Module 6: Cloning and Biotechnology

Question 1

What is Biotechnology?

Answer 1

It is the industrial use of living organisms to produce food, drugs and other products.

Question 2

Why are microorganisms mostly used for biotechnology?

Answer 2

• Ideal growth conditions can be easily created.
• Due to their short life cycle, they grow rapidly, so products can be made quickly.
• Can be grown at any time of the year.
• Can grown on a range on inexpensive materials - makes them economical to use.

Question 3

Describe the types of enzymes used in Biotechnology

Answer 3

Intracellular enzymes: enzymes that are contained within the cells of microorganisms.

Extracellular enzymes: secreted naturally by microorganisms (more cheaper), but others have to be extracted,

Isolated enzymes: enzymes that are not contained within cells.

Question 4

Give an example of an enzymes used in Biotechnology.

Answer 4

Lactase. It is prepared from Aspergillus fungi

-Breaks down lactose.

• Used in the production of lactose- free products.

Question 5

What is the use of microorganisms in biotechnology when making beer? (Brewing)

Answer 5

Yeast is added to a type of grain (barley) and other ingredients.

The yeast respires anaerobically using the glucose from the grain and produces ethanol (alcohol) and CO2.

Process of fermentation.

Question 6

What is the use of microorganisms in biotechnology when baking?

Answer 6

Yeast makes bread rise.

The CO2 produced from fermentation of sugars in the dough makes sure it doesn’t stay flat.

flat breads, tortillas are made without yeast.

Question 7

What is the use of microorganisms in biotechnology when cheese making?

Answer 7

Cheese production relies on a substance called rennet - contains the enzymes ‘chymosin’ which clots the milk.

Chymosin was obtained by extracting rennet from the lining of calves’ stomach.

it is now obtained from yeast cells that have been genetically modified to produce chymosin.

Cheese making involves lactic acid bacteria. This bacteria converts the lactose in milk into lactic acid, which makes it sour and solidifies it.

Production of blue cheese involves the addition of fungi - creates blue veins.

Question 8

What is the use of microorganisms in biotechnology regarding yogurt production?

Answer 8

Involves the use of lactic acid bacteria to clot the milk and cause it to thicken.

Flavours and colours are then added.

Question 9

What is the use of microorganisms in biotechnology regarding penicillin production?

Answer 9

The fungi (from penicillium genus) produce the antibiotic penicillin to stop bacteria from growing and competing for resources.

• Most common antibiotic = produced on a massive scale.

The fungus is grown under stress in industrial fermenters.

Penicillin is collected and processed to be used in medicine.

Question 10

What is the use of microorganisms in biotechnology regarding insulin production?

Answer 10

Is made by genetically modified bacteria, which have had the gene for human insulin production inserted into their DNA.

Their bacteria are grown in an industrial fermenter on a massive scale and the insulin produced is collected and purified.

Question 11

What is the use of microorganisms in biotechnology regarding Bioremediation?

Answer 11

The process of using microorganisms to remove pollutants - oils and pesticides- from contaminated sites.

Pollutant- removing bacteria that occur naturally at a site are provided with extra nutrients and enhanced growing conditions to allow them to multiply and thrive.

These bacteria break down the pollutants into less harmful products, cleaning up the area.

Question 12

What are the advantages of using microorganisms in food production?

Answer 12

-Can be grown quickly, easily, and cheaply.

-Production costs are low as they have simple growth requirements.

-Can be grown on waste products and less land is required in comparison to growing crops/rearing livestock.

-They can be cultures anywhere with the right equipment - this means that a food source can be readily produced in places where growing crops/rearing livestock is difficult. (hot/cold climates). This could help with malnutrition in developing countries.

-Production of single-cell protein could be used as a way of getting rid of waste products.

-Single-celled protein is often considered a healthier alternative to animal protein.

Question 13

What are the disadvantages of using microorganisms in food production?

Answer 13

• Lots of effort has to go into making sure that the food doesn’t get contaminated with unwanted bacteria - could be dangerous to humans or spoil the food.

-People may not like the idea of eating food that has been grown using waste products.

-Single-cell protein doesn’t have the same texture or flavour as real meat.

-If single-cell protein is consumed in high quantities, health problems could be caused due to high levels of uric acid released when the large amounts of amino acids are broken down.

Question 14

What is a culture?

Answer 14

It is a population of one type of microorganism that has been grown under controlled conditions.

They are grown in large containers called fermentation vessels.

Question 15

How do industrial fermenters vessels work?

