Cloning and biotechnology Flashcards
1
Q
define clone
A
genetically identical organisms or cells
2
Q
what’s vegetative propagation (cloning in plants)
A
reproduction from vegetative parts of a plant (parts of a plant that do not reproduce sexually)
3
Q
examples of explants
A
pieces of leaf, stem, root or bud
4
Q
by which biological process are clones produced
A
asexual reproduction in which the nucleus divides by mitosis
5
Q
by which process do yeast reproduce
A
budding
6
Q
advantages of reproduction by cloning
A
- good conditions for growth for parent will be good for offspring
- rapid - population size can increase rapidly
- reproduction can occur with only one parent, also quicker
7
Q
disadvantages of reproduction by cloning
A
- overcrowding - more competition
- no genetic diversity (except those caused by mutation)
- little variation
- if environment changes to be less advantageous, whole population is susceptible
- difference between genetic diversity and genetic variation is that the latter refers to how different two alleles of the same gene are, while the former refers to how many genes there are in a population
8
Q
which tissue allows plants to reproduce by cloning
A
meristematic tissue
9
Q
examples of the vegetative parts of a plant
A
- runners/ stolons
- rhizomes
- suckers
- bulbs
- corms
- leaves
- tubers
10
Q
what are runners/stolons
A
horizontal stems that come from roots on surface of ground
11
Q
what are rhizomes
A
underground horizontal stems that come from roots
12
Q
what’s a corm
A
underground stem with scaly leaves and buds
13
Q
what are suckers
A
new stems that grow from the roots
14
Q
what are bulbs
A
- underground food stores
15
Q
what are tubers
A
underground structures that act as food storage, covered in “eyes”- each eye is able to sprout new plants- potato
16
Q
where do natural clones grow on leaves
A
clones grow on leaf margins (edges)
17
Q
examples of animals produced by natural cloning
A
- identical twins ( zygote splits into two cells)
- water flea and greenfly
18
Q
what’s micropropagation
A
- growing large numbers of new plants from meristem tissue from plant
- takes place after tissue culture
19
Q
what’s tissue culture
A
- growing new tissues, organs or plants from certain tissues cut from any part of sample plant
- first step in micropropagartion
20
Q
how to make cuttings
A
- choose stem with no leaves to reduce transpiration and no flowers to encourage growth of roots as it takes a lot of energy to grow flowers
- stem cut between two nodes
- may need to dip cut end in rooting hormone or remove bark to encourage plant to produce a callus
- cut end placed in moist soil
- don’t overwater compost to allow air to reach roots
21
Q
what’s a callus
A
- soft tissue that forms over a wounded or cut plant surface, leading to healing. A callus arises from cells of the cambium
- (a mass of totipotent cells)
22
Q
other ways of making cuttings
A
- root cuttings
- scion cuttings
- leaf cuttings
23
Q
what are scions
A
dormant woody twigs
24
Q
why is tissue culture (micropropagation) used instead of taking cuttings for large scale cloning
A
- taking cuttings is time consuming and takes up lots of space. Also some plants don’t respond well to taking cuttings
25
outline the stages in micropropagation
1. explant (piece of plant that's placed in growth medium) cut into small pieces. Meristem tissue is often used, as this is always **free from virus infection**.
2. Explants are sterilised using bleach or alcohol.
3. Explants are placed in sterile growth medium (usually agar) contining high concentrations of **auxins and cytokinins** which stimulate the cells of each explant to divide by mitosis to form a **callus**.
4. Once a **callus** has formed it is divided
5. The cells are placed onto mediums containing different ratios of **cytokinin to auxin** which stimulates shoot and root growth
6. Once tiny plantlets are formed they're transferred to a **greenhouse** to be grown in compost or soil until **acclimatised**
26
advantages of artificial cloning/ vegetative propagation **in plants**
- relatively **rapid** method.
- some plants can't respire sexually
- an **unusual/desirable combination of characteristics** due to selective breeding or genetic modification can be retained without the risk of losing combination via sexual reproduction.
- new plants are uniform in their phenotype, making them **easier to grow and harvest**.
