6.2.1 Cloning and Biotechnology

Question 1

What are clones?

Answer 1

• genetically identical copies of cells or whole organisms

Question 2

How are clones produced?

Answer 2

• by asexual reproduction
• nucleus is divided by mitosis
• these cells may not be physically or chemically identical after division, as they may differentiate to form two different types of cell

Question 3

What are the advantages of natural cloning ?

Answer 3

• if conditions for growth are good for the parent then they will be good for offspring
• cloning is relatively rapid
• population can increase quickly to take advantage of suitable environment
• reproduction can be carried out, even if there is only one parent

Question 4

What are the disadvantage of natural cloning?

Answer 4

• offspring become overcrowded
• no genetic diversity, except mutations in DNA replication
• little variation shown in population
• selection is not possible
• if environment changes to less advantageous. the whole population is susceptible

Question 5

What is vegetative propagation?

Answer 5

• a form of natural cloning in plants
• reproduction through vegetative parts of the plant, rather than through specialised reproductive structure

Question 6

What are runners or stolons?

Answer 6

• stems that grown on the surface of the ground

Question 7

What are rhizomes?

Answer 7

• stems that grow underground
• some are thickened over-wintering organs for new growth in spring

Question 8

What are suckers?

Answer 8

• new stems that grow from the roots of a plant

Question 9

What are bulbs?

Answer 9

• an over-wintering mechanism for many perennial monocotyledonous plants
• consists of an underground stem which grows fleshy leaf bases
• there is also an apical bud, which will grow into a new plant in the spring
• often there is more than one apical bud

Question 10

What are corms?

Answer 10

• corms are solid rather tahn fleshy like a bulb
• it is an underground stem with scaly leaves and buds
• corms remain underground over winter
• in spring the buds grown to produce one or more new plants

Question 11

How do leaves reproduce asexually?

Answer 11

• some plants, such as Kalanchoe, have clones grown on the leaf margins
• immature plants drop off leaf and root

Question 12

What are tubers?

Answer 12

• another type of underground stem

Question 13

Give examples of natural cloning in animals

Answer 13

• identical twins in mammals:
• zygote divides as normal, but two daughter cells then split to become two separate individuals
• each cell grows and develops into a new individual
• water flea and greenfly reproduce asexually to produce clones

Question 14

What is a common way to create clones in plants?

Answer 14

• make cuttings
• cut between a node and place in moist soil
• some plants may need rooting hormone to help stimulate root growth
• it may be helpful to remove bark from the cut end of the stem to produce a callus
• can be used to produce large numbers of plants very quickly

Question 15

What other parts of a plant can cuttings be made successfully from, apart from stem?

Answer 15

• root cuttings: a section of the root is buried just below soil surface and the new shoots
• scion cuttings: these are dormant woody twigs
• leaf cuttings: leaf is placed in moist soil

Question 16

What is tissue culture and what is it used for?

Answer 16

• a series of techniques used to grow cells, tissues or organs from a small sample of cells or tissue
• carried out on a nutrient medium under sterile conditions
• application of plant growth substances at the correct time encourage correct differentiation
• cuttings need a lot of space and time and some don’t respond well to them
• it is widely used commercially to increase number of new plants, in micropropagation

Question 17

What is micropropagation?

Answer 17

• taking a small piece of plant tissue (the explant) and using plant growth substances to encourage growth and develop into a whole new plant

Question 18

Describe the steps of micropropagation

Answer 18

1. suitable plant material is selected and cut into small pieces (explants)
   - they can be tiny pieces of leaf, root or bud
   - meristem tissue is often used as they are free of infection
2. explants are sterilised using dilute bleach or alcohol
   - this kills any bacteria and fungi, as these thrive in the conditions that help the plant grow
3. explants are placed on a sterile growth medium containing glucose, amino acids and phosphates
   - usually contains high concentrations of auxin and cytokinin
   - this stimulates the cells of each explant to divide by mitosis to form a callus (a mass of totipotent cells)
4. after a callus is formed, it is divided to produce a larger number of small clumps of undifferentiated cells
5. they are then stimulated to grow, divide and differentiate into different plant tissues by moving them into different growth media
   - each medium contains different ratios of auxin and cytokinin
   - e.g. first ratio is 100 auxin : 1 cytokinin, stimulating formation of roots
   - second is 4:1
6. after tiny plantlets are formed, these are transferred to a greenhouse to be grown in compost

Question 19

What are the advantages of artificial cloning?

