Chapter 22: Cloning and Biotechnology Flashcards
1
Q
4 types of natural cloning in plants:
A
Bulbs, Runners, Rhizomes, Stem Tubers
2
Q
Natural Cloning: Bulbs
A
Leaf bases swell with stored food, buds form internally which develop into new plants after leaf abscission.
3
Q
Natural Cloning: Runners
A
A lateral stem grows away from the parent plant, the plant develops roots where the stem touches the ground. The runner withers away leaving the individual plant.
4
Q
Natural Cloning: Rhizomes
A
A rhizome is a specialised horizontal stem running underground, often swollen with food. Buds develop on the rhizome, forms vertical shoots which becomes independent plants.
5
Q
Natural Cloning: Stem Tubers
A
Tip of an underground stem becomes swollen with stored food to form a tuber or storage organ. Buds on the tuber produce new shoots.
6
Q
Natural Cloning in Horticulture:
A
Splitting up bulbs, removing young runners, cutting up rhizomes,
7
Q
Cloning sugar cane:
A
Fastest growing crop -> Propagated cloning -> cloned using cuttings
8
Q
When is micropropagation used?
A
When the desired plant: does not readily produce seeds, doesn't respond well to natural cloning, is very rare, genetically modified or selectively bred with difficulty, Required to be pathogen-free,
9
Q
Steps of Micropropagation:
A
- Small tissue sample taken from the meristematic tissue under sterile conditions.
- Sample is sterilised by immersing the tissue in sterilising agents.
- The explant is placed in a sterile culture medium and containing a balance of plant hormones to promote mitosis.
- The cells proliferate forming a mass of cells known as a callus.
- The callus is divided up and individual cells or clumps are transferred to new culture mediums containing nutrients for growth,
- The plantlets are potted into compost
- Young plants are planted out to grow into small plants.
10
Q
What is the explant:
A
Sterilised material removed from the plant.
11
Q
+ves of micropropagation:
A
- Culturing meristem tissues produces disease-free plants
- Allows for rapid production of plants with the same genetic make-up
- Reliably increases numbers of rare or endangered plants
- Produces large numbers of seedless plants
- Provides a way of reproducing difficult to grow or infertile plants.
- It’s possible to form viable numbers of GMO’s
12
Q
-ve’s of micropropagation:
A
- Produces a monoculture susceptible to diseases of changes to conditions
- Expensive process
- Plantlets and explants vulnerable to infection during the production process
- Large numbers of plants can be lost
13
Q
Natural Animal Cloning:
A
Animal cloning is most common in invertebrate animals, but can still occur in vertebrates e.g twinning.
14
Q
Natural Cloning in Invertebrates:
A
- Some can generate an entire animal from fragments of the original.
- Hydra produce small ‘buds’ on the side of their body which can develop into genetically identical clones.
15
Q
Natural Cloning in Vertebrates:
A
- Monozygotic twins (identical twins) -> Early embryo splits
- Female amphibians can produce offspring when no male is available -> not genetically identical but arise from same genetic material.
16
Q
Artificial Cloning Animals:
A
Artificial Clones of invertebrates can be formed by extracting fragments.
Two methods for artificial cloning of vertebrates.
17
Q
Artificial Twinning Animals:
A
The early embryo is manually split into two or more pieces and the pieces develop into identical offspring.
18
Q
Steps of artificial twinning:
A
1) Livestock with desired traits treated with hormones to super ovulate (Stimulates ova release)
2) The ova (oocytes) are fertilised naturally or by artificial insemination by a bull with good traits. Early embryo’s flushed out of the uterus. Another method is the mature egg cells are removed and fertilised by semen in lab
4) Usually before day 6 the cells are still totipotent the cells of the early embryo are split to produce smaller embryos.
5) Each embryo is grown in the lab to ensure no complications before being implanted into surrogate mothers.
6) Embryo’s develop and are born naturally.
19
Q
What is an enucleated egg cell?
A
An oocyte which has had its nucleus removed.
20
Q
What are the two methods of artificial cloning vertebrates (reproductive cloning):
A
Artificial Twinning and Somatic Cell Nuclear Transfer
21
Q
What is an oocyte?
A
An immature egg cell in the ovum.
22
Q
Benefits of artificial twinning:
A
Possible to greatly increase the numbers of offspring produced by animals with best genetic stock. Some of the embryos can be frozen and assessed.
23
Q
During SCNT why are different breeds used for the cell donor, egg donor and surrogate mother?
