Biotechnology and gene technologies

Question 1

outline the differences between reproductive and non-reproductive cloning

Answer 1

reproductive cloning is the production of a new individual with the genotype of an existing one. Non-reproductive cloning, or therapeutic cloning, supplies replacement cells whose genotype matches that of an existing organism.

Question 2

Explain the process of vegetative propagation in Elm trees.

Answer 2

‘Suckers’ - shoots that grow from below ground level, usually from a root - are remove from a tree in autumn and grown in a nursery bed. Alternatively Elms can be ‘layered’ - low growing branches are pegged onto the soil in autumn, left until they grow roots, and then separated.

Question 3

Describe the production of artificial clones of plants using tissue culture (micropropagation)

Answer 3

Explants of tissue are taken from young, developing stems that have been surface sterilised with alcohol.
In aseptic conditions, the explants are placed in a growth medium containing sucrose (energy source), amino acids, vitamins, inorganic ions, plant hormones to stimulated mitosis.
Undifferentiated cells in the explant divide to produce a callus which can be subdivided many times to increase the number of plants that can eventually be produced.
Small pieces of callus are transferred to a new growth medium with plant hormones to trigger differentiation into shoots and roots.
The resulting embryoids grow into plantlets, which are transferred via a hardening medium into sterile soil.

Question 4

What are some advantages and disadvantages of plant cloning in agriculture?

Answer 4

advantages:
very many genetically identical plants can be produced from one original plant.
Desired characteristics are preserved.
Plants can be produced at any time of year.
Callus can be genetically engineered.
disadvantages:
genetically identical plants are all susceptible to the same pathogens/changing environmental conditions.
The process is labour-intensive and therefore costly - more difficult to plant plantlets than sow seeds.

Question 5

describe how artificial clones of animals can be produced by embryo splitting?

Answer 5

A female with desired traits is made to superovulate and the eggs are fertilised in vitro/in vivo by sperm from a suitable male.
The resulting embryos are subdivided and implanted in healthy recipient females which act as surrogate mothers.

Question 6

describe how artificial clones of animals can be produced by somatic nuclear transfer?

Answer 6

• somatic cell nucleus is inserted into an enucleate egg cell from another animal by electrofusion
• the resulting embryo is grown in vitro
• early embryo is split
• embryos are replaced into healthy surrogate mothers

Question 7

Discuss the advantages of reproductive cloning of animals

Answer 7

• Breeding of endangered species: a fertile female of an endangered species is not needed for somatic nuclear transfer
• the reproductive rate of a genetically superior animal is increased
• the number of animals with a wanted trait is increased
• the cloned embryo can be sexed/tested for genetic diseases before implantation into a surrogate
• Production of transgenic animals for ‘pharming’ of human chemicals

Question 8

Discuss the disadvantages of reproductive cloning of animals

Answer 8

Ethical objections:
animals concerned are denied their natural instincts and behaviour and are being used by the breeder as a means to an end.
- If a clone is susceptible to disease or changes in environment, then all the clones will be susceptible.
- It will lead to less variation, and less opportunity to create new varieties in the future.

Question 9

What is biotechnology?

Answer 9

the industrial use of living organisms or parts of living organisms to produce food, drugs or other products

Question 10

Why are microorganisms often used in biotechnological processes?

Answer 10

• they are highly versatile, occupying a wide range of habitats including extreme conditions
• they have a rapid growth rate
• proceses occur at low temperature and pressure - cheaper and safer to maintain
• they are small so can be produced in large numbers in a small volume
• they can grow on unwanted food/nutrients
• Can be genetically engineered to produce specific products
• Tend to generate products that are in a more pure form that those generated via chemical processes

Question 11

Describe what the standard growth curve of a microorganism in a closed culture would look like

Answer 11

There are four recognisable phases of population growth:
Lag phase - start of curve as bacteria adjust and growth is slow,
exponential phase where growth is most ideal and curve is steep and no. bacteria double in each unit of time, stationary phase where death rate is equal to division and decline phase where the curve drops/slopes downwards.