- propagation of seedless plants
- quicker than growing from seed
- can be done during any season
- young seedlings less likely to survive
27
disadvantages of artificial cloning **in plants**
- Tissue culture is **labour intensive**.
- **expensive** to set up the facilities to perform tissue culture successfully
- clones are susceptible to the same pests/ diseases
- no genetic variation except that introduced by variation
- cloning of plants requires **COMPLEX ASEPTIC TECHNIQUES**
28
what are the 2 main techniques to achieve reproductive cloning **in animals**
- embryo twinning
- somatic cell nuclear transfer, SCNT (only way to clone adult)
29
what's embryo twinning/splitting
splitting an embryo to create two genetically identical embryos
30
outline embryo splitting/twinning
1. Zygote created by **IVF**, sperm taken from male with desirabe characteristics
2. Cells divide by mitosis in ball of cells, incubated in lab.
3. Cut up ball of cells at an early stage of development. Cells continue to divide.
4. Implant into surrogate.
Offspring = all genetically identical = **clones of each other, not of parents**.
31
what's somatic cell nuclear transfer (SCNT)
transferring nucleus from a somatic cell to an egg cell
32
what's enucleation
removal of the cell nucleus
33
outline SCNT
1. Remove nucleus from egg cell (enucleation).
2. take normal body cell (adult somatic cell) and remove nucleus.
3. Take **complete adult somatic cell or nucleus** and enucleated egg cell and give them an electric shock to fuse them together
5. The cell undergoes mitosis.
6. Young embryo is implanted into a surrogate.
- offsting are **clones of the original somatic cell**
34
advantage of SCNT over embryo splitting/twinning
- in SCNT, phenotype is known before cloning starts
- in embryo splitting, the precise genotype or phenotype of the offspring produce will depend upon the sperm and egg used. Therefore, the precise phenotype not known.
35
which type of cells are used for animal cloning
**totipotent** - cell that can divide and differentiate into all cell types ( only found in embryos in animals)
36
what can reproductive cloning be useful for
producing large numbers of:
- farm animals produced by **selective breeding** or **genetic engineering**
- **genetically-modified** animals with unusual characteristics
37
what's non-reproductive cloning/ theraputic cloning
- The use of cloned cells for purposes other than reproduction to generate cells, tissues or organs or to replace those damaged by disease or accidents
- tissues grown from the patient’s own cells will be genetically identical and so avoid rejection
38
in what 2 ways can animal cloning be used for scientific research
- research into action of **genes that control differentiation**
- used to grow tissues and organs to test effect of drugs
39
arguments against artificial cloning **in animals**
- **Lack of genetic variation** may expose a herd to certain diseases or pests.
- Animals may be produced with **little regard for their welfare** - which may have undesirable side effects
- **low success rate** of adult cell cloning
- method is a lot more expensive than conventional breeding
- poor life expectancy
- Ethical issues regarding how long the embryo survives and **whether it is right to create a life simply to destroy it**
40
arguments for artificial cloning **in animals**
- Can produce a whole heard of animals with a **high yield or maintain an unusual/valuable characteristic**
- desirable characteristics = high milk yield, less fat, produces many eggs
- Using genetically identical embryos and tissues allows **effects of genes and hormones** to be assessed without interference from different genotypes
- Produce cells/ tissues genetically identical to donor- help in situations of disease or due to accidents
- **Testing medicinal drugs** on cloned cells and tissues avoid using animals for testing
- Individuals from **endangered species** can be cloned to increase species numbers
41
what's biotechnology
the use of living organisms in industrial processes. This could be used to produce food, drugs and other products
42
advantages of using microorganisms in biotechnology
- Relatively **cheap and easy** to grow as nutrients are cheap.
- In most cases **saves fuel and reduces costs** - production process takes place at lower temp than would be required to make the molecules by chemical engineering
- better for environment as reduces land for food production
- can be grown in many locations as microorganisms are not affected by climate
- Production takes place at atmospheric pressure - **safer** than using chemical reactions which can require very high pressure
- Production **not dependent on climate**.
- Microorganisms can be **fed by-products/ waste** from other food industries
- Microorganisms have a **short life cycle and reproduce quickly** so there's high yield of product
- Can GM microorganisms relatively easily.
- Fewer ethical considerations.
