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Biology > 22 - Cloning and biotechnology > Flashcards

22 - Cloning and biotechnology Flashcards

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1
Q

what is micropropagation?

A

is the process of making large numbers of genetically identical offspring from a single parent plant using tissue culture techniques

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2
Q

When is it desirable to use micropropagation?

A
  • doesn’t readily produce seeds
  • doesn’t respond to natural cloning
  • is rare
  • difficult to genetically modify or selectively bred
  • is required to be pathogen free
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3
Q

What are the steps of micropropagation?

A
  • get a small sample of the plant you want to clone (e.g. meristem/leaf/steam/root)
  • sterilise the explant (material removed from plant) with beach/sodium hypochlorite/alcohol
  • place on agar or culture medium, this contains things like glucose/ amino acids/nitrates. as well as auxin and cytokinin’s
  • the cells proliferate and form a callus, and divide them and transfer to new culture medium with more hormones and nutrients to stimulate plantlets
  • pot plantlets into soil
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4
Q

what are advantages of micropropagation?

A
  • Desirable genetic characteristics (i.e. flower colour, high fruit production) are always passed on to clones.
    -Quicker / more rapid than sexual reproduction.
  • culturing meristem tissue produces disease-free plants
    -able to produce infertile plants
  • increase numbers of rare plants
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5
Q

what are disadvantages of micropropagation?

A
  • expensive and labour intensive process
    -process can fail as plantlets and explants are vulnerable to infection during the production process
  • little genetic variation
  • all offspring are susceptible to the same pests, disease and environmental factors
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6
Q

Why is it easier to clone plants thyan animals?

A

-It is a lot easier for plants to reproduce via cloning, as they possess meristematic tissue which is pluripotent.
Animal cells are not able to do this, and adult animal stem cells are multipotent at best.

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7
Q

Two types of cloning

A

Natural cloning
Artificial cloning

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8
Q

who does natural cloning?

A
  • mainly invertebrates
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9
Q

State 3 examples of invertebrates that naturally reproduce via cloning

A
  • starfish- regenerate from fragments
  • flatworms
  • sponges
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10
Q

What is parthenogenisis?

A
  • asexual reproduction without fertilisation, the ovum can develop into new individuals without sperm
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11
Q

Give an example of natural cloning in a animals? what is it?

A
  • monozygotic twins
  • when an embryo that has been fertilised by a single egg splits into two separate embryos
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12
Q

Two ways to artificially clone vertebrates

A

Artificial twinning
Somatic cell nuclear transfer

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13
Q

explain artificially twinning a cow?

A
  • give cow with desirable traits hormones so she is super-ovulate
    -fertilise the ova and flush it out
    split the totipotent cells of the embryo to form smaller embryos
  • grow in the lab (embryo divide in-vitro)
  • implant into surrogate mothers
  • embryos develop into foetus and are born normally- producing the same identical offspring
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14
Q

Explain somatic cell nuclear transfer

A
  • One donor in the process donates a somatic cell (A). This has its nucleus removed (enucleated), which is kept, the rest of the cell is thrown away.
  • The second donor in the process donates an oocyte (egg cell). This oocyte (B) is also enucleated, but this time the nucleus is thrown away and the rest kept.
    -The nucleus from A (somatic cell nucleus) is fused with the enucleated oocyte from B. However, nothing will happen unless it is stimulated with an electric shock. This ensure they both properly fuse (electrofusion) and if successful, the cells will divide and develop into an embryo.
    -The newly dividing embryo is placed into the womb of a surrogate, where it develops to term.
  • The offspring that is born is a clone of the animal from which the somatic cell nucleus came from (i.e. A), and is genetically identical to it. It shares no genes with D or B, aside from mtDNA which it inherited from B.
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15
Q

What could cause the offspring to not be genetically identical?

A
  • mutations
  • environmental factors in the womb
  • mitochondrial DNA
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16
Q

State as many similarities between the processes of artificial twinning and somatic cell nuclear transfer.

A
  • implanted into surrogate
  • embryo develops from foetus
  • desirable features
  • invitro
  • identical clones
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17
Q

differences between both artificial twinning and somatic cell nuclear transfer.