Question 12

Explain what is happening to the population of bacteria in the lag phase, exponential phase, stationary phase and decline phase

Answer 12

lag phase - bacteria adjust to new conditions, synthesising carriers to absorb nutrients or enzymes to digest them. May involve switching on genes. Growth is slow - cells increase in size but little division.
exponential phase - no limiting factors in this phase and so growth is exponential (population doubles per unit time) division>death
stationary phase - limiting factors, such as the failing supply of nutrients and oxygen and the build-up of waste products gradually have their effect until the death rate equals the division rate and no population growth occurs.
decline phase - the death rate is greater than the division rate so the population becomes smaller, although the decline may be slowed slightly by breakdown of cells releasing nutrients that other cells can use.

Question 13

What are some factors that prevent the unlimited population growth of bacteria?

Answer 13

• depletion of nutrients
• depletion of oxygen
• accumulation of toxic or acidic waste products
  These actors are density-dependent so the greater the number of individuals in the population the greater the effect of the factor.

Question 14

What are 4 ways in which enzymes can be immobilised?

Answer 14

• Carrier-binding: adsorption or bonding onto an insoluble matrix, such as collagen or cellulose
• Cross-linking: amino groups on the enzyme are cross-linked by strong covalent bonds to an agent
• Membrane separation: enzyme solution held inside a partially permeable membrane such as cellophane whilst substrate solution is passed along the other side
• Entrapment: held in a microcapsule of polyacrylamide or alginate

Question 15

What are some advantages of the use of immobilised enzymes in large-scale production?

Answer 15

• enzymes can be recovered easily and used many times
• the product is not contaminated with the enzyme making the process ideal for continuous culture (does not have to be extracted - cheaper) less DOWNSTREAM PROCESSING
• protection by the immobilising material means the enzyme is more stable in changing temperatures or pH

Question 16

what is an advantage of use of continuous culture over batch culture in the growth of biotechnologically useful microorganisms?

Answer 16

Greater productivity- exponential growth is maintained.
Smaller vessel required for the same yield.
Production is faster as nutrients are continuously added to the fermentation tank.
No need to clean out fermenter.
more efficient, fermenter operates constantly.
very useful for processes involving the production of primary metabolites

Question 17

what are the advantages of use of batch culture over continuous culture in the growth of biotechnologically useful microorganisms?

Answer 17

easier to set up and control

only one batch is lost should the culture become contaminated whereas potential losses from contamination are much greater in continuous culture because productivity is greater
Very useful for processes involving the production of secondary metabolites

Question 18

compare the process of batch culture vs continuous culture

Answer 18

batch:
carried out in a closed fermenter.
nothing is added, only waste gases removed
product is separated from mixture at end of process
the microorganism’s exponential growth phase is short
continuous:
carried out in an open fermenter
nutrients are added continuously
product is tapped off continuously
the microorganisms are kept in the exponential growth phase

Question 19

what is a primary metabolite?

Answer 19

the product of an organism’s essential metabolism (primary metabolism)

Question 20

what is a secondary metabolite?

Answer 20

Substances produced by an organism that are not part of its normal growth. They are often produced by cells that have stopped dividing, and are not directly involved in the normal growth, development, or reproduction of an organism. Penicillin is a secondary metabolite.

Question 21

Describe how the growing conditions in a fermentation vessel may be manipulated in order to maximise the yield of product

Answer 21

The following conditions are manipulated so that they are OPTIMAL for the exponential growth phase:
temperature (use cooling jacket)
pH (use probes)
concentration of oxygen (bubble air in)
water potential
concentration of nutrients
concentration of waste products (tap off, use paddles to stir - prevents settling)

Question 22

Why is asepsis so important in a fermenter/in the manipulation of microorganisms?

