6.2.1 - Cloning and biotechnology Flashcards
(130 cards)
1
Q
What is asexual reproduction a form of ?
A
A form of cloning
2
Q
What does asexual reproduction result in ?
A
It results in offspring produced by mitosis and known as clones
3
Q
What is natural cloning called in plants ?
A
Vegetative propagation
4
Q
What happens in vegetative propagation ?
A
- A structure forms which develops into a fully differentiated new plant
- It is genetically identical to the parent
- It eventually becomes dependent from its parent once it is propagated from the parent
5
Q
What does vegetative propagation often involve ?
A
It involves perennating organs, which enables plants to survive in adverse conditions
6
Q
What are perennating organs ?
A
- Organs that can survive from one germinating season to another
- Especially under unfavourable conditions
- They are also a means of asexual reproduction
7
Q
What do perennating organs store ?
A
They contained stored foods from photosynthesis and can remain dormant in the soil
8
Q
Where does natural plant cloning occur ?
A
- Bulbs
- Runners
- Rhizomes
- Stem tubers
9
Q
How is natural cloning exploited in horticulture ?
A
- Splitting up bulbs
- Removing young plants from runners
- Cutting up rhizomes
10
Q
Why is it natural cloning beneficial in horticulture ?
A
- Increase plant numbers cheaply
- New plants have exactly the same genetic characteristics as their parents
11
Q
Why is rooting hormone added to roots ?
A
It encourages the growth of new roots
12
Q
What are the advantages of using propagation from cuttings rather than seeds ?
A
- It is much faster
- Guarantees the quality of the plants
- Offspring will be genetically identical to the parents
13
Q
What is the main disadvantage of using propagation from cuttings rather than seeds ?
A
Lack of genetic variation in the offspring should any new disease or pest appear
14
Q
How are sugar canes cloned ?
A
Short lengths of around 30cm long, with three nodes, are cut and buried in a clear field in shallow trenches, covered with a thin layer of soil
15
Q
What is micropropagation ?
A
Process of making large numbers of genetically identical offspring from a single parent plant using tissue culture techniques
16
Q
When can micropropagation be used ?
A
- Plant does not readily produce seeds
- Plant is very rare
- Plant is required to be pathogen free by growers
17
Q
What are the basic principles of micropropagation and tissue culture ?
A
- Take a small sample of tissue from the plant you want to clone
- Sterilise the sample, immerse it in ethanol
- The material removed from the plant is now called the explant
- Place the explant in a sterile culture medium containing a balance of plant hormones that stimulate mitosis
- The cells proliferate forming a mass of identical cells known as a callus
- Callus is divided up and individual cells from the callus are transferred to a new culture medium containing hormones and nutrients that stimulate the development of identical plantlets
- Plantlets are potted into compost
- Young plants are planted out to grow and produce a crop
18
Q
Where does micropropagation now occur ?
A
- In bioreactors
- Making artificial embryo plants to be packaged in artificial seeds
19
Q
What are the arguments for micropropagation ?
A
- Allows for the rapid production of large numbers of plants with known genetic make up which will yield good crops
- Culturing the meristem tissue produced disease free plants
- Makes it possible to produce viable numbers of plants after genetic modification of plant cells
- Provides a way of producing very large numbers of new plants that meet consumer needs
- Provides a way of producing plants which are naturally relatively infertile or difficult to grow from seed
- Provides a way of reliably increasing the numbers of rare or endangered plants
20
Q
What are the arguments against micropropagation ?
A
- Produces a monoculture so they are all susceptible to the same diseases or changes in growing conditions
- Relatively expensive process
- Explants and plantlets are vulnerable to infection by the moulds and other diseases during production
- If the source material has a virus, all of the clones will be affected
21
Q
How can cloning take place in invertebrates ?
A
- Regeneration of entire animals from fragments of the original if they are damaged
- Fragment and form new identical animals as part of their normal reproductive process
- Hydra produce buds on the side of their body which develop into genetically identical clones
- Females can produce offspring without mating
22
Q
What is formed due to vertebrate cloning ?
A
The formation of (monozygotic) identical twins
23
Q
What happens in the formation of monozygotic twins ?
