Chapter 22: Cloning and Biotechnology

Question 1

What is the outcome of natural cloning?

Answer 1

• A new structure forms which differentiates into a fully developed plant that is genetically identical from the parent.

Question 2

Function of perennating organs?

Answer 2

• Enable plant to survive adverse conditions.
• Store food from photosynthesis.
• Remain dormant in soil.
• Way of surviving one growing season to the next.
• Means of asexual reproduction.

Question 3

State what is meant by biotechnology and using suitable examples from different areas of biotechnology and explain why microorganisms are used in biotechnological processes (8 marks)

Answer 3

• Biotechnology = involves use of living organisms/enzymes to improve agriculture, animal husbandry, medicine and industry.
• For instance in bread making yeast added to sugar + water to respire anaerobically –> CO2 produced makes bread rise:
• In brewing –> yeast respires anaerobically to produce ethanol.
• In cheese making –> bacteria feed on lactose milk changing texture and taste + inhibiting growth of bacteria that can make milk go off.
• In yoghurt making –> bacteria used to produce extracellular polymers that give yoghurt its smooth thick texture –> e.g. Lactobacillus bulgaricus (forms ethanal) and Streptococcus thermophilus (forms lactic acid).
• Microorganisms used because:
• they reproduce fast + produce proteins faster than animals + plants.
• have high protein content with little fat.
• have no welfare issues.
• can be genetically modified to produce required protein.
• can use a variety of waste materials including human and animal waste, reducing costs.
• production not dependent on weather or breeding costs –> can be maintained all year round.
• grown in low temp conditions as long as O2 + food supplied and waste removed.
• enormous range available.

Question 4

State the advantages of propagation from cuttings over seeds.

Answer 4

• Faster.
• Better quality of plants.
• Genetically identical offspring so they crop better.

Question 5

State the disadvantages of propagation from cuttings over seeds.

Answer 5

• Lack of genetic variation in offspring.
• Reduces gene pool –> no. of alleles in gene pool.
• Less likely to adapt to changing climate, pest pop. and new diseases –> entire species might get wiped out.

Question 6

State how to increase success rate of most cuttings.

Answer 6

• Use non-flowering system.
• Make oblique cuts in stem.
• Use hormone rooting powder.
• Reduce leaves to two or four.
• Keep cuttings well watered.
• Cover cuttings with a plastic bag for few days.

Question 7

Define micropropagation.

Answer 7

• Process of making a large no. of genetically identical offspring from a single parent plant using tissue culture techniques.

Question 8

When do you use micropropagation to produce plant?

Answer 8

Use when desired plant:

• Does not readily produce seeds.
• Does not respond well to cloning.
• Has been GM or selectively bred with difficulties.
• Is very rare.
• Is required to be pathogen free by growers.

Question 9

Outline the basic principles of micropropagation and tissue culture.

Answer 9

1. Take small sample of tissue (usually virus free) from the plant:

Meristem tissue from shoot tips and axial buds often dissected out in sterile conditions to avoid contamination by fungi + bacteria.

2. Sample sterilised –> immerse in sterilising agents –> e.g. bleach, ethanol, sodium dichloroisocyanurate:

• The latter does not need to be rinsed off –> tissue more likely to remain sterile.
• Explant –> material that is removed from plant.

3. Explant placed in sterile conditions containing balance of plant hormones (cytokinins + auxin) which stimulate mitosis:
   - Cells proliferate forming a callus –> bundle of identical cells.
4. Callus divided up or individual cells/clumps from callus transferred to a new culture medium containing different hormones + nutrients:
   - Stimulate development of tiny genetically identical cells.
5. Plantlets potted into compost where they grow into small plants.
6. Young plants –> planted out to grow + produce crops.

Question 10

What are the advantages of micropropagation?

Answer 10

• Culturing meristem will allow you to produce a large no. of disease free plants.
• Allows rapid production of a large no. of plants with known genetic makeup that will yield good crops.
• Makes it possible to produce a viable no. of plants after GM of plants.
• Provides way of growing plants that are naturally infertile or are difficult to grow from seeds (e.g. orchids).
• Provides way of producing large no. of seedless + therefore sterile plants that meet consumer demands –> e.g. bananas.
• Provides way of reliably increasing no. of rare/endangered plants.

Question 11

What are the disadvantages of micropropagation?

Answer 11

• Monoculture –> all plants genetically identical –> all plants susceptible to same diseases or changes in growing conditions.
• Expensive process + requires many skilled workers.
• Explants + plantlets –> vulnerable to risk of infection by moulds and other diseases during production process.
• If source material was infected –> all clones will be infected.
• Large no. of plants lost during process.

Question 12

Give some examples of cloning in invertebrates.