Answer 22

To prevent unwanted microbes from being in the fermenter, which:

• compete with the wanted organisms for resources, reducing the yield
• change the conditions
• contaminate batch/product
• to prevent escape of useful microorganisms

Question 23

Why is important that specific conditions are maintained in a fermenter?
e.g. temp, nutrients, O2, pH

Answer 23

To optimise growth rate of microorganisms and therefore yield.
Temperature - important due to the effects on enzymes: too high will cause denaturation and too low will slow growth.
Nutrients - type needed will be specific to microbe, but must include a source of C and N, essential vitamins and minerals. The timing of nutrients’ addition can e manipulated depending on whether the product is a primary or secondary metabolite.
O2 Concentration - most microorganisms need oxygen for aerobic respiration and rapid growth. Lack of O2 causes anaerobic respiration, producing ethanol/lactate which reduce the growth.
pH - affects enzyme action and therefore growth rate.

Question 24

what is a genome and why is knowing its sequence important?

Answer 24

The whole set of genes in an organism.
It gives us information about the location of genes and the evolutionary links between organisms as it allows genome-wide comparisons to be made between individuals and between species.

Question 25

Outline the steps involved in sequencing the genome of an organism

Answer 25

DNA is made single-stranded.

1. The primer binds to the DNA, allowing DNA polymerase to attach
2. DNA polymerase adds free nucleotides according to complementary base pairing so strand increases in length
3. when a nucleotide with a terminator base is added (‘tagged’ nucleotides), the DNA polymerase is thrown off and the DNA cannot increase further in length
4. Many copies of different lengths of the DNA are made, each with a different coloured tag at its end.
5. the DNA lengths are separated using electrophoresis - shorter lengths move fastest
6. The ‘nested’ fragments (all end at nucleotides with different bases and differ in length by one nucleotide) are passed through a detector which records the ‘tag’ colour and a computer displays the data as a series of peaks allowing the sequence to be read directly.

Question 26

what is meant by the term recombinant DNA?

Answer 26

DNA that has been made by genetic engineering, by joining together pieces of DNA from two or more different sources.

Question 27

What does genetic engineering involve?

Answer 27

The extraction of a gene from one organisms, or the manufacture of genes, in order to place it in another organism of a different species in such a way that the receiving organism expresses the gene.

Question 28

What is the use of restriction endonucleases?

Answer 28

to extract sections of DNA containing a desired gene from a donor organisms
creates ‘sticky ends’ - unpaired bases as it makes a staggered cut through the DNA at specific base sequences

Question 29

Outline the separation of DNA fragments by electrophoresis

Answer 29

Separation by size

1. DNA sample is cut into fragments by restriction endonuclease enzymes
2. Fragments are put into wells at the negative end of a gel plate - by the cathode
3. A direct current is passed through the gel for a set amount of time
4. DNA fragments are negatively charged so move towards the anode
5. The shorter the fragments, the further they travel in the given time. Blue/fluourescent dye that stains DNA is used to show banding pattern.

Question 30

Describe how DNA probes are used

Answer 30

To identify fragments containing specific sequences:
A DNA probe is a short, single stranded piece of DNA that is labelled (either using a radioactive marker that will show up in exposure to x-ray film, or a fluourescent marker that emits a colour when exposed to UV light) . It will bind to any fragment that gas a complementary base sequence to it.
Useful for identifying a gene needed for genetic engineering, to aid in genome comparisons and to identify an allele associated with a genetic disease.

Question 31

Outline the process of PCR

Answer 31

1. DNA sample is mixed with DNA nucleotides and DNA polymerase.
2. DENATURATION: The mixture is heated to 95 degrees C to break the H bonds between bases and so produce single stranded DNA
3. ANNEALING: Short lengths of single-stranded DNA called primers are also added, and the temperature is lowered to 55degrees to allow the primers to bind to the ends of the samples, making short double stranded sections (without which DNA polymerase could not attach)
4. ELONGATION: A type of DNA polymerase called taq polymerase binds to these sections and the temp. is raised to 70degrees (optimum for this enzyme) allowing it to add nucleotides to the strand
5. The whole process is repeated many times automatically so the amount of DNA increases exponentially.

Question 32

What is PCR used for and why?

Answer 32

Used in forensic investigations to replicate the DNA found at a crime scene for genetic fingerprinting as it makes multiple copies of DNA fragments.