A
To ensure that the correct animal has been cloned and to make it easier to identify the original animal at each stage.
24
Q
Stages of SCNT:
A
1) The nucleus is removed from the somatic cell of an adult animal.
2) The nucleus is removed from a mature ovum harvested from a different female animal of the same species.
3) The nucleus from the adult somatic cell is inserted into the ovum and given a mild electric shock so it fuses and begins to divide. In some cases the two cells fuse in the presence of an electric current (electrofusion) and begin to divide.
4) The embryo develops and transferred into the uterus of a third animal.
5) The new animal is a clone of the somatic cell donor.
25
Why is a clone produced by SCNT not a complete genetic match?
- Mitochondrial DNA will come from the egg cell
| - Mutations to the Somatic Cell Nucleic Acid.
26
Uses of SCNT:
Pharming- production of animals genetically engineered to produce therapeutic human (pharmaceutical) proteins.
Produce genetically modified animals to grow human organs for transplants.
27
Pros of Animal Cloning:
- Artificial Cloning - enables high-yielding farms to produce more offspring - Enables the success of a sire (male) at passing on desirable genes to be determined (cloned parents)
- SCNT - enable GM embryos to be replicated and develop, giving many embryos from one procedure -Enables scientists to clone specific animals - Enable rare, endangered, and extinct animals to be reproduced.
28
Cons of Animal Cloning:
SCNT - Inefficient process -> most animals require many eggs to produce a single cloned offspring
Many cloned animal embryos fail to develop, miscarry or produce malformed offspring.
Many animals produced by cloning have reduced lifespans.
SCNT is often unsuccessful in increasing the populations of endangered animals.
29
Biotechnology:
The application of biological organisms or enzymes to the synthesis, breakdown or transformation of materials in the service of people.
30
Bioremediation:
Microorganisms are used to breakdown pollutants and decontaminate soil or water.
31
Why are microorganisms used?
- No welfare issues, only need the optimum conditions for growth.
- Variety of microorganisms capable of a variety of reactions
- Genetic engineering allows for artificial manipulation to carry out reactions not seen naturally.
- Have a short life cycle and rapid growth rate -> quantities can be produced in a short amount of time.
- Simple and cheap nutrient requirements.
- Bioprocesses cheaper than non-biological industrial processes.
32
Indirect food production:
Microorganisms change/alter the food.
33
Direct food production:
Microorganisms create the food.
34
Indirect Food Production: Baking: What microorganism(s)?
Yeast -> mixed with water to respire aerobically -> CO2 produced makes bread rise
35
Indirect Food Production: Brewing: What microorganism(s)?
Yeast -> respires anaerobically to produce ethanol. GM yeast ferments at lower (cheaper) temperatures and flocculates at the end of the process.
36
Indirect Food Production: Cheese-making: What microorganism(s)?
Bacteria - feed on lactose in milk changing the texture and inhibit the growth of unwanted bacteria.
37
Indirect Food Production: Yoghurt-making: What microorganism(s)?
Bacteria - (Lactobacillus bulgaricus) forms ethanal. Streptococcus thermophilus (forms lactic acid). Both produce extracellular polymers that give yoghurt its texture.
38
Indirect Food Production: Baking: Process
- Active yeast added to flour and other ingredients - mixed in warm environment
- Dough is knocked back (remove excess air), kneaded and shaped
- Cooked in hot oven - CO2 bubbles expand the bread and kill yeast cells
39
Indirect Food Production: Brewing: Process
- Malting -> barley germinates producing enzymes that break down starch into sugars to be used by yeast.
- Mashing -> Malt is mixed with hot water and enzymes break down starch to produce wort. Hops are added for flavour and antiseptic qualities.
- Fermentation -> wort is inoculated with yeast and temp maintained. Eventually, yeast is inhibited by decreased pH, lack of oxygen and build-up of ethanol.
- Maturation -> beer is conditioned for a month 2-6'C and then filtered and pasteurised.
40
Indirect Food Production: Cheese-making: Process
-Milk is pasteurised (95'C for 20 seconds) and homogenised
| -
41
Indirect Food Production: Yoghurt-making: Process
- Skimmed milk powder is added to milk and the mixture is pasteurised, homogenised and cooled to 47'C.
- Milk is mixed 1:1 with bacteria and incubated at 45'C for 5 hours.
- At the end of fermentation they can be put into cartons at 10'C
- Thick yoghurts are mixed and ferment in a pot.
42
What is the type of protein produced by microorganisms for direct food production?
SCP - single-cell protein
43
What type of microorganisms produces mycoprotein?