A
  • many offspring produced in artificial twinning
  • undifferentiated cells are used in AT, and differentiated cells in SCNT
  • you remove the nucleus in SCNT
  • two cells are used in SCNT and one cell in AT
  • mDNA is the same in AT then SCNT
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18
Q

Arguments for animal cloning

A
  • Cloning allows for the replication of animals with specific, desirable traits
    -Cloning can contribute to conservation efforts by helping to preserve endangered or extinct species
    -Cloning provides valuable models for biomedical research, enabling scientists to study diseases, test new drugs
  • Cloning can be used to propagate high-quality livestock with desirable agricultural traits, potentially increasing efficiency in food production.
    -Cloning can speed up selective breeding programs by rapidly replicating animals with desirable traits, reducing the time required for traditional breeding.
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19
Q

Arguments against animal cloning

A
  • many cloned animal embryos fail to develop and miscarry of form malformed offspring
  • can make animals with shortened life spans
    -Cloned animals may experience health issues and suffer, and concerns have been raised about the welfare of animals involved in cloning experiments.
  • Cloning can lead to a reduction in genetic diversity within a population, which may increase vulnerability to diseases and environmental changes.
    -Cloning is often an expensive and inefficient process
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20
Q

What is recombinant DNA

A

DNA that has genetic information that has been combined from different sources

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21
Q

Why aren’t offspring produced via parthenogenesis considered to be full clones of the mother?​

A

Embryos are produced by duplication of chromosomes in haploid cell or from fusion of 2 of the mother’s haploid cells. Means that the offspring will not be genetically identical to the mother even though all the DNA came from the mother.

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22
Q

What is biotechnology?

A

is a field where biological organisms or enzymes are used in the synthesis, breakdown, or transformation of materials for people.​

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23
Q

What are examples of biotechnology?

A

Yeast in bread and alcoholic drinks​

Lactobacilli used to make yoghurt​

Rennet used to make cheese​

Genetically modified bacteria that make insulin​

Enzymes that breakdown pollutants

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24
Q

What organisms are used in biotechnology?

A

Fungi (Typically yeasts)​

Or Bacteria​

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25
Q

Why are microorganisms used?

A
  • no welfare issues
  • Large range of microorganisms that can carry out a wide range of different chemical synthesis or degradation, this can be utilised
  • Can genetically engineer microorganisms to carry out synthesis functions that aren’t natural to then e.g., human insulin
  • have a short life cycle and rapid growth rate, with correct environment, a large amount can be produced in a short time
  • their nutrient requirement is often simple and relatively cheap
  • can GM them so they utilise wasted materials
  • bioprocesses relatively cheap- as low temp, supply of food and oxygen
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26
Q

How are microorganisms used in brewing?

A

Alcoholic beverages are the product of the anaerobic respiration of yeast. Wine is made using grapes that naturally have yeast on their skin. Grapes contain the sugars fructose and glucose. When the grapes are crushed, the yeast uses these sugars to produce carbon dioxide and alcohol. Ale or beer is brewed using barley grains that are beginning to germinate. This process is called malting. As the grain germinates it converts stored starch to maltose, which is respired by the yeast. Anaerobic respiration again produces carbon dioxide and alcohol. Hops are used to give a bitter taste to the liquid.

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27
Q

How are microorganisms used in brewing?

A
  • yeast + sugar + water -anaerobically respire> CO2
  • the CO2 made makes the bread rise
  • yeast is mixed with other ingredients and is left in a warm environment
    -cooked and rises
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28
Q
A
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29
Q

How are microorganisms used in yoghurt-making?

A

yoghurt is milk that has undergone fermentation by Lactobacillus bulgaricus and Streptococcus thermophilus. The bacteria convert lactose into lactic acid. The acidity denatures the milk protein, causing it to coagulate. The bacteria partially digests the milk, making it easier to digest for those that consume it. Fermentation also produces the flavours characteristic of yoghurt. Other bacteria such as L. acidophilus, L, subsp casei, and Bifidobacterium may be added to probiotics to benefit human health by improving digestion of lactose, aiding gastrointestinal function and stimulating the immune system.

30
Q

What are the advantages for using microorganisms to produce human food?

A
  • they reproduce fast and produce proteins faster than animals
  • have a high protein content
  • they can use a wide variety of waste materials, reducing cost
  • they can be genetically modified
  • production isn’t dependant in weather or breeding cycles
  • no welfare issues
31
Q

What are the disadvantages for using microorganisms to produce human food?

A
  • some can produce toxins in non-optiumum conditions
  • must be separated from nutrient broth and processed
  • need sterile conditions, adding to costs
  • people have concerns when eating GM foods
  • protein must be purified
32
Q

How can microorganisms be cultured?

A

In a lab, using aseptic technique.