Answer 15

The fermenters are made from stainless steel (which does not corrode) and are sterilised using hot steam to remove any contaminating microorganisms which may kill or compete the bacteria or fungi.

The air inlet provides a supply of oxygen to allow the microorganisms to respire aerobically and paddles to distribute the nutrients and oxygen evenly.

The temperature can be carefully controlled using a thermostat and a water jacket.

pH is also kept constant using a pH sensor which ensures that the pH is at an optimum value for enzymes within bacteria and fungi to function efficiently.

Question 16

What are cultures grown in fermentation vessels?

Answer 16

1) To obtain lots of microorganisms (for the production of single celled protein)

2) To collect lots of a useful product that microorganism makes.

Question 17

What are the 2 main methods for culturing microorganisms?

Answer 17

Batch fermentation

Continuous fermentation.

Question 18

What is Batch fermentation?

Answer 18

This is where microorganisms are grown in individual batches in a fermentation vessel.

When one culture ends it is removed and then a different batch of microorganism is grown in the vessel - closed culture.

Question 19

What is continuous fermentation?

Answer 19

This is where microorganisms are continually grown in a fermentation vessel without stopping.

Nutrients are put in and waste products taken out at a constant rate.

Question 20

Why must the conditions inside the fermentation vessels be kept at optimum for growth?

Answer 20

As this maximises the yield of microorganisms and desirable products.

Question 21

What are the 5 main factors that need to be controlled in a fermentation vessel?

Answer 21

pH

Temperature

oxygen supply

Contamination

Nutrient concentration

Question 22

Why/how must pH be controlled in a fermentation vessel?

Answer 22

The pH is monitored by a pH probe and is kept at the optimum level.

This increases the product yield as enzymes can work efficiently - the rate of reaction is kept as high as possible.

Question 23

Why/how must temperature be controlled in a fermentation vessel?

Answer 23

Temperature is kept at the optimum level by a water jacket that surround the vessel.

This increases the product yield as enzymes can work efficiently - rate of reaction is kept as high as possible.

Question 24

Why/how must oxygen supply be controlled in a fermentation vessel?

Answer 24

The volume of oxygen is kept at the optimum level for respiration by pumping in sterile air when needed.

This increases the product yield because microorganisms can always respire to provide the energy for growth.

Question 25

Why/how must nutrient concentration be controlled in a fermentation vessel

Answer 25

Microorganisms are kept in contact with fresh medium by paddles that circulate the medium around the vessel. This increases product yield because microorganisms can always access the nutrients needed for growth.

Question 26

Why/how must contamination be controlled in a fermentation vessel

Answer 26

Vessels are sterilised between uses with the superheated steam to kill any unwanted organism and make sure the next culture isn't contaminated. This increases the product yield as the microorganisms are not competing with other organisms.

Question 27

What is a closed culture and give an example of when it is used?

Answer 27

This is when growth takes place in a vessel that is isolated from the external environment - extra nutrients are not added and waste products are not removed from the vessel during growth. E.g. Batch Fermentation.

Question 28

What are the 4 phases from a standard growth curve, in a closed culture.

Answer 28

1. Lag phase 2. Exponential (log) phase. 3. Stationary phase 4. Decline phase

Question 29

What happens in the Lag phase?

Answer 29

The population size increases very slowly as the microorganisms have to make enzymes and other molecules before they can reproduce. This means the reproduction rate is low.

Question 30

What happens in the Exponential (log) phase?

Answer 30

The population size increases quickly because the culture conditions are at their most favourable for reproduction - lots of food and little competition. The number of microorganisms doubles at regular intervals.

Question 31

What happens in the stationary phase?

Answer 31

The population size stays the same - death rate of the microorganisms equals their reproductive rate. Microorganisms die because there is not enough food and poisonous waste products build up.

Question 32

What happens in the decline phase?

Answer 32

The population size falls because the death rate is greater than the reproductive rate. This is because food is very scarce and waste products are at toxic levels

Question 33

How can you work out how many cells are present in a population?

Answer 33

Use this equation: N= N0 x 2n N0= is the initial number of cells. 2n= n is the number of divisions.

Question 34

How are cultures of microorganisms grown in the lab? (practical)

Answer 34

Use a agar plate- a sterile Petri dish containing agar jelly. Microorganism = liquid broth (a mixture of distilled water and nutrients). To culture the microorganisms, use a sterile implement like a wire inoculation loop to transfer some of the sample to the plate. A pipette could also be used to transfer the sample, which could then be gently spread across the whole surface of the agar using a glass or a plastic spreader (which is sterilised by using a Bunsen burner) The plates then need to be incubated at 25 degrees to allow the microorganisms to grow.