Question 33

What are the advantages of using taq polymerase in PCR?

Answer 33

As a heat-stable DNA polymerase it is not destroyed by the denaturation step so does not have to be replaced each cycle.
Its high optimum temperature means that the temperature does not have to be dropped below that of the annealing process, so efficiency is maximised.

Question 34

explain how isolated DNA fragments can be placed in plasmids

Answer 34

If both plasmid and DNA are cut by the same restriction endonuclease, sticky ends will be complementary So H bonds will form between bases on sticky ends of DNA fragment and plasmid. DNA ligase enzyme restores the covalent bonds in the sugar-phosphate backbone. Recombinant DNA is formed.

Question 35

What are some commonly used vectors into which fragments of DNA may be incorporated?

Answer 35

• liposomes: tiny circles of phospholipid bilayer surround the required gene. The liposome fuses with a cell membrane and so carries the gene into a cell.
• bacterial plasmids: cut with same restriction enzyme as gene, produces complementary sticky ends
• modified viruses - viral transfer: insert their DNA into a cell
• Electroporation = high voltage pulse disrupts membrane increasing porosity
• heat shock/Ca2+ ion treatment to increase membrane permeability
• Microinjection = DNA injected using a micro-pipette
  Ti plasmid = circular DNA in bacteria that infects a plant’s genome

Question 36

how can recombinant plasmids containing a new gene then be made to express a desired protein?

Answer 36

bacterial cells take up plasmids to produce a transgenic microorganism that can express a desired gene product (e.g. insulin protein).

Question 37

What are the advantages to microorganisms of their capacity to take up plasmid DNA from the environment? (transformation)

Answer 37

• exchange of genetic material
• use in natural selection and evolution (selective advantage in some conditions)
• passing on of genes for antibiotic resistance

Question 38

outline how genetic markers in plasmids can be used to identify the bacteria that have taken up a recombinant plasmid;
using the example of the E.coli bacteria and insulin gene

Answer 38

replica plating
Original plasmids have antibiotic resistance genes (used as a genetic marker) for the antibiotics ampicillin and tetracycline.
In the genetically engineered plasmids, the insulin is inserted in the middle of the tetracycline gene meaning no more resistance to tertracycline.
Bacteria is then grown on agar plates: first ampicillin agar, then tetracycline agar.
Both bacteria with the recombinant plasmid and those with the original plasmid will grow on ampicillin agar as the resistance gene is undamaged.
Only those which have the intact tetracyline-resistance gene - the non-recombinant colonies - will be able to grow on the tetracycline agar, allowing identification of the recombinant colonies from the previous plate.

Question 39

outline the process involved in the genetic engineering of bacteria to produce human insulin

Answer 39

1. Preparation of insulin gene: mRNA for human insulin is extracted from pancreas cells. Reverse transcriptase uses it as a template to make single-stranded cDNA that is made double stranded by DNA polymerase. Cut by restriction endonuclease = sticky ends/
2. Preparation of vector: plasmid is cut open with same restriction endonuclease as above to produce complementary sticky ends. Mixed together with insulin gene so sticky ends form base pairs. DNA ligase links sugar-phosphate backbones.
3. Formation of genetically engineered bacteria: plasmids are mixed with bacteria in the presence of calcium ions for increased porosity. Bacteria take up plasmids and multiply to form clones. Genetically engineered bacteria are able to transcribe and translate the human gene to produce human insulin.

Question 40

outline the process involved in the genetic engineering of ‘Golden RiceTM’

Answer 40

1. 2 genes are isolated using an restriction enyme, one gene from a daffodil and the other from soil which together produce beta-carotene.
2. A Ti-plasmid is removed from the ‘Agrobacterium tumefaciens’ bacterium using the same restriction enzyme.
3. The 2 genes as well as a marker gene are inserted into the plasmid and DNA ligase links the sugar-phophate backbone of the complementary sticky ends.
4. The recombinant plasmid is reinserted into the bacterium.
5. Transformed bacteria are incubated with normal rice cells, which they infect, inserting their DNA and creating transformed plant cells.
6. All cells are transferred to a selective medium on which only cells containing the marker gene can grow, so only plants producing golden rice grow.