Fungi
44
What is homogenisation in yoghurt and cheese making?
The even distribution of fat droplets.
45
Example of direct food production:
Quorn (mycoprotiens)
46
Advantages of using microorganisms to produce human food:
- More quickly reproduce and synthesise protein
- High protein content with low-fat content
- Microogranisms can use a wide variety of waste materials -> reduces cost
- GMO can produce required protein
- Not dependant on weather or breeding cycles - constant supply.
- No welfare issues
- Can taste like anything
47
Disadvantages of using microorganisms to produce human food:
- Some microorganisms can also produce toxin when not in optimum conditions
- Have to be separated from nutrient broth and processed.
- Need carefully controlled sterile conditions (increased cost)
- GMO stigma
- Protein needs to be purified
- People dislike thought of recycled waste
- Little natural flavour (needs additives)
48
Conditions for penicillin production:
- Small fermenters because oxygen levels difficult to control in large bioreactors.
- Continuously stirred to keep oxygenated
- Rich nutrient medium
- Growth medium contains a buffer to maintain pH at around 6.5
- Bioreactors are maintained around 25-27'C
49
Issues with using animal insulin:
- Supply changed seasonally as demand for meat changed
- Diabetics often allergic to animal insulin (due to its impurity)
- Delay in activity of several hours.
- Unsuitable for certain faith groups
50
Pros of genetically engineered human insulin:
- Constant and greater supply
- Less wellfare issues
- Pure insulin that is less likely to trigger allergies
- Faster acting and more efficient
- Cheaper overall
51
Naturally occurring microorganisms in Bioremediation:
- Algae clean sewage water
- Bacteria bioremediate foul water
- Crude oil spills can be speeded with nutrients to encourage bacteria
52
Genetically Modified microorganisms in Bio Remediation:
-GM bacteria that will breakdown or accumulate contaminants.
53
Microbial Colonies:
Visible cluster of microorganisms growing on the surface of or inside a solid growth medium.
54
Are microbial colonies clones?
Yes, except for rare mutations.
55
How are genetically identical organisms or pure strains obtained?
Organisms are spread on a culture plate and start a new stock from a single resulting colony.
56
What are the two inoculation Aseptic Techniques:
Nutrient Broth and Agar Plates
57
Nutrient Broth:
inoculated with a small sample of a liquid culture and a scraping of a colony taken from an agar plate. Stoppered Cotton wool covered with aluminum foil.
Incubated at a suitable temperature in a shaking aerator to keep oxygenated.
58
Agar Plates:
Inoculating loop is sterilised in a Bunsen flame, allowed to cool and dipped in a liquid culture or use to scrape some of a colony onto an agar plate.
- Zigzag streaks on the surface of the plate with the inoculating loop. Sterilise the loop after each stage to achieve a paattern of dilution.
- Plates stored upside down and at suitable temperature in an incubator.
59
Why are agar plates stored upside down?
To prevent water the accumulation of water and lessen risk of air-born particles landing on the colony.
60
Nutrient medium:
Medium containing nutrients to allow for the growth of microorganisms.
- Allow rapid growth of bacterial colonies on agar plates
- Sterilised before use.
61
Why must health and safety procedures be followed when growing a culture?
- Mutation -> may cause a harmless microorganism to mutate and become pathogenic.
- Contamination -> The culture may be harmless but pathogenic microorganisms from the environment may also be cultured.
62
What factors effect the growth of a culture?
Temperature - Changes efficiency of enzyme activity (denature)
pH - Affects the bonding in the enzyme -> can denature
Nutrient availability - Competition
Accumulation of Harmful Waste - May damage cells
O2 availability
63
What are the 4 phases of a bacterial growth curve?
Lag Phase, Exponential (log) phase, Stationary Phase, Death Phase
64
Lag Phase:
Microorganisms adapt to environment - genes and ribosomes produce proteins to deal with new environment - Birth rate = deathrate
65
Exponential (log) Phase:
Rate of reproduction is increasing rapidly - Birthrate > Death Rate
66
Stationary Phase:
Reproduction rate = death rate - accumulation of waste and limited nutrients
67
Death Phase:
Death Rate > reproduction rate - Limiting factors outweigh factors benefitting growth - reproduction rate decreases.