Question 35

Why are aseptic techniques used?

Answer 35

They are used to prevent contamination of cultures by unwanted microorganisms, which may affect the growth of the microorganism being cultured.

Question 36

What do contaminated cultures result in?

Answer 36

They result in imprecise results and may be hazardous to health. Contamination on a industrial scale may be very costly because entire cultures may have to be thrown away.

Question 37

What are some important aseptic techniques?

Answer 37

1. Regularly disinfect work surfaces to minimise contamination. 2. Work near a Bunsen flame. Hot air rises, so any microorganism in the air should be drawn away from your culture. 3. Sterilise the apparatus used to transfer cultures before and after each use - passing it through a Bunsen burner for 5 seconds. This will kill any microorganisms. 4. If using broth, briefly pass the neck of the broth container through a Bunsen burner flame just after it is opened and before it is closed- this causes air to move out of the container, preventing unwanted organisms from falling in. 5. Minimise the time that the agar plate is open and put the lid on as soon as possible. This reduces the chance of airborne microorganisms contaminating the culture - could even work in an inoculation cabinet. 6.Sterilise all glassware before and after use using an autoclave - a machine that steams equipment at a high pressure. 7. Wear lab coat, googles and tie long hair back.

Question 38

Describe the practical investigating the factors that affect microorganism growth for temperature.

Answer 38

Use a sterile pipette to transfer a fixed volume of bacterial broth to six agar plates. Use a sterile plastic spreader to spread the broth across the entire surface of the agar. Put the lid on the agar plates and lightly tape them shut. Incubate half of the plates in the fridge (at 4oC) and the other half in a 25oC incubator (or left out at room temperature). Place the plate upside down to prevent condensation from dropping onto the agar. Place another agar plate (without bacteria) in the fridge, and another in the warm incubator. These are your negative controls – any bacteria found growing on them indicates possible contamination. Incubate the plates for a set time period (e.g. 48 hours). You should see bacterial colonies on the six plates (but not the two controls). Count the number of colonies formed on each plate and calculate the mean number of colonies at each temperature. You’d expect to find a higher mean number of colonies on the plates incubated at 25oC.

Question 39

How can you investigate the effect of pH and nutrients on the growth of microorganism?

Answer 39

You can investigate the pH by adding buffers at different pH levels to the broth You can investigate the effect of nutrient availability by using different preparations of agar, which contain different nutrients.

Question 40

How could you investigate the growth of microorganisms directly in the broth?

Answer 40

You could use a spectrophotometer. This is a machine that measures the turbidity (cloudiness) of the broth. Higher turbidity means that more cells are present, and therefore, more replication has taken place.

Question 41

Why are immobilised enzymes used in industry?

Answer 41

Isolated enzymes used in industry can become mixed with the products of a reaction. The products need to be separated from this mixture, which can be costly and complicated. This is avoided in a large-scale production by using immobilised enzymes- are enzymes that are attached to an insoluble material so they cannot be mixed with the products.

Question 42

What are the 3 main ways that enzymes are immobilised?

Answer 42

1. They are ENCAPSULATED in jelly-like alginate beads, which acts as a semi-permeable membrane. 2. ENTRAPPED in a silica gel matrix. 3. COVALENTLY BONDED to cellulose or collagen fibres

Question 43

How do immobilised enzymes work in industry?

Answer 43

In industry, the substrate solution for a reaction is run through a column of immobilised enzymes. The active sites of the enzymes are still available to catalyse the reaction but the solution flowing out of the column will only contain the desired product.

Question 44

What are the advantages of using immobilised enzymes in industry?

Answer 44

1. Columns of immobilised enzymes can be washed and reused - this reduces the cost on a industrial scale as you don't need to keep buying new enzymes. 2. The product is not mixed with the enzymes - no money or time is spent separating them out. 3. They are more stable than free enzymes - they are less likely to denature in high temperatures or extremes of pH.

Question 45

What are the disadvantages of using immobilised enzymes in industry?

Answer 45

1. Extra equipment is required- can be expensive to buy. 2. Immobilised enzymes are more expensive to buy than free enzymes. So the sum of costs including equipment suggests that they are not always economical for use in smaller scale production. 3. The immobilisation of the enzymes can sometimes lead to a reduction in the enzyme activity because they cannot freely mix with their substrate. (reaction rates are lower)

Question 46

Explain the conversion of lactose to glucose and galactose using immobilised enzymes.