Question 41

Why was Golden Rice developed?

Answer 41

To control Vitamin A deficiency (which can cause blindness) in parts of the world where rice is a staple food.

Question 42

What are some concerns people have about Golden Rice?

Answer 42

• reduce rice genetic diversity
• clone may suffer from disease / environmental change ;
• hybridisation with wild rice / spread genes to wild populations ;
• seeds expensive. need to be bought each year ;
• rice may not grow in all areas where needed ;
• idea of doubts whether vitamin A content sufficient ;

Question 43

outline how animals can be genetically engineered for xenotransplantation

Answer 43

Pigs can be genetically engineered to reduce the immune response in the recipient human - human genes coding for a cell surface protein can be transferred to pig genes, resulting in the production of pig organs lacking particular glycoproteins that human antibodies bind to and recognise as foreign in the immune response, reducing the problem of organ rejection.

Question 44

What is gene therapy?

Answer 44

the treatment of a genetic disorder by altering an individual’s genotype. When the disorder is a recessive condition (e.g. cystic fibrosis) the therapy aims to add the normal, dominant allele of the defective gene to the genotype.
The therapy can by somatic cell or germ line cell.

Question 45

What are the differences between somatic cell and germ line cell therapy?

Answer 45

Somatic: body tissue cell used, allele is added in target cells only, added allele is not passed to offspring.
Germ line: egg, sperm or fertilised egg cells used, added allele in every cell of the body, added allele passes to offspring via gamete

Question 46

Outline the ethical concerns raised by the genetic manipulation of microbes

Answer 46

• microbes could escape into the environment and pass genes to pathogenic bacteria, with unknown effects
• genetic engineering using plasmids with genetic markers of antibiotic resistance could be passed to other bacteria, leading to more antibiotic resistance

Question 47

Outline the ethical pros and cons raised by the genetic manipulation of plants

Answer 47

Pros:
- golden rice could solve problem of vitamin A deficiency
- Genetically engineered plants that are pest resistant reduce the amount of pesticides needed, decreasing pollution and increasing yields
Cons
- Genes put into crop plants could pass to wild relatives by wind/insect pollination
- genes for pesiticide resistance could pass to weeds, making them resistant and hard to control
- reduction of genetic diversity of crops = potential difficulties in future due to smaller gene pool
- GM plants could be toxic to other organisms

Question 48

Outline the ethical pros and cons raised by the genetic manipulation of animals

Answer 48

Pros:
- GM animals can make useful products in their milk - made to express proteins/medicines
- animals can be genetically engineered for increased yields
- gm animals made for xenotransplantation ro humans
- can be modified to require fewer ressources and produce less waste, to impact the environment less
Cons:
- GM animals may suffer adverse lifestyles
- use of some animals is against some religions

Question 49

Outline the ethical pros and cons raised by the genetic manipulation of humans

Answer 49

Pros:
- gene therapy can treat some genetic disorders
Cons:
- germ line cell gene therapy results are unpredictable, and all offspring are affected (currently banned in the UK)
- germ line cell gene therapy could be used to enhance favourable characteristics - ‘designer babies’ - in eugenics

Question 50

What are some ethical concerns relating to the use of somatic gene therapy?

Answer 50

• virus vector may cause viral disease
• procedure may be, invasive / dangerous
• temporary - needs to be repeated
• immune system / rejection problems
• animal testing concerns

Question 51

compare the advantages and disadvantages of PCR (in vitro) vs. in vivo cloning

Answer 51

PCR:
- quicker
- uses less equipment, takes up less space
- less labour intensive, easier to perform
- safer - uses only DNA, cells aren’t contaminated
- can use lower quality DNA, amplifying old/prehistoric/forensic samples.
IN VIVO:
- less expensive
- less prone to mutation
- less technically complex - conditions less critical
- can replicate whole chromosomes/genome as opposed to just short sections