- Products are often released from microorganisms into the surrounding medium- makes the product **easier to harvest**.
- Product often **more pure or easier to isolate** than in conventional chemical engineering processes - lower downstream processes.
- suitable for vegans and healthier as its high in protein/ fibre but low in cholesterol
-
43
microorganism in production of yoghurt
- Bacteria convert lactose in pasturised milk to lactic acid
- Acidity causes milk protein to denature which causes it to coagulate
- Bacteria partially digests milk making it easy to digest
44
microorganisms in production of cheese
- lactic acid bacteria convert lactose in milk into lactic acid
- Once it is acidified, the milk is mixed with rennet (enzyme chymosin). Chymosin coagulates the milk protein (casein) in the presence of Ca2+.
- Curd (coagulated milk) is pressed into moulds. Cheese ripened and matured.
- Production of blue cheese involves the addition of fungi.
45
microorganisms in baking
- Yeast saccharomyces cerevisiae
1. ingredients mixed by kneading = dough
2. Proving / fermenting = warm place for 3 hrs, yeast respires anaerobically CO2 causes dough to rise.
3. Cook the risen dough - Alcohol evaporates
46
microorganisms in brewing
- Brewing uses yeast species, eg. Saccharomyces cerevisiae, Saccharomyces pastorianus to respire sugars from **barley malt** and produce **ethanol and CO2**
- The process is anaerobic, and is known as **fermentation**
- Ethanol is the primary product, and CO2 is a by-product
- Some alcoholic drinks have a higher alcohol content than fermentation alone can produce, so these drinks are first fermented, then **distilled** to concentrate the alcohol
47
microorganisms in penicillin production
- Species of mould can be cultured in industrial fermenters
- The technique = deep-tank fermentation
- In **times of stress**, fungi from the genus Penicillium produce the antibiotic Penicillin, to stop bacteria growing and competing for resources.
- Extraction and purification of the product produces large volumes of the drug for therapeutic use
- Made by batch culture
- penicillin = secondary metabolite
48
microorganisms in insulin production
- Previously, diabetics had to be treated with pig insulin, which is hard to isolate, expensive and not as effective as human insulin
- Genetic engineering can incorporate the gene for human insulin into the genome of the bacterium, E.coli
- Recombinant bacteria are grown in batch fermenters, and each bacterial cell expresses insulin
- Insulin is released into the batch medium and can be purified for medicinal use at a later stage
49
microorganisms in bioremediation
- Remediating polluted land can remove the pollutants and restore the land to its natural state
- Examples are oil spills, industrial accidents
- Naturally occurring microorganisms perform aerobic digestion of the contaminants and release non-polluting products
50
Advantages of bioremediation
- Uses natural substances
- Less labour
- **In situ** treatment
- Fewer waste products
- **Less exposure** for workers to danger from materials (eg oil spill workers)
51
microorganisms in making single-cell protein (mycoprotein production)
- Mycoprotein is a meat substitute product
- Mycoprotein is **low-fat and high in fibre**
- This could play a part in ensuring that a growing human population eats enough protein
- Fusarium venenatum fungus used
- A source of **glucose** is added to the tank
- **Oxygen** is also supplied to ensure **aerobic respiration** can occur, helps growth of hyphae
- Nitrogen is introduced in the form of ammonia ( **for protein production?**)
- The product in mycoprotein is the fungus itself
52
advantages of using microorganisms for production of single-cell protein (SCP)
- Protein produced faster
- Biomass high in protein 45 – 85%
- Production inc/ decr to demand
- No animal welfare issues
- Microorganisms are a good source of protein
- No animal fat or cholesterol
- Microorganisms can be easily genetically modified to adjust aa content
- **Seasonally independent**
- Not much **land** required.
53
disadvantages of using microorganisms for production of SCP
- People may not want to eat food grown on waste.