Answer 46

Some peoples are unable to digest lactose as they don't produce enough/any of the enzyme lactase. Lactase breaks down lactose down into glucose and galactose via a hydrolysis reaction. Industrially, fresh milk can now be passed over immobilised lactase to produce lactose-free milk for the use in the production of lactose-free dairy products.

Question 47

Production of semi-synthetic penicillin using immobilised enzymes.

Answer 47

Some bacteria have become penicillin resistant. Semi- synthetic penicillin can now be produced, which have the same antibiotic properties as natural penicillin, but are effective against penicillin-resistant organisms. Immobilised penicillin acylase enzyme is used in their production.

Question 48

Conversion of dextrins to glucose using immobilised enzymes.

Answer 48

Glucose can be derived from starchy foods, such as corn and potatoes, with the help of immobilised enzymes. Starch breaks down into dextrins (carbohydrate products), which are then broken down into glucose by the immobilised enzyme glucoamylase.

Question 49

Conversion of glucose to fructose using immobilised enzymes.

Answer 49

fructose is used as a sweetener in food - using fructose rather than glucose means that less sugar is needed to obtain the same level of sweetness in our foods. Immobilised glucose isomerase is used to convert glucose to fructose on an industrial scale.

Question 50

What is cloning?

Answer 50

It is the process of producing genetically identical cells or organisms from the cells of an existing organism. - Can occur naturally or artificially.

Question 51

What is Vegetative propagation?

Answer 51

This is the production of plant clones from non-reproductive tissues (natural) E.g. - roots, leaves and stems. It is a type of asexual reproduction

Question 52

How do Rhizomes produce natural clones by vegetative propagation?

Answer 52

Rhizomes - are stem structures that grow horizontally underground away from the parent plant. They have 'nodes' from which new shoots and roots can develop. Example - bamboo.

Question 53

How do Runners produce natural clones by vegetative propagation?

Answer 53

Also called Stolons. They grow above the ground, on the surface of the soil horizontally. New shoots and roots can either develop from nodes or form at the end of the runner. Example - strawberry.

Question 54

How do Suckers produce natural clones by vegetative propagation?

Answer 54

These are shoots that grow from sucker buds, which are undeveloped shoots that are present on the shallow roots of a parent plant. Example - elm tree.

Question 55

How do Tubers produce natural clones by vegetative propagation?

Answer 55

These are large underground plant structures (swollen stem) that act as a food store for the plant. Buds form on the surface on the tubers. Buds turn into plantlets Each plantlet is able to sprout and form a new plant. Example - potato.

Question 56

How do Bulbs produce natural clones by vegetative propagation?

Answer 56

They are underground food stores used by some plants. New bulbs are able to develop from the original bulb and form new individual plants. This is because withing the bulb, buds are formed that allow new stem to grow. Example - onion.

Question 57

How do Horticulturists induce vegetative propagation to produce natural clones of the parent plant?

Answer 57

1. They can take cuttings. 2. They can use grafting - joining the shoot of one plant to the growing stem and root of another plant. 3. They can use layering - bending a stem of a growing plant downwards so it enters the soil and grows into a new plant.

Question 58

Explain the procedure on how farmers would use cutting to produce natural clones. (practical)

Answer 58

Use a scalpel to cut a shoot (5-10 cm) from the parent plant. Remove the leaves from the lower end of the shoot but leave one at the shoot tip. Dip the end of the shoot in rooting powder – this contains hormones which promote root growth. Plant the cutting in compost and keep warm (either by placing in a propagator or by covering the pot with a plastic bag). After a few weeks of growth, the cutting is ready to plant. It will be genetically identical to the parent plant.

Question 59

What is tissue culture?

Answer 59

It is an artificial way of cloning plants.

Question 60

How is tissue culture carried out?

Answer 60

Using a sterile scalpel, scrape off some cells from the parent plant. You’ll want to do this from the stem and root tips (meristems) as this is where the stem cells are found. (tissue samples are scraped) Cells are sterilised to kill any microorganisms, such as bacteria or fungi, which would compete with the plant and reduce its growth. Cells are transferred to a culture medium containing growth hormones (e.g. auxin) and organic nutrients (e.g. glucose and amino acids). This is carried out under aseptic conditions. The cells divide to produce a mass of undifferentiated cells. Once the cells have grown into a small plant, they are planted in compost. They will all be clones (genetically identical) to the parent plant.

Question 61

Why is tissue culture used? Give an example.

Answer 61

It is used to clone plants that do not readily reproduce or are endangered or rare. It is also used to grow whole pants from genetically engineered plant cells. E.g. British orchid species are now endangered in the UK. It is very difficult to reproduce orchids using seeds as it takes a long time for plants to produce flowers - they have a specialised mechanism of pollination where seeds require a fungus in order to germinates. Many have been successfully reproduced using tissue culture.

