Chapter 22: Cloning and Biotechnology Flashcards
1
Q
What is the outcome of natural cloning?
A
- A new structure forms which differentiates into a fully developed plant that is genetically identical from the parent.
2
Q
Function of perennating organs?
A
- 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.
3
Q
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)
A
- 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.
4
Q
State the advantages of propagation from cuttings over seeds.
A
- Faster.
- Better quality of plants.
- Genetically identical offspring so they crop better.
5
Q
State the disadvantages of propagation from cuttings over seeds.
A
- 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.
6
Q
State how to increase success rate of most cuttings.
A
- 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.
7
Q
Define micropropagation.
A
- Process of making a large no. of genetically identical offspring from a single parent plant using tissue culture techniques.
8
Q
When do you use micropropagation to produce plant?
A
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.
9
Q
Outline the basic principles of micropropagation and tissue culture.
A
- 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.
- 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.
- Explant placed in sterile conditions containing balance of plant hormones (cytokinins + auxin) which stimulate mitosis:
- Cells proliferate forming a callus –> bundle of identical cells. - 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. - Plantlets potted into compost where they grow into small plants.
- Young plants –> planted out to grow + produce crops.
10
Q
What are the advantages of micropropagation?
A
- 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.
11
Q
What are the disadvantages of micropropagation?
A
- 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.
12
Q
Give some examples of cloning in invertebrates.
A
- 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.
13
Q
Give some examples of cloning in vertebrates.
A
- 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.
14
Q
What are the stages of artificial cloning in vertebrates?
A
- Cow with desirable traits treated with hormones so she super-ovulates releasing more mature ova than normal.
- 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. - Alternatively –> mature eggs removed + fertilised by top quality bull in a lab.
- 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.
- 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. - Embryos develop into foetuses + are born naturally so a no. of identical cloned animals produced by different mothers.
15
Q
Define somatic cell nuclear transfer.
A
- The process of taking a nucleus from an adult somatic cell and transferring it to an enucleated egg cell.
16
Q
Outline the steps of somatic cell nuclear transfer.
A
- Nucleus removed from somatic cell of adult animal.
- Nucleus removed from mature ovum harvested in different female animal of the same species (it is enucleated).
- 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. - Embryo that develops is transferred into the uterus of a third animal, where it develops to term.
- 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.
17
Q
What are the pros of animal cloning?
A
- 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.
18
Q
What are the cons of animal cloning?
A
- 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.
19
Q
Why are microorganisms used in production of human food?
A
- 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
20
Q
Define bioremediation.
A
- The use of microorganisms to break down pollutants + contaminants in soil/water.
21
Q
State some disadvantages of using human microorganisms to produce human food.
A
- 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.
22
Q
Outline the process of brewing.
A
- 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.
- Mashing = malt mixed with hot water + enzymes break down starches to produce wort:
- Hops added for flavour + antiseptic qualities.
- Wort sterilised and cooled.
- Fermentation = wort inoculated with yeast + temp maintained for optimum anaerobic respiration:
- Eventually yeast inhibited by falling pH, build up of ethanol + lack of O2. - Maturation = beer conditioned for 4-26 days in tanks.
- Finishing = beer filtered, pasteurised, then bottled or caned with addition of CO2.
23
Q
Outline process of baking.
A
- Active yeast mixture added to flour + other ingredients -> mixed + left in warm environment to rise.
- Dough knocked back (excess air removed), kneaded, shaped and left to rise again.
- Cooked in hot oven –> CO2 bubbles expand so bread rises more + yeast cells killed during cooking.
24
Q
Outline process of cheese making.
A
- Milk pasteurised (heated to 95 degrees for 20 seconds to kill off natural bacteria) + homogenised (fat droplets evenly distributed through milk).
- Mixed with bacterial cultures + sometimes chymosin enzyme + kept until milk separates into solid curds + liquid whey.
- For cottage cheese, curds separated from whey, packaged and sold.
- For most cheese –> curds cut + cooked in whey then strained through draining moulds or cheese cloth –> whey used for animal feed.
- Curds put into steel or wooden drums + pressed:
- Left to dry, mature + ripen before eating as bacteria continue to act for weeks-years.
25
Q
Outline process of yoghurt-making.