On an industrial scale, using a fermenter.
​

33
Q

What do microorganisms need to be cultured

A

Correct temp​

Nutrient medium​

Oxygen​

pH​

Water

34
Q

The procedure for an aseptic technique?

A

Disinfecting the working area​

Washing hands​

Lab technicians wear clean lab coats, cover hair and wear gloves​

All equipment sterilised before and after use by heating in flame until glowing, UV light, autoclave (121oC for 15 minutes)​

Work carried out in fume cupboard or updraft of Bunsen burner so air circulation carries away airborne contaminants​

Passing the neck of an open vessel through the Bunsen burner flame once opening and again before closing​

Cultures kept closed wherever possible​

In more sensitive applications, “clean rooms” are used.​

35
Q

What is an aseptic technique?

A

Any measure taken during a biotechnological process to ensure unwanted microorganisms do not contaminate culture being grown or products extracted

36
Q

What is meant by ‘aseptic’?

A

Free of microorganisms

37
Q

Describe what would happen if a single cell of a pathogen entered a culture dish.

A

Growth and cell division would continue until a colony containing millions of cells would be visible.

38
Q

Explain why agar is suitable as a culture medium for growing bacteria.​

A

Agar can be sterilised without affecting it chemically; it can be melted & poured then set. It contains no nutrients, and it is inert, meaning microbial enzymes cannot digest it so it remains solid.

39
Q

Describe the streak plate method

A

1- Light Bunsen burner
2- Work closely to the flame under the sterile zone
3- Loosen cap on bottle but don’t remove
4- ​Sterilise wire loop by heating​
red hot in flame
5- ​Allow loop to cool for 10​ seconds without touching​
anything
6- Remove top of bottle, dip loop into sample ​
7- ​Remove lid from agar plate a little and sweep loop across agar
8- Re-sterilise & repeat swiping a few times to produce a distinct pattern.​
9- Seal Petri dish with adhesive tape, Store it upside down

40
Q

What is continuous fermentation?

A
  • The nutrient medium is continually added so it does not run out.
  • Waste products are removed.
  • Culture broth is continually harvested (i.e. product is removed, along with microorganisms) which ensures a constant culture volume in the fermenter.​
  • Main application is culture of SCP (single cell protein).
41
Q

What is batch fermentation? When are products harvested?

A

Microorganisms are grown in a fixed volume of medium.​

This means (as time passes) nutrients are used up and waste products build up.​
- at the end of the stationart phase as this when imporatant metabolites are at the optimum

42
Q

What can be harvested from fermentation?

A

Metabolites

43
Q

What are metabolites?

A
  • intermediate products of metabolic pathways catalysed by enzymes
  • primary
  • secondary
44
Q

What are primary metabolites?

A

-essential chemicals formed as part of normal growth
- Amino acids, proteins, nucleic acids and ethanol

45
Q

What are secondary metabolites?

A
  • are non-essential chemicals that form later in growth, once the microorganism is established in the medium
  • antibiotics
  • common in bath cultures
46
Q

How and why is pH controlled in fermentation?

A
  • By adding acidic or alkaline buffers to the broth​
  • creates the optimum pH environment for the microorganisms so they reproduce at the highest possible rate. (or produce more desired metabolites)
47
Q

How and why is temperature controlled in fermentation?

A
  • Cooling system present – whole fermenter is surrounded by a water jacket
    -Creates the optimum temperature environment for the microorganisms so they reproduce at the highest possible rate. (or produce more desired metabolites). Also prevents denaturation of enzymes
48
Q

How and why is access to nutrients controlled in fermentation?

A
  • An impeller / stirring paddle rotates constantly, mixing the broth
  • Ensures the fermenter is well mixed, meaning microorganisms always have access to their nutrients
49
Q

How and why is Volume of O2 controlled in fermentation?

A
  • Monitored by an oxygen probe. Sterilised air is pumped into the tank via a sparger, which produces bubbles oxygenating the tank.
  • Microorganisms are provided with oxygen for aerobic respiration, to produce ATP needed for many metabolic processes. (if aerobic microbes)
50
Q

How and why is asepsis controlled in fermentation?

A
  • Superheated steam is used to sterilise the chamber and nutrient broth / culture medium
  • Kills any unwanted microorganisms that would compete with those being cultured
51
Q

How does a fermenter work?