Question 62

What is Micropropagation?

Answer 62

This is when tissue culture is used to produce lots of cloned plants very quickly. The cells are taken from developing cloned plants and are grown on another fresh medium culture (subcultured) - repeating this process creates large numbers of clones. This technique is used extensively in horticulture and agriculture - to produce fields full of crops that have been genetically engineered to be pest-resistant.

Question 63

What are the arguments for artificial plant cloning?

Answer 63

1. Desirable genetic characteristics are always passed on to clones - this doesn't always happen when plants reproduce sexually. 2. Less space is required by tissue culture than would be needed to produce the same number of plants by conventional growing methods. 3.Tissue culture allows plants to be reproduced in any season as the environment is controlled. 4. It produces lots of plants quickly compared to the time it would take to grow them from seeds.

Question 64

What are the arguments against artificial plant cloning?

Answer 64

1. Undesirable genetic characteristics such as producing fruits with lots of seeds, are always passed onto clones. 2. cloned plant populations have no genetic variability meaning that a single disease could kill them all. 3. Production costs of tissue cultures are very high due to high energy use and the training of skilled workers, so therefore it is unsuitable for small scale production. 4. Contamination by microorganisms during tissue cultures can be disastrous and result in complete loss of plants being cultured.

Question 65

How can animal clones be produce naturally?

Answer 65

During sexual reproduction - once the egg has been fertilised, it can split and develop into multiple embryos with the same genetic information. The embryo can develop as normal to produce offspring that are all genetically identical - identical twins.

Question 66

What are the 2 main ways animals clones can be made artificially?

Answer 66

1. Artificial embryo twinning. 2. Somatic cell nuclear transfer methods.

Question 67

Describe how artificial embryo twinning is carried out.

Answer 67

An egg cell is extracted from a female animal and fertilised in vitro (i.e. by mixing the egg and sperm in a Petri dish). The zygote divides to form an embryo, from which individual cells are removed and placed into a separate Petri dish. Each cell is left to grow and form an embryo, which can be implanted into surrogate mothers to produce multiple clones. The embryos continue to develop inside the surrogate cows, and eventually the offspring are born. They are all genetically identical to each other.

Question 68

Describe how Somatic cell nuclear transfer (SCNT) is carried out to produce animal clones.

Answer 68

It involves taking a somatic cell (any cell that is not a sex cell) from an animal and extracting the nucleus. We then take an oocyte (an immature egg cell) from another animal and discard the nucleus to form what’s called an enucleated oocyte. The nucleus from the somatic cell is inserted into the enucleated oocyte. They’re fused together using a technique called electrofusion, which involves applying an electric current. This also stimulates the egg to divide, forming an embryo. The embryo is implanted into a surrogate mother and eventually the animal born is a clone of the animal which provided the nucleus.

Question 69

Why is animal cloning used/carried out?

Answer 69

1. Scientists use cloned animals for research purposes - they can test new drugs on cloned animals. They are all genetically identical, so the variables that come from genetic differences (e.g. the likelihood of developing cancer) are removed. 2. Can be used in agricultures where farmers can increases the number of animals with desirable characteristics to breed from. For example a cow with high milk production could be cloned. 3. Cloning could be used to save endangered animals from extinction by cloning new individuals. 4. Some scientists only want cloned embryonic stem cells - they can be used to replace damaged tissues in a range of diseases. For examples - heart diseases, spinal cord injuries and Parkinson's disease. If replacement tissue is made from cloned embryonic stem cells that are genetically identical to the patient's own cells, it wont be rejected by their immune system.

Question 70

What are the arguments for animal cloning?

Answer 70

1. Desirable characteristics are always passed on to clones. 2. Infertile animals can be reproduced via cloning 3. Animals can be cloned at any time - no need to wait until a breeding season to get new animals. 4. Increasing the population of endangered species helps preserve biodiversity. 5. Cloning can help us develop new treatments for disease, which could mean less suffering for some people.

Question 71

What are some arguments against animal cloning?

Answer 71

1. It is very difficult, time-consuming and expensive. 2. There's no genetic variability in cloned populations, so undesirable genetic characteristics (weak immune system) are always passed on to clones. A single disease could kill them all. 3. Clones may not live as long as natural offspring - unethical. Dolly died as the age of 6 after developing a lung disease and arthritis. 4. Using cloned human embryos as a source of stem cells is controversial. The embryos are destroyed after obtaining the stem cells - some people believe that doing this is destroying a human life.