Answer 19

• a relatively rapid method of producing new plants compared with growing plants from seed
• it can be carried out where sexual reproduction is not possible
• e.g. plants that are unable to breed sexually e.g. bananas
• plants that are hard to be grown from seed
• plants selected will all be genetically identical to parents:
• they will display the same desirable characteristics
• e.g. high yield, resistance to a common pest etc
• if the original plants had an unusual combination of characteristics that wants to be kept the same
• new plants are all uniform in their phenotype, making them easy to grow and harvest
• using the apical bud (meristem) as an explant for tissue culture ensures the new plants are free from viruses

Question 20

What are the disadvantages of artificial cloning?

Answer 20

• labour intensive
• expensive to set up facilities
• can fail due to microbial contamination
• genetically identical offspring means they are susceptible to the same pests/diseases
• monoculture allows rapid spread of a disease or pest
• no genetic variation, except that introduced by mutation

Question 21

Why may reproductive cloning be useful in animals?

Answer 21

• elite-farm animals produced by selective breeding or genetic modification
• genetically-modified animals developed with unusual characteristics

Question 22

What are the two main techniques to achieve reproductive cloning?

Answer 22

• embryo twinning
• somatic cell nuclear transfer (SCNT)

Question 23

What is embryo splitting?

Answer 23

• mammals can produce identical offspring if an embryo splits very early in development
• this has given rise to an artificial technique used since the 1970s
• it has been used to clone elite farm animals or animals for research
• however, the precise genotype and phenotype depends on the sperm and egg so it is unknown until the animals are born

Question 24

Describe the process of embryo twinning

Answer 24

1. a zygote is created by IVF
2. zygote is allowed to divide by mitosis to form a small ball of cells
3. the cells are separated and allowed to continue dividing
4. each small mass of cells is placed into the uterus of a surrogate mother

Question 25

What is somatic cell nuclear transfer (SCNT)?

Answer 25

• the only way to clone an adult
• advantage is that the phenotype is known before cloning starts
• it was first performed successfully on Dolly the sheep in 1996

Question 26

Describe the process of SCNT

Answer 26

1. an egg cell is obtained and its nucleus removed, known as enucleation
2. a somatic cell from the adult to be cloned is isolated and may have the nucleus removed
3. the complete adult somatic cell or its nucleus is fused with the empty egg cell by applying an electric shock
4. the shock also triggers the egg cell to start developing, as if they had just been fertilised
5. the cell undergoes mitosis to produce a small ball of cells
6. the embryo is placed into the uterus of a surrogate mother

Question 27

What is non-reproductive cloning and give some examples

Answer 27

• the production of cloned cells and tissues for purposes other than reproduction
• therapeutic cloning
• cloning for scientific research

Question 28

Describe therapeutic cloning

Answer 28

• new tissues and organs can be grown as replacement parts for people who are not well:
• skin can be grown in vitro to act as a graft over burned areas
• cloned cells have been used to repair damage to the spinal cord of a mouse and to restore the capability to produce insulin in the pancreas
• there is the potential to grow whole new organs to replace diseased organs
• tissues grown from the patient’s cells will be genetically identical and so avoid rejection

Question 29

Explain cloning for scientific research

Answer 29

• it can be used for research into the action of genes that control development and differentiation
• they can be used to grow specific tissues or organs for use in tests of the effects of medicinal drugs

Question 30

What are the arguments for and against artificial cloning?

Answer 30

Question 31

What are the four main areas in which microorganisms are used in biotechnology? What organisms are used?

Answer 31

Question 32

What are the advantages of using microorganisms in biotechnology?