A
- Early embryo splits to form two separate embryos
- Cause of this is unknown
24
Q
Even though identical twins are genetically identical why might they look different at birth ?
A
Different positions and nutrition in the uterus
25
What are the two main methods used to produce artificial clones of vertebrates ?
- Artificial twinning
- Somatic cell nuclear transfer
26
After an egg is fertilised what does it divide to form ?
- A ball of totipotent cells
- As this continues, the embryo becomes a hollow ball of cells
- After this the embryo can no longer divide successfully
27
What does totipotent mean ?
It has the potential to form an entire new animal
28
What happens in natural twinning ?
An early embryo splits and two foetuses go on to develop from the two halves of the divided embryo
29
What happens in artificial twinning ?
The split in the early embryo is done manually and then the foetuses go on to develop from these two halves of the divided embryo
30
How many pieces can the early embryo be split in, in artificial twinning ?
More than two pieces
31
What are some uses of artificial twinning ?
Used by the farming community to produce the maximum offspring from particularly good dairy or beef cattle or sheep
32
What are the stages of artificial twinning ?
- A cow with desirable traits is treated with hormones so she super-ovulates
- The ova may be fertilised naturally by a bull with particularly good traits. The early embryos are flushed out of the uterus
- Around day 6 when the cells are still totipotent, the cells of the early embryo are split into many individual totipotent cells
- Each of these individual embryos is grown in the lab before it is implanted into the surrogate mother.
- Each embryo is in an individual mother, this is safer
- Embryo develops into a foetus and are born normally
- So a number of identical cloned animals are produced by different mothers
33
What is a benefit of artificial twinning ?
Makes it possible to greatly increase the numbers of offspring produced by the animals with the best genetic stock
34
Why may some embryos be frozen ?
Allows the success of a particular animal to be assessed and if the stock is good, remaining embryos can be implanted and born
35
Describe the process of SCNT
- Taking the nucleus from an adult body cell and transferring it to an enucleated egg cell (nucleus is removed)
- A tiny electric shock is used to fuse the egg and the nucleus, stimulate the combined cell to divide, and form an embryo that is a clone of the original adult
36
What does SCNT allow to happen ?
Form clones of an adult animal
37
What are the stages of SCNT ?
- The nucleus is removed from a somatic cell of an adult animal.
- The nucleus is removed from a mature ovum harvested from a different female animal of the same species (it is enucleated).
- The nucleus from the adult somatic cell is placed into the enucleated ovum and given a mild electric shock so it fuses and begins to divide.
- In some cases, the nucleus from the adult cell is not removed - it is simply placed next to the enucleated ovum and the two cells fuse (electrofusion) and begin to divide under the influence of the electric current.
- The 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.
38
What is SCNT also known as ?
Reproductive cloning, because live animals are the end result
39
In what ways can SCNT be used ?
- It is used in pharming (the production of animals which have been genetically engineered to produce therapeutic human proteins in the milk)
- To produce GM animals which grow organs that have the potential to be used in human transplants
40
What are the arguments for animal cloning (Artificial twinning) ?
- AT enables high yielding farm animals to produce many more offspring than normal
- Enables the success of a male animal at passing on desirable genes to be determined
- If the first cloned embryo results in a successful breeding animal, more identical animals can be reared from the remaining frozen clones
41
What are the arguments for animal cloning (SCNT) ?
- Enables GM embryos to be replicated and to develop, many embryos from one procedure
- Important process in pharming
- Enables scientists to clone specific animals
- Has the potential to enable rate, endangered or even extinct animals to be reproduced
42
What are the arguments against animal cloning ?
- Many cloned embryos fail to develop and miscarry or produce malformed offspring
- Many animals produced by cloning have shortened lifespans
- SCNT is a very inefficient process, may take many eggs to produce one offspring
- SCNT has been relatively unsuccessful so far in increasing the populations of rare organisms or allowing extinct species to be brought back to life
43
What does biotechnology involve ?
Applying biological organisms or enzymes to the synthesis, breakdown, or transformation of materials in the service of people
44
How is biotechnology used now ?
