelling effects of changes to DNA can be carried out – predicting the effects of a
mutation on humans by testing the effects on an organism carrying the same gene
(a) Yeast is a haploid organism
(b) Mutation to a gene is always shown in the phenotype
(c) Studies have tested effects on genes obtained from yeast
(d) Genes are also found in the human genome
(5) Identification of specific genes or base-‐pair sequences causing a disease – comparing
genomes from pathogenic and similar but non-‐pathogenic organisms
(a) Specific genes targeted to develop more effective drug treatments and vaccines
(6) Analysis of DNA of individuals – reveals mutant alleles, shows presence of alleles
associated with increased risk of a disease eg
...
A plasmid with DNA from two different organisms or sources
iv) Explain that genetic engineering involves the extraction of genes from one organism, or
the manufacture of genes, in order to place them in another organism (often of a
different species) such that the receiving organism expresses the gene product (HSW6a)
(1) Required gene is obtained
(a) From extraction from organism
(i) mRNA produced from the transcription of the gene can be obtained from cells
in an organism where that gene is expressed
(ii) mRNA used as a template to make a copy of the gene
(iii) Eg
...
flush ends
(b) Sticky ends – staggered cut through DNA by restriction enzymes which forms a
short run of unpaired, exposed bases at the end of the cut section of the DNA
(i) Overhanging single stranded DNA
(ii) Majority of restriction enzymes cut DNA to form sticky ends
(iii) EcoR1 cuts at every GAATTC between the G and the A
vi) Outline how DNA fragments can be separated by size using electrophoresis (HSW3)
(1) Gel electrophoresis – separation of DNA fragments of different lengths in a mixture
as the negatively charged fragments move towards the cathode at different speeds
(a) Similar to chromatography
(b) Used for identification and analysis
(c) Separates fragments of DNA produced by restriction enzymes according to size
(d) Accurate enough to separate fragments that are different by one base in length
(e) Shorter fragments pass through the gel more easily and so move further in a
fixed period of time
(2) Process
(a) Treat DNA samples with restriction enzymes to cut them into fragments
(b) Make gel ‘plate’ or ‘slab’ – purified form of agar containing agarose sugar
(i) Covered in buffer solution
(ii) Gel acts like a sieve
(iii) Electrodes attached to each end of the gel so a current can pass through it
(c) Special comb used to cut wells into the negative electrode end of the gel
(d) Mix DNA fragments with loading dye
(e) Load DNA samples into the wells in the gel
(f) Immerse gel in a tank of buffer solution
(g) Electrical current is applied to the electrodes
(h) Pass electric current through the solution for a fixed period of time eg
...
Cools so that primer hybridises to the DNA
(5) Stage 1 – separation of DNA
(a) Place all DNA fragments to be copied, free-‐floating DNA nucleotides and DNA
polymerase into the thermocycler
(b) Heat to 95°C – heat does the job of DNA helicase enzymes in nature
(c) Heat breaks hydrogen bonding holding the complementary strands together
(d) Strands of DNA separated and made single-‐stranded
(6) Stage 2 – annealing (joining)
(a) Add primers to the thermocycler
(b) Cool mixture to 55°C
(c) Primers allowed to anneal to the complementary bases at the end of the DNA
fragments via hydrogen bonding
(d) Small sections of double-‐stranded DNA at either end of the DNA sample formed
(e) Primers provide double-‐stranded sections for DNA polymerase to bind and work
(7) Stage 3 – synthesis of DNA
(a) Increase temperature to 72°C
(b) Optimum temperature for DNA polymerase
(c) DNA polymerase starts to add free, complementary DNA nucleotides along each
of the separated DNA strands
(d) Double stranded sections of DNA provided by the primers are extended
(e) This will continue until the DNA polymerase reaches the end of the chain
ix) Explain how isolated DNA fragments can be placed in plasmids, with reference to the
role of ligase
(1) DNA ligase enzyme – catalyses a condensation reaction which joins the sugar-‐
phosphate backbones of the DNA double helix together
(2) Used in natural DNA replication to seal DNA nucleotides together to form new DNA
(3) Both DNA fragments need to have originally been cut with the same restriction
enzyme to be joined together by DNA ligase
(a) Ensures nucleotide bases of sticky ends are complementary to one another
(b) Bases of sticky ends can anneal – pair up and hydrogen bond together
(c) DNA ligase can seal the sugar-‐phosphate backbone to form recombinant DNA
x) State other vectors into which fragments of DNA may be incorporated
(1) Choice of cloning vector depends upon the nature of the experiment undertaken
(2) Naturally occurring vectors include…
(a) Bacteriophages – viruses which act as parasites, infecting and replicating inside a
bacteria, requiring much preparation before being used as cloning vehicles
(b) Plasmids – small (relative to major chromosome) double-‐stranded circular pieces
of DNA found in many bacteria, separate from the main bacterial chromosome
(i) Capable of self-‐replication independent of the host cell chromosome – more
than one can be found in a single bacterial cell
(ii) Carry genes needed only under special circumstances eg
...