Answer 32

• relatively cheap and easy to grow
• in most cases, the process takes place at a lower temperature than would be required to make it chemically
• saves fuel and reduces costs
• the production process can occur and normal atmospheric pressure, which is safer than chemical reactions with higher pressures
• not dependent on climate
• can be fed by-products from other food industries
• microorganisms have a short life cycle and reproduce quickly so a large population can grow very quickly inside the reaction vessel (fermenter)
• microorganisms can be GMed easily, allowing specific production process to be achieved
• fewer ethical considerations to worry about
• products are often released from the microorganism into the surrounding medium, easy to harvest
• product is often more pure or easier to isolate than in chemical engineering

Question 33

What other organisms apart from microoorganisms can be used in biotechnology?

Answer 33

• genetically-modified mammals
• e.g. sheep, goats, cows to produce useful proteins
• in some mammals, proteins are incorporated into milk and can be easily harvested
• e.g. goats that are GMed to secrete spider silk in milk
• e.g. some protein is secreted into the blood

Question 34

What are some other forms of biotechnology?

Answer 34

• gene technology
• genetic modification and gene therapy
• selective breeding
• cloning by embryo splitting and micropropagation
• use of enzymes in industrial processes
• immunology

Question 35

What is yoghurt and how is it made?

Answer 35

• milk that has undergone fermentation by Lactobacillus bulgaricus and Streptococcus thermophilus
• this converts lactose to lactic acid
• the acidity denatures the milk protein, causing it to coagulate
• it is easy to digest because the bacteria partially digest the milk
• other bacteria, such as L. acidophilus, L. subsp. Casey and Bifidobacterium may be added as probiotics

Question 36

How is cheese made?

Answer 36

• milk is usually pre-treatteed to Lactobacillus that can produce lactic acid from lactse
• once it is acidified, the milk is mixed with rennet
• rennet contains the enzymes rennin (chymosin)
• it coagulates the milk protein (casein) in the presence of calcium ions
• Kappa-casein: keeps the casein in solution, is broken down, making casein insoluble
• the casein is precipitated by the action of calcium ions, which bind the molecules together
• the resulting solid is separated from the whey
• bacteria continue to grow, producing more lactic acid
• it is then moulded and ripening, maturing etc occurs

Question 37

How is bread made?

Answer 37

• mixture of flour, water and salt with some yeast Saccharomyces cerevisiae
  1. Mixing
  2. Proving: yeast expires anaeobically, produces CO₂
  3. Cooking: alcohol evaporates during cooking process

Question 38

How are alcoholic beverages made?

Answer 38

• also the product of the anaerobic respiration of yeast (S. cerevisiae)
• wine is made using grapes because it naturally has yeast on the skin and it contains fructose and glucose
• ale or beer is brewed using barley grains to germinate, called malting
• as the grain germinates, it converts stored starch to maltose, which is respired by the yeast

Question 39

How is single-cell protein (SCP) used in food?

Answer 39

• the fungus Fusarium venenatum is most frequently used
• this fungal protein or mycoprotein is also called a single-celled protein (SCP)
• e.g. Quorn
• there is also a huge potential in SCP production using microorganisms such as Kluyveromyces, Scytalidium, Candida
• these can produce protein with similar amino acid profile to animal and plant protein
• and they can grow on almost any organic substrate, including waste material such as paper and whey

Question 40

What are thadvantages of using microorganisms in food?

Answer 40

• production of protein can be many times faster than that of animal or plant protein
• biomass produces has very high protein content (45-85%)
• production can be increased and decreased according to demand
• there are no animal welfare issues
• microorganisms provide a good source of protein
• protein contains no animal fat or cholesterol
• microorganisms can be easily GMed to adjust amino acid content of the protein
• SCP production could be combined with the removal of waste products
• production is independent of seasonal variations
• not much land is required

Question 41

What are the disadvantages of using microorganisms in food?

Answer 41

• some may not want to eat fungal protein or food that has been grown on waste
• isolation of protein: they need to be separated from the material on which they grow
• protein has to be purified to ensure it is uncontaminated
• microbial biomass can have a high proportion of nucleic acids, which must be removed
• amino acid profile may be different from traditional animal protein, especially if deficient in methionine
• infection: conditions are ideal for pathogen growth; care must be taken
• palatability: protein does not have taste or texture of traditional protein sources

Question 42

How can the production of drugs be scaled up in commercial production?