- Need to isolate protein from fermented products
- Protein has to be purified to ensure it's not contaminated
- Microbial biomass has a high proportion of **nucleic acids which need to be removed**
- amino acid profile may be different from traditional protein eg lacking in methionine
- Infection - care must be taken not to allow pathogens to grow
- Palatability.. Does not taste like meat
54
in commercial drug production, which conditions are controlled in fermenters to ensure the best possible yield of product
- Temperature
- Nutrients available
- O2 availability
- pH
- Concentration of product
55
why is temp controlled in fermenters
too hot - enzymes denatured. Too cool - growth limited
56
why is nutrient availability controlled in fermenters
nutrients required for growth and synthesis of products
57
why is O2 availability controlled in fermenters
most microorganisms respire aerobically
58
why is product concentration controlled in fermenters
product build up may affect synthesis process (inhibitor?)
59
what are primary metabolites
- products made during normal metabolism when microorganisms are actively growing
- products will be collected from a fermenter during the **log (exponential) phase**. In a fermenter, the population is not kept in a closed culture, but conditions are maintained for optimal growth.
60
what's a closed culture
- A culture which has no exchange of nutrients or gases with the external environment.
61
what are secondary metabolites
- normally produced during the **stationary phase** of growth.
- The population must be kept in a **closed culture** and the metabolites can be collected at the end of the stationary phase or during the decline phase.
62
features of continuous culture
- Primary metabolites are continuously released from the cells and can be extracted continuously from the fermenting broth.
- Broth is simply topped up with nutrients as these are used by microorganisms.
- **Broth removed regularly** to extract the product and remove cells from the broth - otherwise the **population becomes too dense**.
- THIS IS CONTINUOUS CULTURE - keeps culture growing at a **specific growth rate**.
63
features of batch culture
- Other products are produced ONLY when the cells are placed under **stress**- such as high population density or limited nutrient availability.
- These are secondary metabolites
- Culture is set up with **limited quantity of nutrients** and allowed to ferment for a specific time.
- Then the fermenter is emptied- culture extracted and process set up again- BATCH CULTURE.
- batch culture used to make penicillin is cultured from penicillium fungus
64
batch vs continuous culture
Batch:
- Growth = slower. Less nutrients with time as they're used up.
- Easy to set up / maintain.
- Contamination = only that batch affected.
- Less efficient = fermenter is not constantly in use.
Continuous:
- Growth = quicker. Constant nutrients for growth.
- Harder to maintain because constant growing conditions have to be maintained for optimum growth.
- If contaminated, huge volumes of product will be affected.
- More efficient. Fermenter is constantly in use.
65
why is asepsis essential in cultures
- Unwanted microorganism= contaminant
- might compete with culture microorganism for nutrients/ space
- reduce useful products from culture.
- cause product spoilage
- may also produce toxic products
- may destroy culture organism and its products
66
what are the two types of growth medium
- nutrient broth
- agar in petri dishes
67
aseptic technique procedure
1. wash hands
2. disinfect working area
3. Bunsen burner operating nearby, causes air to rise preventing pathogens from settling and sterilises air
4. open vessel and pass neck of bottle over flame to prevent entry of bac
5. open petri dish enough to allow introduction of desired microorganism
6. glassware/metal passed over flame before and after contact with organism
68
3 main steps in growing microorganisms
1. sterilisation
2. inoculation
3. incubation
69
outline sterilisation
- medium is heated in an autoclave
- allow it to cool then poor into petri dish and leave to set
- keep lid on to prevent infection
70
outline inoculation
- the introduction of microorganisms to the sterile medium:
- streaking: using a sterile wire inoculating loop to transfer liquid medium
- seeding: sterile pipette used
- spreading: sterile glass spreader
- cotton bud
71
outline incubation
- label petri dishes and taped at bottom
- **don't seal completely** to prevent anaerobic bac which are pathogenic
- petri dish placed in warm environment (incubator), upside down to prevent condensation falling onto surface and prevents agar drying out too quickly
- temp **shouldn't be above 25C** to prevent growth of pathogenic bac
- petri dishes completely sterilised after and use and before disposal
- wash hands
72
how will liquid broth look different when bac has grown
turns cloudy
73
why might serial dilutions need to be carried out
Numbers of individual organisms in a broth can be high – if plated, there may be too many colonies to count.