01% of bacterial cells contain the desired gene – inefficient
xii) Describe the advantage to microorganisms of the capacity to take up plasmid DNA from
the environment
(1) Conjugation – process whereby copies of plasmid DNA are passed between bacteria,
thus they are exchanging genetic information
(a) Sometimes plasmid DNA is exchanged between different species of bacteria
(b) Conjugation tube forms between a donor and a recipient
(c) Enzyme in donor cell makes a nick in the plasmid
(d) Plasmid DNA replication starts as the transferred DNA strand starts moving
through the conjugation tube
(e) The transferred DNA reseals back up to form a circular piece of DNA
(f) Cells move apart
(g) Circular DNA plasmids (one in each cell) reform
(2) Advantage of conjugation – contribute to genetic variation and survival…
(a) Speeds the spread of antibiotic resistance between bacteria populations –
plasmids often carry genes associated with antibiotic resistance
(b) MRSA – resistant strain of bacteria commonly found on human skin
(i) Transfer of bacteria from the skin to a wound can lead to a serious infection
(ii) New antibiotics continually being looked for to target such organisms
xiii) Outline how genetic markers in plasmids can be used to identify the bacteria that have
taken up a recombinant plasmid
(1) Gene markers – fluorescent proteins or enzymes that produce visible products
(a) Radioactive marker – radioactive 32P present in the phosphoryl groups forming
the strand can be revealed by exposure to photographic film
(b) Fluorescent marker – usually used in automated DNA sequencing where
nucleotides will emit a colour on exposure to UV light
(c) Antibiotic resistance genes
(2) Reasons for very low efficiency of transformation
(a) Only 1% of bacteria will take up the plasmid
(b) Some bacteria will contain an ‘empty’ plasmid
(i) Plasmid’s sticky ends have joined back together
(ii) The plasmid has sealed up on itself to reform the original plasmid
(iii) The plasmid has not taken up and sealed in the gene during gene insertion
(c) Only 0
...
E
...
pro vitamin A – precursor molecule which is converted to
active vitamin A in the human gut
(c) Fat-‐soluble – lipids needed in diet if vitamin A is to be absorbed properly
(d) Deficiency is significant in poorer populations where rice is the staple food –
vitamin and other food supplements to target groups has made little impacts on
the devastating effects of malnutrition on these populations
(2) Functions of vitamin A – central role in maintain integrity of the immune system
(a) Eyesight – forms part of the visual pigment rhodopsin
(b) Cell growth and development – involved in synthesis of many glycoproteins
(c) Epithelial tissue – needed for maintenance and differentiation of epithelial cells,
helps reduce the risk of infection
(d) Bones – essential for bone growth
(3) Rice plant (Oryza sativa) – contain genes that code for production of beta carotene
(a) Green tissues – inedible part of the plant that produces beta carotene
(i) Photosynthetic pigment molecule codes for the production of beta carotene
(b) Endosperm – grain, edible part of the seed that does not produce beta carotene
(i) All required genes to produce beta carotene are present in the grain
(ii) But some of these genes are turned off during development
(c) Outer coat of dehusked grains – contains valuable nutrients but no beta carotene
(i) Eg
...