Answer 12

• Starfish = regenerate entire animal from fragments if the original is damaged.
• Hydra = produce small buds on side of body that develop into genetically identical clones.
• Sponges + flatworms = fragment + form new identical animals as a part of their reproductive process.
• Some insects = females produce offspring without mating.

Question 13

Give some examples of cloning in vertebrates.

Answer 13

• Formation of monozygotic twins (identical twins) –> may look different due to position + nutrition in uterus
• Early embryo splits to form two separate embryos.
• Female amphibians + reptiles –> produce offspring when no males available –> often male offspring that are not clones of their mothers.

Question 14

What are the stages of artificial cloning in vertebrates?

Answer 14

1. Cow with desirable traits treated with hormones so she super-ovulates releasing more mature ova than normal.
2. The ova may be fertilised naturally or by artificial insemination, by a bull with particularly good traits:
   a. Early embryos gently flushed out of uterus.
3. Alternatively –> mature eggs removed + fertilised by top quality bull in a lab.
4. Before/around 6 days –> when cells still totipotent –> cells of early embryo split to produce several smaller embryos each capable of growing on to form healthy full-term calf.
5. Each of the split embryos grown in lab for few days before implanted into surrogate mother:
   a. Each embryo implanted into different mother as single pregnancies carry fewer risks than twin pregnancies.
6. Embryos develop into foetuses + are born naturally so a no. of identical cloned animals produced by different mothers.

Question 15

Define somatic cell nuclear transfer.

Answer 15

• The process of taking a nucleus from an adult somatic cell and transferring it to an enucleated egg cell.

Question 16

Outline the steps of somatic cell nuclear transfer.

Answer 16

1. Nucleus removed from somatic cell of adult animal.
2. Nucleus removed from mature ovum harvested in different female animal of the same species (it is enucleated).
3. Nucleus from adult somatic cell placed into enucleated ovum + given mild electric shock so it fuses + begins to divide:
   a. In some cases nucleus from adult cell not removed –> placed next to enucleated ovum + the two cells fuse (electrofusion) + begin to divide under influence of electric current.
4. Embryo that develops is transferred into the uterus of a third animal, where it develops to term.
5. The new animal is a clone of the animal from which the original somatic cell is derived, although the mitochondrial DNA will come from the egg cell.

Question 17

What are the pros of animal cloning?

Answer 17

• Artificial twinning enables increased offspring production compared to normal reproduction.
• Artificial twinning enables success of sire at passing on desirable genes to be determined –> if successful more identical animals reared from frozen clones.
• SCNT allows GM embryos to be replicated + develop producing many embryo from one procedure.
• SCNT allows cloning of specific animals.
• SCNT can potentially reproduce rare/endangered/extinct animals.

Question 18

What are the cons of animal cloning?

Answer 18

• SCNT is very inefficient –> can take many eggs to produce a single cloned offspring.
• Many cloned offspring fail to develop and miscarry or produce malformed offspring.
• Many animals produced by cloning have shortened lifespans.

Question 19

Why are microorganisms used in production of human food?

Answer 19

• they reproduce fast + produce proteins faster than animals + plants.
• have high protein content with little fat.
• have no welfare issues.
• can be genetically modified to produce required protein.
• can use a variety of waste materials including human and animal waste, reducing costs.
• production not dependent on weather or breeding costs –> can be maintained all year round.
• grown in low temp conditions as long as O2 + food supplied and waste removed.
• enormous range available.
• can be made to taste like anything

Question 20

Define bioremediation.

Answer 20

• The use of microorganisms to break down pollutants + contaminants in soil/water.

Question 21

State some disadvantages of using human microorganisms to produce human food.

Answer 21

• Some can produce toxins if conditions are not maintained at optimum.
• Microorganisms have to be separated from nutrient broth and processed to make food.
• Need sterile conditions that are carefully controlled adding to costs.
• Often involve GM organisms and people have ethical concerns about eating GM food.
• Protein has to be purified to ensure it contains no toxin or contaminants.
• Many people dislike the thought of eating microorganisms grown on waste.
• Has little natural flavour –> needs additives.

Question 22

Outline the process of brewing.

Answer 22

1. Malting = barley germinates producing enzymes that break down starch molecules to sugars which yeast can use –> seeds then killed by slow heating but enzyme activity retained to produce malt.
2. Mashing = malt mixed with hot water + enzymes break down starches to produce wort:

• Hops added for flavour + antiseptic qualities.
• Wort sterilised and cooled.

3. Fermentation = wort inoculated with yeast + temp maintained for optimum anaerobic respiration:
   - Eventually yeast inhibited by falling pH, build up of ethanol + lack of O2.
4. Maturation = beer conditioned for 4-26 days in tanks.
5. Finishing = beer filtered, pasteurised, then bottled or caned with addition of CO2.