A
- Skimmed milk powder added to milk + mixture is pasteurised.
- Milk mixed with 1:1 ratio of Lactobacillus bulgaricus and Streptococcus thermophilus + incubated at around 45 degrees for 4-5 hours.
- At end of fermentation yoghurt put into cartons at temp of 10 degrees as plain yoghurt or mixed with previously sterilised fruit.
- Thick-set yoghurts mixed + ferment in pot.
26
Q
Why are some people allergic to non-human insulin?
A
- 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.
27
Q
Outline stages of penicillin growth + conditions needed.
A
- 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.
- First stage = fungus grows.
- Second stage = produces penicillin.
- Third stage = drug extracted from medium + purified.
28
Q
How to culture microorganisms using an inoculating broth?
A
- Make suspension of bacteria to be grown.
- Mix known volume with sterile nutrient broth in flask.
- Stopper the flask with cotton wool to prevent contamination from air.
- Incubate at suitable temp, shaking regularly to aerate broth providing O2 for growing bacteria.
29
Q
How to culture microorganisms using inoculating agar?
A
- 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. - 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.
- 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.
30
Q
What are the four stages of a exponential growth curve for bacterial pop?
A
- Lag phase = bacteria adapting to new environment:
- Growing, synthesising enzymes they need + are not yet reproducing at theoretical maximum. - Log/Exponential phase = rate of bacterial reproduction close to or at theoretical maximum –> natality rate > mortality rate.
- Stationary phase = total growth rate is 0 –> mortality rate = natality rate.
- Decline/Death phase = reproduction has almost ceased + mortality rate > natality rate + death rate keeps increasing.
31
Q
What are the limiting factors that prevent exponential growth of bacteria culture?
A
- Nutrients available,
- O2 levels.
- Temperature.
- Waste build-up –> toxic material inhibit further growth.
- Change in pH.
32
Q
Define primary metabolites and give examples.
A
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.
33
Q
Define secondary metabolites and give examples.
A
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.
34
Q
Outline steps of batch fermentation.
A
- Microorganisms inoculated into fixed volume of medium.
- As growth takes place, nutrients used up + both new biomass + waste products build up.
- As culture reaches stationary phase:
a. Overall growth ceases.
b. Microorganisms carry out biochemical changes to form desired end product.
- Process stopped before death phase + products harvested.
- Whole system cleaned + sterilised and new batch culture started up.
35
Q
Outline steps of continuous culture.
A
- Microorganisms inoculated into sterile nutrient medium and start to grow.
- Sterile nutrient medium added continually to culture once it reaches the exponential point of growth.
- Culture broth continually removed medium, waste products, microorganisms, and product keeping vol. in bioreactor constant.
36
Q
How is temp used to control bioreactors?
A
- 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.
37
Q
How are nutrients and O2 used to control bioreactors?
A
• O2 + nutrient medium added in controlled amounts to broth when probes or sample tests indicate levels are dropping.
38
Q
How does the mixing mechanism control bioreactors?
A
- 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
39
Q
How does asepsis control bioreactors?
A
- 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.
40
Q
State the advantages of using isolated enzymes.
A
- More specific.
- Maximises efficiency.
- Lowers downstream producing –> pure product produced.
- More efficient.
- Less wasteful.
41
Q
Advantages of using extracellular enzymes over intracellular enzymes as isolated enzymes?
A
- Cheaper.
- Easy to identify + isolate required enzyme.
- More robust –> adapted to greater variations in temp + pH.
42
Q
Advantages of using immobilised enzymes?
A
- 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.
43
Q
Disadvantages of using immobilised enzymes?
A
- Reduced efficiency.
- Higher initial costs of materials.
- Higher cost of bioreactor.
- More technical issues.
44
Q
State the advantages and disadvantages of surface immobilisation by adsorption to inorganic carriers.
A
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.
45
Q
State the advantages and disadvantages of surface immobilisation by covalent or ionic bonding to inorganic carriers.
A
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.
46
Q
State advantages + disadvantages of immobilising enzyme by entrapping it within a matrix
A
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.
47
Q
State the advantages + disadvantages of immobilising enzyme by entrapping it in microcapsules or behind a semi-permeable membrane.
A
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.
48
Q
Outline a few uses of immobilised enzymes.
A
- 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.