Answer 42

• it uses large stainless steel containers called fermenters

Question 43

What conditions need to be controlled in fermenters?

Answer 43

• temperature: for enzymes
• nutrients available: sources of carbon, nitrogen, minerals and vitamins are needed for microorganisms
• oxygen availability: most microorganisms respire aerobically
• pH: affect enzyme activity and hence growth
• the concentration of product: if the product builds up, it may affect the synthesis process

Question 44

Describe the fermenter and label

Answer 44

• it is sterilised using superheated steam

Question 45

What are primary metabolites?

Answer 45

• some products are synthesised by the microorganism during normal metabolism when they are actively growing
• these products are continuously released from the cells and can be extracted from the fermenting broth
• it is topped up with nutrients as they are used by the microorganisms
• some of the broth is removed to extract the product and remove cells from the broth
• this stops the population from becoming too dense
• this is continuous culture
• it keeps the microorganism growing at a specific growth rate

Question 46

What are secondary metabolites?

Answer 46

• products produced only when cells are placed under stress, such as high population density or limited nutrient availability
• the culture is set up with a limited quantity of nutrients and allowed to ferment for a specific time
• after, the fermenter is emptied and the product can extracted from the culture
• this is known as batch culture

Question 47

Why is asepsis so important?

Answer 47

unwanted microorganisms:

• compete with the cultures microorganisms for nutrients and space
• reduce the yield of useful products
• spoil the product
• produce toxic chemicals
• destroy the cultures microorganism and their products

Question 48

Briefly explain the production of penicillin

Answer 48

• Penicillium chrysogenum is selectively bred to be more productive than early strains in modern times
• it is a secondary metabolite, so is only produced once the population has reached a certain size
• therefore, penicillin is manufactured by batch culture

Question 49

How is penicillin made?

Answer 49

• the fermenter is run for six to eight days
• the culture is then filtered to remove the cells
• the antibiotic is precipitated as crystals by the addition of potassium compounds
• it may then be modified by the action of other microorganism or chemicals
• the antibiotic is then mixed with inert substances and prepared for administration in tablet form, as a syrup or in a form for injection

Question 50

How is modern insulin made?

Answer 50

• synthetic human insulin was developed by genetically modifying a bacterium
• it was then combined with a plasmid to act as a vector, to be inserted into E.coli
• this produces vast quantities of human insulin at a relatively low cost
• insulin is manufactured by continuous culture

Question 51

What is bioremediation?

Answer 51

• the use of microorganisms to clean the soil and underground water on polluted sites
• the organisms convert the toxic pollutants to less harmful substances
• it involves stimulating the growth of suitable microbes that use the contaminants as a source of food

Question 52

What are the right conditions for the growth of microorganisms for bioremediation?

Answer 52

• available water
• a suitable temperature
• suitable pH
• if conditions are not quite suitable, additional substances may be added
• e.g. molasses, pumped oxygen

Question 53

What are the advantages and disadvantages of bioremediation?

Answer 53

Advantages:

• uses natural systems
• less labour/equipment is required
• treatment in situ
• few waste products
• less risk of exposure to clean-up personnel

Disadvantages:

• may only be suitable for certain products
• heavy metals such as calcium or lead cannot be treated

Question 54

What type of growth medium are microorganisms grown in?

Answer 54

• broth: a soup-like liquid in bottles or tubes
• agar: melted and poured into Petri dishes

Question 55

What is in agar?

Answer 55

• peptones (from the enzymatic breakdown of gelatine)
• yeast extract
• salts
• water
• may also contain glucose and blood

Question 56

Describe aseptic techniques

Answer 56

Question 57

What are the three main steps of growing microorganisms on agar plates?