beta-‐carotene
(i) Beta carotene accumulates in the endosperm
(ii) Complex metabolic pathway to synthesise beta carotene reactivated in the
endosperm cells (within the grain) with minor intervention
(iii) Beta carotene made the rice grains yellow-‐orange in colour
(b) Phytoene synthetase gene isolated and extracted from daffodil plants – enzyme
converts a variety of precursor molecules, including GGP, into phytoene
(c) Crt 1 gene isolated and extracted from soil bacterium (Erwinia uredovora) –
enzyme cocatalyses the conversion of phytoene to lycopene
(i) Lycopene – precursor molecule for the carotenoids
(ii) Enzymes are present in rice endosperm to convert lycopene to beta carotene
(d) Both genes inserted into plasmid found in Agrobacterium tumifaciens
(e) Agrobacterium incubated with rice embryos
(f) Agrobacterium tumifaciens naturally infected the rice plants and by doing so, also
transferred the genes that encode the instructions for making beta carotene
(g) Genes inserted near a specific promoter sequence in the rice genome that
switches on the genes associated with endosperm development
(h) Genes were thus expressed as the endosperm grew
(i) Result – genetically engineered rice which had beta carotene in the grain
(5) Crossbred GM rice with natural varieties
(a) Survival in local climate conditions ensured – GM rice did not grow well in harsh
conditions of the paddy fields
(b) Golden Rice 2 – Syngenta produced a variety which accumulated 20 times more
beta carotene in the endosperm in 2005
(c) Field trials are still currently taking place
(6) Lack of success
(a) Never been commercially produced
(b) Still contain very little beta carotene – would have to eat large amounts of rice to
take in sufficient amounts of beta carotene
(c) Humanitarian Use Licences – allow farmers in third world countries to keep and
replant crop seeds without having to pay a licence fee
(i) Given free of charge by researchers and biotechnology companies that have
produced Golden Rice
(ii) Critics accuse these companies of only doing this to gain public acceptance of
GM crops
(d) Greenpeace argue GM crops are still unacceptable
(i) Lead to a reduction in biodiversity
(ii) Human food safety of engineered rice is unknown
(iii) GM rice would breed with wild types and contaminate wild rice populations
xvi) Outline how animals can be genetically engineered for xenotransplantation (HSW6a, 6b)
(1) Xenotransplantation – transplantation of cells, tissues or organs between animals of
different species
(2) Xenograft – a tissue graft or organ transplant from a donor of a different species
from the recipient
(3) Allotransplantation – transplantation between animals of the same species
(4) Purpose – allows for more organ transplants into humans, saving lives
(a) Failure of a particular organ results in the need for an organ transplant
(b) Worldwide shortage of donor organs – 60% of patients awaiting replacement
organs die whilst on the waiting list
(c) Non-‐self transplanted organs can trigger an immune response – rejection of
transplanted tissue
(5) Pigs – donor animals of choice
(a) Physiology is similar to humans
(b) Distant enough from humans in evolutionary terms that humans do not share a
lot of pathogens with pigs
(c) Practical to breed them in quantities required for human transplantation –
produce large litters that mature very quickly
(6) Problems
(a) Acute rejection – immune system produces antibodies against donated organ
(i) 2003 – pigs genetically engineered to lack alpha 1-‐3 transferase, an enzyme
antibodies are targeted against and therefore a key trigger for graft rejection
(ii) 2006 – insertion of human nucleotidase (E5’N) gene into pig cells in culture
reduced immune cell activities involved in xenotransplant rejection
(b) Physiological problems
(i) Differences in size of organs
(ii) Premature ageing of xenograft – eg
...
Cell surface antigens produced will make the cells vulnerable to attack by
the immune system
(d) Targeted, somatic cells treated with the functioning allele
(e) Difficulties in getting the allele into the genome in a functioning state
(i) Use of ex vivo therapy – specific, somatic cells removed, treated and replaced
(ii) Genetically modified viruses used as a vector – host becomes immune to them
so cells will not accept the vector on subsequent treatments
(iii) Liposomes used as an alternative vector – inefficient
(f) Short-‐lived treatment – regular doses of the functional gene are required for it to
be expressed
(g) Specialised cells containing the functional allele will not pass on the allele –
functional gene is restricted to these targeted, somatic cells
(h) Genetic manipulations are restricted to the actual patient
(3) Problems with gene therapy
(a) Individuals resulting from germline cell gene therapy would have no say in
whether their genetic material should have been modified
(b) Unknown level of risk on future generations
(c) Inadvertent modification of DNA in germline cell – can’t tell whether the allele
has been successfully introduced without unintentional changes to the embryo
(i) Could create a new human disease
(ii) Eugenics could interfere with human evolution – germline cell gene therapy
taken advantage of to enhance favourable characteristics
(d) Could interfere with expression of another gene due to where it is inserted
(i) Severe combined immunodeficiency (SCID) – recessive disease leading to
complete dysfunction of the immune system
(ii) One of ten forms due to presence of defective gene for adenosine deaminase
(iii) Lack of ADA – accumulations of metabolites toxic to T lymphocytes
(iv) Insertion of functional allele in X-‐linked SCID patient near LM02 gene has led
to cases of leukaemia
(e) Inactivated virus used as a vector to carry the healthy genes into the patient’s
cells is not as safe as researchers had once thought
(4) Future research
(a) Ways of getting genes into cells without relying on a virus
(i) Liposomes
(ii) Microinjection – ‘naked’ DNA directly injected into host nucleus using a very
fine micropipette
(iii) Artificial human chromosomes
(b) Ensure that a transferred gene goes into the cell’s genome at the same position
as the already mutated gene
xix) Discuss the ethical concerns raised by the genetic manipulation of animals (including
humans), plants and microorganisms
(1) Transgenic animals – new genes inserted into animals by microinjection of egg cells
or early embryos, then reinserted back into the mother’s uterus/surrogate mother
(a) Long-‐life tomatoes
(i) Tomatoes that make less PG ripen more slowly but retain flavour
(ii) Flavr Savr – antisense technology silenced gene for enzyme PG involved in
softening of tomatoes when ripening
(iii) Zeneca tomato – disrupted PG enzyme gene
(b) Insect-‐resistant crops
(i) Genes for powerful protein toxins transferred from Bacillus thuringiensis (BT)
to crop plants eg
...