- DNA manipulation to produce genetically engineered microorganisms
- Synthesising drugs such as insulin and antibiotics
- Use of biological systems to remove soil and water pollution in a process known as bioremediation
45
What are the most commonly used organisms in biotechnology processes ?
- Fungi, particularly the yeasts
- Bacteria
46
What does most biotechnology involve ?
- Using enzymes in a manufacturing process
- The most stable, convenient, and effective form of the enzymes is often a whole microorganism.
47
What are the reasons as to why microorganisms are ideal for bioprocesses ?
- There are no welfare issues to consider
- There is an enormous range of microorganisms that can be used
- Genetic engineering allows us to artificially manipulate microorganisms to carry out synthesis reactions that they would not do naturally, for example, to produce human insulin.
- Microorganisms have a very short life cycle and rapid growth rate.
- The nutrient requirements of microorganisms are often very simple and relatively cheap.
- The conditions which most microorganisms need to grow include a relatively low temperature, a supply of oxygen and food, and the removal of waste gases. This makes bioprocesses relatively cheap.
48
What indirect effect do microorganisms have on food production ?
- It is their actions on other food that is important
- When you eat bread you are mainly eating flour, when you eat
yoghurt or cheese it is mainly milk.
49
What are the few disadvantages of using microorganisms in food production ?
- The conditions are not ideal (e.g., too hot or too cold) the microorganisms do not grow properly and so they do not work efficiently.
- Conditions that are ideal for the microorganisms can also be ideal for microorganisms that cause the food to go off or cause disease and so the processes have to be sterile.
- Increasingly the microorganisms used in food production have been genetically engineered, and some people have ethical issues with the use of GM organisms, although this is generally much less the case with microorganisms than with animals and plants.
50
What is single-cell protein (SCP) ?
Microorganisms being used to directly produce protein you can eat
51
How are SCP’s made ?
- A single celled fungus is grown in large fermenters using glucose syrup as a food source
- Microorganisms are combined with albumen (egg white) and then compressed and formed into meat substitutes.
52
How else can protein be made ?
Yeasts, algae, and bacteria can be used to grow proteins that match animal proteins found in meat as well as plant proteins.
53
What are some advantages of using yeast, bacteria and algae to form protein ?
- Can be grown on almost anything, are relatively cheap and low in fat
- BUT none of the alternative protein sources has proved successful so far.
54
What are the advantages of using microorganisms to produce human food ?
-
- Microorganisms reproduce fast and produce protein faster than animals
and plants
- Microorganisms have a high protein content with little fat
- Microorganisms can use a wide variety of waste materials including human and animal waste, reducing costs
- Microorganisms can be genetically modified to produce the protein
required
- Production of microorganisms is not dependant on weather, breeding cycles etc - it takes place constantly and can be increased or decreased to match demand
- No welfare issues when growing microorganisms
- Can be made to taste like anything
55
What are the disadvantages of using microorganisms to produce human food ?
- Some microorganisms can also produce toxins if the conditions are not maintained at the optimum
- The microorganisms have to be separated from the nutrient broth and processed to make the food
- Need sterile conditions that are carefully controlled adding to costs
- Often involve GM organisms and many people have concerns about eating GM food
- The 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
56
What was the first ever effective antibiotic ?
Penicillin
57
What are the two stages of producing penicillin ?
- First stage of the production process allows the fungus to grow
- Second stage is when the penicillin is produced
- finally the drug is extracted from the medium and purified
58
What conditions does P.chrysogenum need to grow well ?
- High oxygen levels
- Rich nutrient medium to grow well
- Sensitive to pH and temperature
59
What are the key points regarding the production of penicillin ?
- Process uses relatively small fermenters because it is very difficult to maintain high levels of oxygenation in very large bioreactors
- The mixture is continuously stirred to keep it oxygenated
- There is a rich nutrient medium
- The growth medium contains a buffer to maintain the pH around 6.5
- Bioreactors are maintained at about 25-27 degrees
60
How was insulin produced back in the day ?
- Extracted from the pancreas of animals
- Supply was erratic as it depended on the demand for meat - fewer animals killed meant less insulin was available
61
What were the problems regarding how insulin was initially produced ?