Answer 57

• sterilisation
• inoculation
• incubation

Question 58

Describe sterilisation

Answer 58

• agar medium and any equipment must be sterilised
• it is sterilised by heating in an autoclave at 121 degrees Celsius for 15 mins
• this kills all living organisms, including any bacterial or fungal spores
• when it has cooled, it is poured into sterile Petri dishes and left to set
• the lid must be kept on the Petri dish to prevent infection

Question 59

What is inoculation and describe it

Answer 59

• the introduction of microorganisms to the sterile medium
• it can be achieved in a number of ways:
• streaking: a wire inoculating loop is used to transfer a drop of liquid medium onto the surface of the agar
• the dfrop is drawn out into a streak by dragging the loop across the surface
• seeding: a sterile pipette can be used to transfer a small drop of liquid medium to the surface of the agar or to the Petri dish before the agar is poured in
• spreading: a sterile glass spreader may be used to spread the inoculated drop over the surface of the agar
• a small cotton swab or cotton bud can be moistened with distilled water and used to collect microorganisms from a surface and then carefully wiped over the surface of the agar medium

Question 60

Describe incubation of agar

Answer 60

• Petri dish must be labelled and tapped using two strips of tape
• do not seal Petri dish completely, to avoid selection of anaerobic bacteria that is more likely to be pathogenic
• place in a suitable environment such as an incubator
• place upside down to prevent drops of condensation falling onto the surface of the agar and to present medium from drying out too quickly
• examine cultures after 24-36 hours
• they must be sterilised after use and before disposal

Question 61

Why do you use a liquid medium?

Answer 61

• a liquid broth is initially clear but will turn cloudy when bacteria have grown
• it can be useful to increase numbers of microorganisms before transferring to agar plates for counting or identification
• aseptic techniques must be applied when using a broth
• a liquid broth can be used to investigate population growth

Question 62

Why may you need to do a serial dilution?

Answer 62

• the number of individual microorganisms in a broth can be high
• there may be too many colonies which merge together on an agar plate, making it impossible to count
• it is then essential to reduce the population density

Question 63

How do you perform a serial dilution?

Answer 63

• the broth is diluted by a factor of 10 at each step
• follow the picture
• a drop of each dilution can be used to inoculate an agar plate
• one of them will produce a culture plate in which the number of colonies can be counted

Question 64

What is a closed culture?

Answer 64

• a population in which all the conditions are set at the start and there is no exchange with the external environment
• the population growth will follow a predictable pattern
• a small population of microorganisms in a closed culture that contains all the nutrients required for growth will undergo population growth

Question 65

Describe the graph for the growth of a population of microorganisms in a closed culture

Answer 65

Question 66

Describe the lag phase for the growth of microorganisms in a closed culture

Answer 66

• in the early part of population growth, the population does not grow quickly
• partly because population is still small
• also because organisms are adjusting to their new environment
• which may involve:
• taking up water
• cell growth
• activating certain genes
• synthesising specific enzymes

Question 67

Describe the log phase for microorganism growth

Answer 67

• the organisms have adjusted to their environment
• they each have enzymes needed to survive
• each individual has sufficient nutrients and space to grow rapidly and reproduce
• population doubles with each generation
• some can double once every 20-30 mins

Question 68

Describe the stationary phase

Answer 68

• the increasing numbers of organisms use up nutrients and produce increasing amounts of waste products such as carbon dioxide and other metabolites
• rate of population growth declines and number of individuals dying increases until the reproduction rate equals the death rate
• there is no population growth

Question 69

Describe the death phase

Answer 69

• nutrients run out and the concentration of waste products may become lethal
• more individuals die than are produced and the population begins to fall
• eventually all organisms will die

Question 70

What are primary metabolites?

Answer 70

• primary metabolites produced during the normal activities of the microorganisms will be collected from a fermenter during lof phase
• in a fermenter, the population is not kept in a closed culture, but conditions are maintained for optimal growth

Question 71

What are secondary metabolites?

Answer 71

• they are produced in the stationary phase
• population must be kept in closed culture
• the metabolites can be collected at the end of the stationary phase or during the decline phase

Question 72

What are immobilised enzymes?

Answer 72

• an enzyme that is held in place and not free to diffuse through the solution
• they do not freely mix with the substrate

Question 73

What are the advantages of immobilised enzymes?