chemical insecticides
1
...
Specific vs
...
Minimum damage to the environment vs
...
Avoids bioaccumulation of chemical insecticide
(c) Nitrogen-‐fixing crops
(i) Transfer 15 genes required for nitrogen fixation from Rhizobium (nitrogen
fixing bacteria) into cereals and other crop plants
(ii) Crops can then fix their own atmospheric nitrogen
(iii) No fertilisers needed
(d) Tick-‐resistant sheep
(i) Enzyme chitinase – kills ticks by digesting their exoskeletons
(ii) Chitinase enzyme gene transferred from plants to sheep
(iii) Sheep immune to tick parasites
(iv) No sheep dip needed – hazardous to those who come into contact with it
(2) Arguments for genetic engineering
(a) Production of ‘unnatural’ organisms has already been done for centuries
(i) Selective breeding
(ii) Organisms with valuable traits have been bred over many generations
(iii) Domesticated varieties produced are far removed from their ancestral wild
relatives
(b) Media hype often has no scientific background
(i) Use of the term ‘Frankenfoods’ suggests that transfer of DNA into the human
genome could occur from eating food containing DNA
(ii) Absurd suggestion that DNA as part of the diet is something unnatural
(c) Useful products can be produced
(i) Human insulin and human growth hormone – GE microorganisms
(ii) Pharmaceutical chemicals – alpha anti-‐trypsin in milk of female transgenic
sheep used to treat hereditary emphysema
(d) Combat vitamin A deficiency in third world countries – accumulation of beta
carotene in endosperm of seeds of rice plants
(e) Increase crop yields – resistance to herbicides allows application of weedkillers,
resistance to pesticides allows application of them
(f) Gene therapy – treat genetic disorders such as AIDS and X-‐linked SCID
(3) Ethical arguments against genetic engineering
(a) Research into genetic engineering is harming current living organisms
(i) Trials using inactivated viruses in germline cell gene therapy to carry healthy
genes into the patient’s cells is not as safe as researchers had once thought
(ii) Inadvertent modification of DNA in germline cell – can’t tell whether allele has
been successfully introduced without unintentional changes to the embryo
(b) Lack of long-‐term knowledge – unknown level of risk on future generations
(i) Expression of a gene influenced by presence of other genes and environment
(ii) Risk means genetically manipulating animals for any reason is unethical
(c) Reduction in genetic variation
(i) GE crop plant passes on genes to wild relatives
(ii) GE organism competes with the natural species which is then lost
(d) Widespread resistance to antibiotics – genetic engineering often uses antibiotic
resistance genes as markers which could be passed to other microorganisms
(i) E
...
coli (a bacteria which forms part of the natural
fauna in the human gut) could enter humans
(e) Mutated genes transferred to pathogenic microorganisms – GE microorganism
producing useful products may escape from containment and transfer mutations
(f) Hybrid crops produced are less useful – produced as GE crop plant and wild
relatives share genes
(g) Super-‐weeds – herbicide resistance could be passed to weeds so stronger
chemicals would need to be developed to remove the weed
(h) Super-‐pests – pesticide resistance could be passed to pests so stronger chemicals
would need to be developed to remove the pest
(i) More rapid evolution of attack mechanisms in pathogens – to counteract more
plants becoming resistant to the pathogen
(j) Stability of ecosystems could be affected – pesticide resistance could be passed
to pests, affecting many other organisms in the associated food chains
(k) GM plants may be toxic to other organisms
(l) GM plants may lead to allergic responses in humans
(m) Drugs produced by GE animals could contaminate milk/meat supplies
(n) Large companies get patents for GE organisms and exploit farmers in the 3rd
world eg
Title: BIOLOGY OCR F215
Description: OCR Board A2 Level Biology F215 SECTION 2: BIOTECHNOLOGY AND GENE TECHNOLOGIES
Description: OCR Board A2 Level Biology F215 SECTION 2: BIOTECHNOLOGY AND GENE TECHNOLOGIES