- Some people were allergic to the animal insulin
- Peak activity of animal insulin is several hours after injecting, made it difficult to calculate when to eat meals
- Using pig products is not permitted in some religions
62
How is insulin produced now ?
Bacteria are grown in a fermenter and downstream processing results in a constant supply of pure human insulin
63
What is bioremediation ?
Microorganisms being used to break down pollutants and contaminants in the soil or in water
64
What are the two different approaches to bioremediation ?
- Using natural organisms
- Using GM organisms
65
How are natural organisms used for bioremediation ?
- Many microorganisms naturally break down organic material producing CO2 and H2O
- If these microorganisms are supported, they will break down and neutralise many contaminants
66
How are GM organisms used for bioremediation ?
Scientists are trying to develop GM bacteria which can break down or accumulate contaminants which they would not naturally encounter
67
Give an example of GM organisms being used for bioremediation
- Bacteria have been engineered that can remove mercury from water
- Mercury is toxic and accumulates in food chains
- The aim is to develop filters containing these bacteria to remove mercury from contaminated sites
68
Where does bioremediation normally take place ?
At the site of contamination but sometimes material is removed for decontamination
69
What normally works better, natural organisms or GM organisms ?
- Natural organisms outperform GM ones
- May be possible to use bio remediation more effectively in the future
70
Why must health and safety procedures be followed when culturing microorganisms ?
- There is always the risk of a mutation taking place making the strain pathogenic
- There may be contamination with pathogenic microorganisms from the environment
71
What do microorganisms need to be cultured ?
- Food
- Correct temperature, oxygen and pH
72
What is nutrient medium ?
- The food provided for the microorganisms
- Can be in liquid form or solid form (broth or agar)
73
Why are nutrients added to the agar ?
To provide a better medium for microbial growth
74
What is the medium normally enriched with ?
- Blood, yeast extract or meat
- These are all good protein sources
75
What do enriched nutrient media allow for ?
Allows for samples containing a very small number of organisms to multiply rapidly
76
What condition must the nutrient medium be kept in ?
It must be kept sterile
77
What is inoculation ?
Adding the bacteria to the nutrient
78
How do you inoculate broth ?
- Make a suspension of the bacteria to be grown
- Mi a known volume with the sterile nutrient in the flask
- Stopper the flask to prevent contamination from the air
- Incubate at a suitable temperature, shaking regularly to aerate the broth
79
How do you inoculate agar ?
- The wire inoculating loop must be sterilised by holding it in a Bunsen flame until it glows red hot.
- It must not be allowed to touch any surfaces as it cools to avoid contamination.
- Dip the sterilised loop in the bacterial suspension. Remove the lid of the Petri dish and make a zig-zag streak across the surface of the agar.
- Replace the lid of the Petri dish. It should be held down with tape but not sealed completely so oxygen can get in, preventing the growth of anaerobic bacteria.
- Incubate at a suitable temperature
80
What are the four stages to the bacteria growth curve ?
- Lag phase
- Exponential phase
- Stationary phase
- Death phase
81
Lag phase of bacteria growth curve
- When bacteria are adapting to their new environment
- They are growing, synthesising the enzymes they need and are not yet reproducing at their max rate
82
Exponential phase of bacteria growth curve
This is when the rate of bacterial reproduction is close to or at its theoretical max
83
Stationary phase of bacteria growth curve
Occurs when the total growth rate is zero
84
Death phase of bacteria growth curve
Comes when reproduction has almost ceased and the death rate of the cells is increasing
85
What are the limiting factors which prevent exponential growth ?
- Nutrients available
- Oxygen levels
- Temperature
- Build up of waste
- Change in pH
86
How can the nutrients available be a limiting factor preventing exponential growth ?
- As the numbers of microorganisms grows, the nutrient medium is used up
- It will then become insufficient to support further growth
87
How are the oxygen levels a limiting factor preventing exponential growth ?
As the population grows, the demand for oxygen also increases, so the oxygen levels can become limiting
88
How is temperature a limiting factor preventing exponential growth ?