68
Requirements of a microorganism to be cultured industrially:
- Synthesise or digest required chemical
- Work quickly enough to meet commercial demand
- Provide a good yield (increase profits)
- Use relatively cheap nutrient (reduce cost)
- Not Require extreme conditions (less expensive)
- Not produce poisons that could contaminate the final product
- Not mutate easily to form non-functioning variants
69
Primary Metabolites:
Chemicals produced from the normal functioning of a cell, e.g ethanol from respiration
70
Secondary Metabolites:
Chemical produced that are produced from non-essential functioning, e.g penicillin
71
What are the three things that could be wanted when using an industrial culture:
The microorganism, primary metabolites, secondary metabolites
72
When are primary metabolites produced?
During active growth period
73
When are secondary metabolites produced?
When max biomass has been reached in stationary phase.
74
What are the two types of industrial scale processes:
Batch Fermentation, Continuous Culture
75
Batch Fermentation: - Closed - secondary metabolites
- Inoculation into a fixed volume of nutrient medium
- Nutrients consumed during microbial growth
- Stationary phase - secondary metabolites produced
- Process halted before the death phase - products harvested and culture vessel is cleaned and sterilised
76
Continuous Culture: - Open - primary metabolites
Microorganism is inoculated into the nutrient medium.
Sterile nutrient medium is added continually during the exponential phase.
The nutrient medium is continually removed, at the same rate at which new nutrient medium is added.
77
Example Question: Explain why batch culture encourages the production of penicillin:
Penicillin is a secondary metabolite and-so the culture is prepared to reach stationary phase as more secondary metabolites are produced.
78
Advantages of using isolated enzymes:
- Less wasteful than using microorganisms
- More efficient than microorganisms
- No unwanted products (specific reactions)
- Greater production of product (Enzymes can operate in their optimum conditions)
- (Immobilised enzymes) Less down stream processing, pure product produced rather than mixture with microorganisms
79
Two types of enzymes:
Extracellular and intracellular
80
Intracellular enzymes:
- Huge range of enzymes within a single cell
- Range allows for a more specific enzyme extracted
- Higher cost of isolating the intracellular enzyme.
- Less robust
81
Extracellular enzymes:
- Secreted by cells -> easier to isolate
- More limited range of extracellular enzymes per cell-> identification and isolation easier
- More robust and can cope with greater range of conditions.
82
Advantages of immobilised enzymes:
- Enzymes reused - Cheaper (long term)
- Cheaper - less easily denatured
- Catalytic properties can be altered to fit bioprocess
- Increased reliability due to inert support system -> cheaper
- Enzyme doesn't contaminate the product (when operational)
83
Disadvantages of Immobilised enzymes:
- Reduced efficiency -> immobilisation can reduce rate of reaction
- Higher initial costs compared to microorganisms
- Higher set up costs of bioreactor
- Increased technical problems
84
Immobilised Enzymes:
Enzymes that have been extracted from cells and immobilised by attaching them to an inert support system.
85
What are the different methods of immobilising enzymes?
Surface immobilisation and entrapment
86
What are the two types of surface immobilisation?
Surface adsorption and ionic/covalent bonding to inorganic carriers.
87
What are the two types of entrapment?
Matrix and Encapsulation.
88
Matrix:
- Enzymes arranged in an matrix (net),
- More stable than surface immobilisation
- Enzyme on inert material, substrate flows across.
89
Encapsulation:
Enzymes in microcapsules or inside a semi-permeable membrane
- relatively simple
- Small effect on enzyme activity
- relatively expensive
- diffusion of substrate to and product from the active site can slow the rate of reaction
90
Surface Adsorption:
- Enzymes adsorped to inorganic carriers (e.g cellulose)
- Weak attachment can cause leakage - waste - less efficient
- Weak attachment makes the structure easy to alter and extract.
91
Covalent/ ionic enzyme immobilisation:
- enzymes covalently or ionically bonded to inorganic carriers
- strongly bound -> less likely to leak
- Enzymes accessible to substrate
- pH and substrate conc. have a little effect on the enzyme activity
- cost varies
- Active site of the enzyme may be modified.
92
Immobilised Penicillin Acylase
Develops semi-synthetic penicillin from natural penicillin -> create more effective drugs
93
Immboilised Glucose isomerase
Convert glucose into fructose -> produce sweeteners
94
Immobilised Lactase
Hydrolyses lactose into glucose and galactose -> make lactose free milk
95
Immobilised aminocyclase
Produces samples of L-amino acids -> used in pharmaceuticals
96
Immobilised nitrile hydratase
Catalyses the hydration of acrylonitrile into acrylamide -> production of plastics
97
Immobilised glucoamylase
Breaks down dextrins (starch brokendown by amylase) into glucose