74
example of serial dilution where broth is diluted by a factor of 10 each time
1. take 1cm3 sample broth and add 9cm3 of distilled water
2. take 1cm3 sample of this diluted broth and add 9cm3 distilled water
- label each test tube
- ensure mixing after each step
- make sure fresh pipette is used at each step
- The number of colonies observed on the agar must be multiplied to calculate the population density in the broth. ( no of colonies in 1st multiplied by 10 and 2nd test tube multiplied by 100 for example)
- The volume of the drop used to inoculate must also then be used to calculate a colony forming unit (cfu) per cm3.
75
stages in a standard growth curve of a microorganism
- lag phase
- log (exponential) phase
- stationary phase
- death phase
76
what's the lag phase
- Small number of individuals reproduce
- Organisms are adjusting to their new environment: taking up water, cell growth, switching on certain genes, synthesising specific proteins.
77
what's the log phase (exponential)
- Ever-increasing number of individuals reproduce. The population doubles with each generation
- Organisms have adjusted to their environment. They have enzymes needed to survive. Individuals have sufficient nutrients and space to grow rapidly and reproduce
78
what's the stationary phase
- Population declines until it remains stable with cyclic fluctuations.
- Increasing numbers of organisms use up nutrients and produce increasing amounts of waste products. Reproduction rate = death rate.
79
what's the death phase
- Nutrients run out, waste builds up. Eventually all will die. More individuals die than are produced. Population falls.
- death rate above reproduction rate
80
what are immobilised enzymes
an enzyme that is held in place and not free to **diffuse** through the solution
81
Why use immobilised enzymes?
- They don’t mix with the product so extraction costs are lower
- The enzymes can be easily reused
- No cells requiring nutrients or releasing waste products
- Enzymes are surrounded by the immobilising matrix so are protected from extreme conditions (high temps and wide pH range).
82
Methods to immobilise enzymes
- Adsorption
- Covalent bonding
- Entrapment
- Membrane separation
83
outline adsorption
- Enzymes are bound to a supporting surface (e.g. clay, porous carbon, glass beads, resins). by hydrophobic interactions and ionic links.
- Active sites stay exposed to substrates.
Disadvantages:
- when substarate binds it can distort active site by additional interactions - reducing activity.
- Bonding forces not always strong and enzymes can become detached and leak into the reaction mixture.
84
outline covalent bonding in immobilised enzymes
- Enzymes bonded onto a supporting surface such as clay using strong covalent bonds.
- Enzymes are bonded using a cross-linking agent, which may also link them in a chain.
- Not likely to become detached and leak into reaction mixture.
Disadvantage:
- Expensive, can distort active site - reducing activity.
85
outline entrapment
- Enzymes trapped in a matrix that does not allow free movement.
- Active sites remains fully active.
- Substrate must diffuse into the entrapment matrix.
Disadvantages:
- Product must be able to diffuse out so can only be used when substrate and product molecules are small.
86
Outline membrane separation
- Enzymes are separated from reaction mixture by a partially permeable membrane.
Disadvantage:
- As in entrapment, substrate and product must be small enough to pass through the membrane by diffusion which may limit reaction rate.
87
5 examples of immobilised enzymes used in industrial processes
- Glucose isomerase
- Penicillin acylase
- Lactase
- Aminoacylase
- Glucoamylase
88
what's glucose isomerase (immobilised enzymes) used for in industry
- Converts glucose to fructose.
- Produces high fructose corn syrup - much sweeter than sucrose.
- Often used in diet foods.
- Cheaper than sucrose.
89
what's penicillin acyclase (immobilised enzymes) used for in industry
- Formation of semi-synthetic penicillins e.g. amoxicillin and ampicillin.
- Some penicillin resistant microorganisms are not resistant to these.
90
what's lactase (immobilised enzyme) used for in industry
- Lactose -> glucose and galactose by hydrolysis
- Used to produce lactose free milk.
91
what’s aminoacyclase (immobilised enzyme) used for in industry
- Used to produce pure samples of L-amino acids
- L-amino acids are used to make:
Pharmaceutical compounds
92
what’s glucoamylase (immobilised enzyme) used for in industry
- Converts dextrins to glucose.
- During the hydrolysis of starch, short polymers of glucose (dextrins) are formed.
93
equation for final no of bac
- N = No x 2^n
- N = final no of bac
- No = initial no of bac
- n = no of divisions