Question 23

Outline process of baking.

Answer 23

1. Active yeast mixture added to flour + other ingredients -> mixed + left in warm environment to rise.
2. Dough knocked back (excess air removed), kneaded, shaped and left to rise again.
3. Cooked in hot oven –> CO2 bubbles expand so bread rises more + yeast cells killed during cooking.

Question 24

Outline process of cheese making.

Answer 24

1. Milk pasteurised (heated to 95 degrees for 20 seconds to kill off natural bacteria) + homogenised (fat droplets evenly distributed through milk).
2. Mixed with bacterial cultures + sometimes chymosin enzyme + kept until milk separates into solid curds + liquid whey.
3. For cottage cheese, curds separated from whey, packaged and sold.
4. For most cheese –> curds cut + cooked in whey then strained through draining moulds or cheese cloth –> whey used for animal feed.
5. Curds put into steel or wooden drums + pressed:
   - Left to dry, mature + ripen before eating as bacteria continue to act for weeks-years.

Question 25

Outline process of yoghurt-making.

Answer 25

1. Skimmed milk powder added to milk + mixture is pasteurised. 2. Milk mixed with 1:1 ratio of Lactobacillus bulgaricus and Streptococcus thermophilus + incubated at around 45 degrees for 4-5 hours. 3. At end of fermentation yoghurt put into cartons at temp of 10 degrees as plain yoghurt or mixed with previously sterilised fruit. 4. Thick-set yoghurts mixed + ferment in pot.

Question 26

Why are some people allergic to non-human insulin?

Answer 26

- Traces of impurities in animal insulin. - Its different protein structure may mean it's recognised as non-self. - Lymphocytes with antigen binding sites complementary to insulin may be activated + immune response initiated.

Question 27

Outline stages of penicillin growth + conditions needed.

Answer 27

- Needs high O2 levels + rich nutrient medium to grow. - pH + temp sensitive. - Process uses small fermentors --> difficult to maintain high level of oxygen saturation in large bioreactors. - Mixture continuously mixed to keep it oxygenated. - Growth medium contains buffer to maintain pH at around 6.5. 1. First stage = fungus grows. 2. Second stage = produces penicillin. 3. Third stage = drug extracted from medium + purified.

Question 28

How to culture microorganisms using an inoculating broth?

Answer 28

1. Make suspension of bacteria to be grown. 2. Mix known volume with sterile nutrient broth in flask. 3. Stopper the flask with cotton wool to prevent contamination from air. 4. Incubate at suitable temp, shaking regularly to aerate broth providing O2 for growing bacteria.

Question 29

How to culture microorganisms using inoculating agar?

Answer 29

1. Wire inoculating loop --> sterilised --> hold in Bunsen burner flame until it grows red hot: - Not allowed to touch any surfaces until it cools to avoid contamination. 2. Dip sterilised loop in bacterial suspension: - Remove lid of petri dish and make zig-zag streak across surface of agar. - Avoid loop digging into agar by holding it almost horizontal. - Many streaks applied --> surface of agar must be kept intact. 3. Replace lid of petri dish: - Should be held down with tape but not sealed completely so O2 can get in, preventing growth of anaerobic bacteria. - Incubate at suitable temp.

Question 30

What are the four stages of a exponential growth curve for bacterial pop?

Answer 30

1. Lag phase = bacteria adapting to new environment: - Growing, synthesising enzymes they need + are not yet reproducing at theoretical maximum. 2. Log/Exponential phase = rate of bacterial reproduction close to or at theoretical maximum --> natality rate > mortality rate. 3. Stationary phase = total growth rate is 0 --> mortality rate = natality rate. 4. Decline/Death phase = reproduction has almost ceased + mortality rate > natality rate + death rate keeps increasing.

Question 31

What are the limiting factors that prevent exponential growth of bacteria culture?

Answer 31

- Nutrients available, - O2 levels. - Temperature. - Waste build-up --> toxic material inhibit further growth. - Change in pH.

Question 32

Define primary metabolites and give examples.

Answer 32

Primary Metabolites = substances are wanted which are formed as an essential part of the normal functioning of a microorganism: * Yeast --> product of anaerobic respiration in yeast. * Ethanoic acid. * Range of amino acids + enzymes.

Question 33

Define secondary metabolites and give examples.

Answer 33

Secondary Metabolites = organisms produce substances which are not essential for normal growth, but are still used by cells: * Many pigments. * Toxic chemicals plants produce to protect against herbivore attacks. * Organism would not suffer in short term without them.

Question 34

Outline steps of batch fermentation.