A

A fermenter must be sterilised using superheated steam.
Then be filled with all the components required for growth and supplied
A pressure vent in the fermenter prevents any gas build-up.
An air inlet provides sterile oxygen to the aerobic fermenters, which enter the culture through air outlets which are often in a ring and allow the bubbles to mix into the culture (sparging).
A motor rotates mixing blades/impellers to mix the culture evenly. An inlet is present for the addition of nutrients, and the fermenter is surrounded by a water jacket with an inlet and outlet to allow circulation of water around the fermenter to regulate the temperature. Electronic probes measure oxygen, pH, and temperature levels. There is also an outlet tap for draining the fermenter. All inlets and outlets are fitted with filters to prevent contamination.
Primary metabolites are products that are only synthesised during normal

52
Q

There are two main ways of growing organisms in a fermenter;

A

Batch Fermentation​

Continuous Fermentation.

53
Q

how are microorganisms cultured in a lab

A

using aseptic technique.

54
Q

how are microorganisms cultured On an industrial scale

A

fermenter / bioreactor

55
Q

Primary metabolites

A

are essential chemicals formed as part of normal growth;​

Amino acids / proteins / nucleic acids / ethanol​

56
Q

Secondary metabolites

A

are non-essential chemicals that form later in growth, once the microorganism is established in the medium;​

Antibiotics / certain enzymes​

Common in batch culture (as culture reaches stationary phase), much rarer in continuous. ​

57
Q

4 stages of the growth curve

A

lag
log/exponential
stationary
decline/ death curve

58
Q

lag phase

A

bacteria are adapting to the new environment, they are growing synthesising enzymes
- are not reproducing at their maximum rate

59
Q

log phase

A

rate of bacteria reproduction id close to its theoretical maximum

60
Q

stationary phase

A

total growth = 0
death = new growth

61
Q

decline / death stage

A

when reproduction has nearly ceased- and death rate is increasing

62
Q

limiting factors preventing exponential growth of bacteria

A

nutrients available
oxygen levels
temperature
build-up of waste
change in pH

63
Q

Advantages of using isolated enzymes instead of whole organisms;​

A

less wasteful, because none of the Substrate is used for Growth and Reproduction, as enzymes alone do not undergo these processes.

maximise the efficiency of the process, as the conditions can be tailored specifically to the Enzyme, which often differ from those that would be considered for the Whole Microorganism.

less Downstream Processing. This is because you have made a Pure product and do not need to spend time and money Isolating it from the rest of the mixture.​

64
Q

Use of extracellular enzymes;

A

Extracellular enzymes are Adapted to work outside of the cell they are made in. This generally means they are more Robust than intracellular enzymes, and are able to tolerate greater variations in Temperature and pH than intracellular enzymes.

microorganisms produce Fewer extracellular enzymes in comparison to intracellular, making it easy to identify and Isolate the enzyme.​

despite their disadvantages, intracellular enzymes are still sometimes used. This is because there is a Larger range of them and therefore they sometimes provide the perfect enzyme for a process. A key example is the use of the enzyme Penicillin Acylase for converting natural penicillin into a semi-synthetic penicillin, which some penicillin resistant organisms are not resistant to.

65
Q

why are intracellular enzymes used over other options

A

despite their disadvantages, intracellular enzymes are still sometimes used.

This is because there is a Larger range of them and therefore they sometimes provide the perfect enzyme for a process.

A key example is the use of the enzyme Penicillin Acylase for converting natural penicillin into a semi-synthetic penicillin, which some penicillin resistant organisms are not resistant to.

66
Q

Four main methods of immobilising enzyme

A

Encapsulation​
Entrapment within a silica gel matrix​
Covalently bonded to a surface​
Adsorption to a surface​

67
Q

Encapsulation​

A

The enzyme is contained in jelly like beads, which act as a selectively-permeable membrane.​

does have a downside in that the substrate must diffuse to the enzyme and the product away from it… this can slow down the rate of reaction.

68
Q

Entrapment within a silica gel matrix​

A

This physically holds individual enzymes in place.​

It is applicable to many processes, but the process is more difficult to accomplish in comparison to encapsulation

69
Q

Covalently bonded to a surface​

A

No issues with access to substrate, unlike encapsulation and entrapment.​

However, the process can sometimes alter the active site, slowing the rate of reaction.

70
Q

Adsorption to a surface​

A

Enzymes adhere to a surface Due to electrostatic forces)​

Versatile, simple, and cheap process​

Activity is not affected​

However enzymes can be dislodged more easily ​

(often only held in place by weak van der waals forces)

71
Q
A

Biology (24 decks)

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