If the temperature gets too high it will denature the enzymes and kill the microorganisms
89
How is a build up of waste a limiting factor preventing exponential growth ?
The build up of toxic material may inhibit further growth and can even kill the culture via poisoning
90
How is a change in pH a limiting factor preventing exponential growth ?
As CO2 produced increases, pH of the culture falls until a point where the low pH affects enzyme activity and inhibits population growth
91
What are primary metabolites ?
Substances that are wanted which are formed as an essential part of the functioning of a microorganism
92
What are secondary metabolites ?
- Substances that an organism produces which are not essential for normal growth but are still used by the cells
- The organism will not suffer in the short run without them
93
What are secondary metabolites known to be ?
- The required product in a bioprocess
- e.g. penicillin
94
What are the main ways of growing microorganisms ?
- Batch fermentation
- Continuous fermentation
95
How does batch fermentation take place ?
- The microorganisms are inoculated into a fixed volume of medium.
- As growth takes place, nutrients are used up and both new biomass and waste products build up
- As the culture reaches the stationary phase, overall growth ceases but during this phase the microorganisms often carry out biochemical changes to form the desired end products
- The process is stopped before the death phase and the products harvested
96
How does continuous fermentation take place ?
- Microorganisms are inoculated into sterile nutrient medium and start to grow.
- Sterile nutrient medium is added continually to the culture once it reaches the exponential point of growth.
- Culture broth is continually removed - the medium, waste products, microorganisms, and product - keeping the culture volume in the bioreactor constant
97
What does continuous culture allow to happen ?
It enables continuous and balanced growth, with constant levels of nutrients, pH and metabolic products
98
Where is continuous culture usually used ?
Continuous cultivation is largely used for the production of single-celled protein and in some waste water treatment processes.
99
What can be maximised when growing microorganisms ?
Maximum production of biomass or the maximum production of the primary or secondary metabolites.
100
What are most systems adapted for ?
Maximum yield of metabolites
101
What does a bioreactor produce a mixture of ?
Nutrient broth, microorganisms, primary metabolites, possibly secondary metabolites, and waste products.
102
What must happen for the useful part of the bioreactor mixture to be separated ?
- Downstream processing
- Most difficult and expensive part of the whole process
103
What factors must be controlled when using a bioreactor ?
- Temperature
- Nutrients and oxygen
- Mixing things up
- Asepsis
104
What happens when the temperature of the bioreactor is too low ?
Microorganisms will not grow quickly enough.
105
What happens when the temperature of the bioreactor is too high ?
Enzymes start to denature and the microorganisms are inhibited or destroyed.
106
How do bioreactors maintain optimum temperature ?
They often have a heating and/or a cooling system linked to temperature sensors and a negative feedback system
107
How can the amount of nutrients and oxygen be controlled in a bioreactor ?
Can be added in controlled amounts to the broth when probes or sample tests indicate that levels are dropping.
108
Why do bioreactors have a mixing mechanism and continuously stir the mixture ?
- To ensure that the microorganisms receive enough food and oxygen
- To ensure that the whole mixture is kept at the right temperature
109
Why are most bioreactors sealed aseptic units ?
- To stop the bioprocess from being contaminated by microorganisms from the air or from workers
- This is because it can seriously affect the yield
110
Why are most bioreactors sealed aseptic units ?
- To stop the bioprocess from being contaminated by microorganisms from the air or from workers
- This is because it can seriously affect the yield
111
What are some advantages of using isolated enzymes rather than whole organisms in bioprocesses ?
- They are less wasteful
- More efficient - work at much higher concentrations
- More specific - no unwanted enzymes are present
- Maximise efficiency - Isolated enzymes can be given ideal conditions for maximum product formation
- Less downstream processing - pure product is produced
112
What are isolated enzymes an example of ?
Extracellular enzymes produced by microorganisms
113
What are isolated enzymes said to be compared to intracellular enzymes ?
Easier and cheaper to use than intracellular enzymes
114
What are some benefits of using extracellular enzymes rather than intracellular enzymes ?