Answer 34

1. Microorganisms inoculated into fixed volume of medium. 2. As growth takes place, nutrients used up + both new biomass + waste products build up. 3. As culture reaches stationary phase: a. Overall growth ceases. b. Microorganisms carry out biochemical changes to form desired end product. 4. Process stopped before death phase + products harvested. 5. Whole system cleaned + sterilised and new batch culture started up.

Question 35

Outline steps of continuous culture.

Answer 35

1. Microorganisms inoculated into sterile nutrient medium and start to grow. 2. Sterile nutrient medium added continually to culture once it reaches the exponential point of growth. 3. Culture broth continually removed  medium, waste products, microorganisms, and product  keeping vol. in bioreactor constant.

Question 36

How is temp used to control bioreactors?

Answer 36

* Temp too low --> microorganisms will not grow quickly enough. * Too high --> enzymes denature + microorganisms inhibited/destroyed. * Bioreactors have heating/cooling system linked to temp sensors + negative feedback system to maintain optimum conditions.

Question 37

How are nutrients and O2 used to control bioreactors?

Answer 37

• O2 + nutrient medium added in controlled amounts to broth when probes or sample tests indicate levels are dropping.

Question 38

How does the mixing mechanism control bioreactors?

Answer 38

* Large vol of liquid in bioreactor  thick + viscous due to microorganism growth. * Simple diffusion not enough to ensure all microorganisms receive enough food + O2 or that whole mixture is kept at right temp: o Most bioreactors have mixing mechanism + many are stirred continuously

Question 39

How does asepsis control bioreactors?

Answer 39

* If bioprocess contaminated by microorganisms from air or from workers yield is affected. * Bioreactors are sealed, aseptic units. * If process involves genetically engineered organisms --> they should be contained within bioreactor and not released into environment.

Question 40

State the advantages of using isolated enzymes.

Answer 40

- More specific. - Maximises efficiency. - Lowers downstream producing --> pure product produced. - More efficient. - Less wasteful.

Question 41

Advantages of using extracellular enzymes over intracellular enzymes as isolated enzymes?

Answer 41

- Cheaper. - Easy to identify + isolate required enzyme. - More robust --> adapted to greater variations in temp + pH.

Question 42

Advantages of using immobilised enzymes?

Answer 42

- Reusable --> cheap. - Less downstream processing --> easily separated from reactants + products of reaction. - More reliable --> insoluble support provides stable microenvironment for immobilised enzymes. - Greater temp tolerance --> less easily denatured by heat. - Ease of manipulation.

Question 43

Disadvantages of using immobilised enzymes?

Answer 43

- Reduced efficiency. - Higher initial costs of materials. - Higher cost of bioreactor. - More technical issues.

Question 44

State the advantages and disadvantages of surface immobilisation by adsorption to inorganic carriers.

Answer 44

Advantages: - Simple + cheap to do. - Can be used with many different processes. - Enzymes very accessible to substrate + their activity is virtually unchanged Disadvantages: - Enzymes can easily be lost from the matrix.

Question 45

State the advantages and disadvantages of surface immobilisation by covalent or ionic bonding to inorganic carriers.

Answer 45

Advantages: - Cost varies. - Enzymes bound strongly + so are difficult to be lost. - Enzymes are very accessible to substrate. - pH + substrate conc. often have v.low effect on enzyme activity. Disadvantages: - Cost varies. - Active site of enzyme may be modified in process making it less effective.

Question 46

State advantages + disadvantages of immobilising enzyme by entrapping it within a matrix

Answer 46

Advantages: - Widely applicable to different processes. Disadvantages: - May be expensive. - Can be difficult to entrap. - Diffusion of substrate to + from active site may be slow and hold up the reaction. - Effect of entrapment on enzyme activity is variable, depends of the matrix.

Question 47

State the advantages + disadvantages of immobilising enzyme by entrapping it in microcapsules or behind a semi-permeable membrane.

Answer 47

Advantages: - Simple to do. - Little effect on enzyme activity. - Widely applicable to many processes. Disadvantages: - Relatively expensive. - Diffusion of substrate to + from active site may be slow + hold up the reaction.

Question 48

Outline a few uses of immobilised enzymes.

Answer 48

- Immobilised penicillin acylase = production of semi-synthetic penicillin from natural penicillin. - Immobilised glucose isomerase = produce fructose from glucose. - Immobilised lactase = produce lactose free milk by hydrolysing lactose to glucose and galactose. - Immobilised aminoacylase = production of pure L-amino acids. - Immobilised glucoamylases = catalyses breakdown of dextrins to glucose in after amylase breaks down starch to dextrins. - Immobilised nitrile hydratase = enzyme involved in conversion of acrylonitrile to acrylamide in hydration reactions.