Answer 73

• enzymes do not mix with the product. so extraction costs are lower
• enzymes can be easily reused
• a continuous process is made easier, as there are no cells requiring nutrients, reproducing and releasing waste products
• enzymes are surrounded by the immobilising matrix, which protects them from extreme conditions
• so higher temperatures or wider pH range can be used without denaturing

Question 74

What are disadvantages of immobilised enzymes?

Answer 74

• they are more expensive to set up
• they can be less active than free enzymes, making the process slower

Question 75

What are the brief methods to immobilise enzymes?

Answer 75

• adsorption
• covalent bonding
• entrapment
• membrane separation

Question 76

Describe how adsorption allows enzymes to be immobilised

Answer 76

• enzyme molecules are bound to a supporting surface by a combination of hydrophobic interactions and ionic links
• suitable surfaces include clay, porous carbon, glass beads and resins
• the enzyme molecules are bound with the active site exposed and accessible to the substrate
• however, the active site may be slightly distorted by the additional interaction affecting enzyme activity
• the bonding forces are not always strong and enzymes can become detached and leak into reaction mixture

Question 77

Describe how covalent bonding allow enzymes to be immobilised

Answer 77

• enzyme molecules are bonded to a supporting surface such as clay using strong covalent bonds
• the enzymes are bonded using a cross-linking agent, which may also link them in a chain
• the production of covalent bonding can be expensive and can distort the enzyme active site, reducing activity
• however, enzymes are much less likely to become detached and leak into the reaction mixture

Question 78

How does entrapment allow enzymes to be immobilised?

Answer 78

• enzyme molecules are trapped in a matrix that does not allow free movement
• they are unaffected by entrapment and remain fully active
• however, the substrate molecules must diffuse into the entrapment matric and the product molecules must be able to diffuse out
• so, the method is only suitable for processes where the substrate and product molecules are relatively small
• calcium alginate beads are often used in schools to immobilise enzymes by entrapment
• industrial processes may use a cellulose mesh

Question 79

How does membrane separation allow enzymes to be immobilised?

Answer 79

• enzyme molecules are separated from the reaction mixture by a partially permeable membrane
• the substrate and product molecules must be small enough to pass through the partially permeable membrane by diffusion
• this access to the enzymes may limit the reaction rate

Question 80

How is the immobilised enzyme glucose isomerase used?

Answer 80

• converts glucose to fructose
• very commonly used
• used to produce high fructose corn syrup
• much sweeter than sucrose
• used in diet foods and for diabetics
• cheaper so also replaces sucrose

Question 81

How is the immobilised enzyme penicillin acylase used?

Answer 81

• formation of semi-synthetic penicillins, such as amoxicillin and ampicillin
• some penicillin-resistance microorganisms are not resistant to these

Question 82

How is the immobilised enzyme lactase used?

Answer 82

• converts lactose to glucose and galactose by hydrolysis
• used to produce lactose-free milk
• milk is an important source of calcium etc

Question 83

How is the immobilised enzyme aminoacylase used?

Answer 83

• a hydrolase used to produce pure samples of L-amino acids by removing the acyl group from the nitrogen of an N-acyl-amino acid
• L-amino acids are used as building blocks for synthesis of a number of pharmaceutical and agrochemical compounds
• they may be used as additive for human food and animals feed

Question 84

How is the immobilised enzyme glucoamylase used?

Answer 84

• converts dextrins to glucose
• it can be immobilised on a variety of surfaces and used to digest sources of starch such as corn and cassava
• the enzyme is used in a wide range of fermentation processes
• it converts starch pulp to alcohol used to produce gasohol, an alternative fuel for motor vehicles
• also used to make high fructose corn syrup

Question 85

How is the immobilised enzyme nitrile hydratase used?

Answer 85

• converts nitriles to amides, including acrylonitrile to acrylamide
• acrylamide can then be polymerised to form polyacrylamide, which is a plastic used as a thickener
• polyacrylamide can be used in the treatment of water
• it sticks many small contaminants together, so they are precipitated and filtered
• it is also used in paper-making and to make gel for electrophoresis