- They are easier to isolate as they are secreted
- Each microorganism produces relatively few extracellular enzymes, making it easy to identify and isolate the required enzyme
- They tend to be more robust compared to intracellular enzymes, they are able to adapt to cope with greater variations in temperature and pH
115
Why can intracellular enzymes sometimes be used instead of extracellular enzymes ?
There is a bigger range of intracellular enzymes so in some cases they provide the ideal enzyme for the process
116
Give an example of intracellular enzymes being used for isolated enzymes
- Glucose oxidase for food preservation
- Penicillin acylase for converting natural penicillin into semi synthetic drugs
117
What type of enzymes are being more used nowadays over isolated enzymes ?
Immobilised enzymes
118
What is an immobilised enzyme ?
Enzyme that is attached to an inert support system over which the substrate passes and is converted to a product
119
Why can immobilised enzymes be recovered after being used in the reaction procedure ?
Because they are held stationary during the catalytic processes
120
Is downstream processing required for immobilised enzymes ?
Less downstream processing is needed because the enzymes do not contaminate the end product
121
What are the advantages of using immobilised enzymes ?
- They can be reused which is cheaper
- Easily separated from the reactants and products which is cheaper
- More reliable as there is a high degree of control over the process
- Greater temperature tolerance
- Ease of manipulation - the catalytic properties of the enzymes can be altered to fit a particular process
122
What are the disadvantages of using immobilised enzymes ?
- Reduced efficiency - the process of immobilising an enzyme may reduce its activity rate
- Higher initial costs of materials - immobilised enzymes are more expensive than free enzymes or microorganisms
- Higher initial costs of bioreactor
- More technical issues
123
What are the advantages of whole organisms being immobilised rather than just the enzymes ?
Avoids the expensive and time consuming process of extracting the pure enzyme and immobilising it before the process starts
124
What are the disadvantages of whole organisms being immobilised rather than just the enzymes ?
Whole organism needs food and oxygen, and a carefully controlled environment to work at their optimum rate
125
What are some examples of immobilised enzymes being used ?
- Immobilised penicillin acylase used to make semi synthetic penicillins from naturally produced penicillins
- Immobilised glucose isomerase used to produce fructose from glucose
- Immobilised lactase used to produce lactose-free milk
- Immobilised glucoamylase, which can be used to complete the breakdown of starch to glucose syrup.
- Immobilised aminoacylase used to produce pure samples of L-amino acids used in the production of pharmaceuticals, organic chemicals, cosmetics, and food
- Immobilised nitrile hydratase, an enzyme which is playing an increasing role in the plastics industry.
126
Immobilised penicillin acylase used to make semi synthetic penicillins from naturally produced penicillins
- Many types of bacteria have developed resistance to naturally occurring penicillins so they are no longer very effective drugs.
- Fortunately, many bacteria are still vulnerable to the semi-synthetic penicillins produced by penicillin acylase so they are very important in treating infections caused by bacteria resistant to the original penicillin.
127
Immobilised glucose isomerase used to produce fructose from glucose
Glucose is produced from cheap, starch-rich plant material. Glucose isomerise is then used to turn the cheap glucose into very marketable fructose
128
Immobilised lactase used to produce lactose-free milk
- Some people are intolerant of lactose (milk sugar).
- Immobilised lactase hydrolyses lactose to glucose and galactose, giving lactose-free milk.
129
Immobilised glucoamylase, which can be used to complete the breakdown of starch to glucose syrup.
Amylase enzymes break starch down into short chain polymers called dextrins. The final breakdown of dextrins to glucose is catalysed by immobilised glucoamylase.
130
Immobilised nitrile hydratase, an enzyme which is playing an increasing role in the plastics industry.
- Acrylamide is a very important compound which is used in the production of many plastics. It is made by the hydration of acrylonitrile.
- Traditionally the hydration of acrylonitrile to acrylamide was done using sulphuric acid with a reduced copper catalyst, but the conditions needed are extreme and therefore expensive.
- Furthermore, unwanted by-products form and the yield is poor.
- Using immobilised nitrile hydratase the conversion takes place under moderate conditions so the process is cheaper and it also gives a 99% yield and no unwanted by-products.
