StemUp: OCR A A level Biology 6.2.1 Cloning and biotechnology

What is cloning? (2)

- The process of producing genetically identical cells or organisms from the cells of an existing organism

- Can occur naturally in plants/animals or be carried out artificially

What is vegetative propagation? (1)

The production of plant clones from non-reproductive tissues (e.g. roots, stems, leaves)

What are rhizomes? (3)

- Stem structures that grow horizontally underground, away from the parent plant.

- Have nodes from which new shoots and roots can develop

- Example plant: Bamboo

What are stolons/runners? (3)

- Similar to rhizomes, but they grow above ground on the surface of the soil

- New roots and shoots can develop from the nodes or form at the end of the stolon.

- Example plant: Strawberries

What are suckers? (2)

- Shoots that grow from sucker buds (undeveloped shoots) present on the shallow roots of a parent plant

- Example plant: Elm trees

What are tubers? (3)

- Large underground plant structures that act as a food store for the plant

- Covered in eyes, each eye can sprout and form a new plant

- Example plant: Potatoes

What are bulbs? (3)

- Underground food stores used by some plants

- New bulbs can develop from the original bulb to form new individual plants

- Example plant: Onions

What are some other methods of vegetative propagation used by horticulturists to produce clones? (3)

- Taking cuttings

- Grafting (joining the shoot of one plant to the growing stem and root of another plant)

- Layering (bending the stem of a growing plant downwards so it enters the soil and grows into a new plant)

What are the steps for taking and growing a cutting from a stem? (6)

1. Use a scalpel to take a cutting, 5cm - 10cm long, from the end of a stem of the parent plant

2. Remove leaves from the lower end, leaving just one leaf at the tip

3. Dip the lower end in rooting powder (contains auxins to induce root formation)

4. Plant the cutting in a pot containing a suitable growth medium, e.g. well-drained compost

5. Provide a warm, moist environment

6. Once the cutting has formed roots and is strong enough, it can be planted elsewhere

How can you provide a warm and moist environment when taking and growing a cutting from a stem? (2)

- Putting it in a propagator

- Covering the pot with a plastic bag

What are the steps for taking and growing a cutting from a root? (5)

1. Cut a piece of root from the plant using a scalpel

2. Remove the uncut end of the root with a slanted cut

3. Dip the end of the root in rooting powder

4. Plant the cutting in a suitable growth medium

5. Provide a warm, moist environment (bag or propagator)

What are the steps for taking and growing a cutting from a leaf (split vein cutting)? (4)

1. Remove a complete leaf and score the large veins on the lower leaf surface using a scalpel

2. Place the leaf on top of the growth medium with the broken veins facing down

3. Provide a warm, moist environment (bag or propagator)

4. New plants will form from each break in the veins

What is tissue culture and why is it used? (2)

- Used to clone plants that don't readily reproduce or are endangered/rare (e.g. British orchids)

- Also used to grow whole plants from genetically engineered plant cells

What are the steps for micropropagation and tissue culture? (6)

1. Cells are taken from the original plant

2. Cells from the stem and root tips are used because they are stem cells

3. Cells are sterilised to kill microorganisms (bacteria and fungi)

4. Cells are placed on a culture medium containing nutrients (e.g. glucose) and growth hormones (e.g. auxins)

5. Once the cells have divided and grown into a small plant, they are removed from the medium and planted in soil

6. The plants develop into genetically identical clones of the original plant.

Describe micropropagation? (4)

- Micropropagation is when tissue culture is used to produce large numbers of cloned plants very quickly

- Cells are taken from developing cloned plants and subcultured (grown on fresh culture medium).

- Repeating this process creates large numbers of clones

- Used in horticulture and agriculture to produce crops (e.g. pest-resistant varieties) quickly

What are the arguments for artificial cloning in plants? (4)

- Desirable genetic characteristics (e.g. high fruit production) are always passed on to clones

- Tissue culture allows plants to be reproduced in any season since the environment is controlled

- Less space is needed for tissue culture compared to conventional growing methods

- Tissue culture produces lots of plants quickly compared to growing from seeds

What are the arguments against artificial cloning in plants? (4)

- Undesirable genetic characteristics (e.g. fruit with lots of seeds) are always passed on to clones

- Cloned plant populations lack genetic variability, making them vulnerable to a single disease

- Production costs of tissue culture are high and require skilled workers

- Contamination by microorganisms during tissue culture can result in complete loss of the plants being cultured

How do invertebrates naturally clone themselves? (2)

- Some invertebrates can regenerate entire animals from fragments (e.g. starfish)

- Some animals fragment and produce clones of the original as part of their normal reproductive process

How do vertebrates naturally clone themselves? (2)

- Cloning occurs in vertebrates via the formation of monozygotic twins (identical twins)

- In this case, the early embryo splits to form two identical embryos

What happens in artificial embryo twinning within animals? (6)

1. An egg cell is extracted from a female animal and fertilised in vitro (in a Petri dish)

2. The fertilised egg divides, forming an embryo in vitro

3. The individual cells of the embryo are separated and each put into a separate Petri dish

4. Each cell divides and develops normally, forming an embryo in each dish

5. The embryos are implanted into female surrogates

6. Offspring are born, and all are genetically identical to each other

Describe the process of Somatic Cell Nuclear Transfer (SCNT) in animal cloning

1. A somatic cell is taken from animal A. the nucleus is extracted and kept

2. An oocyte (immature egg cell) is taken from animal B, and its nucleus is removed to form an enucleated oocyte

3. The nucleus from animal A is inserted into the enucleated oocyte from animal B

4. The nucleus and enucleated oocyte are fused together and stimulated to divide by e.g. electrofusion (electric current applied)

5. An embryo is produced, implanted into a surrogate mother, and a clone of animal A is born

What are five uses of animal cloning? (5)

- Research purposes

- Endangered species

- Agriculture

- Genetically modified animals

- Stem cell harvesting

What are the arguments for artificial cloning in animals? (5)

- Desirable genetic characteristics are always passed on

- Infertile animals can be reproduced

- Endangered species population can be increased, helping to preserve biodiversity

- Cloning can be done any time, not limited by breeding season

- Cloning can help develop new treatments for diseases, reducing suffering

What are the arguments against artificial cloning in animals? (4)

- Cloning is difficult, time-consuming, and expensive

- No genetic variability: undesirable traits and susceptibility to diseases are passed on

- Shorter lifespan: cloned animals may not live as long as natural offspring, raising ethical concerns

- Ethical issues: using cloned human embryos for stem cells is controversial since the embryo is destroyed

Why are microorganisms used in biotechnological processes? (4)

- Easily controlled growth conditions (right nutrients, temperature, pH, etc.)

- Short life cycle: rapid growth under the right conditions allows quick production

- Can be grown on inexpensive materials, making the process economical

- Can be grown year-round, regardless of season

How are microorganisms used in the brewing industry? (3)

- Yeast is added to grain and other ingredients

- Anaerobic respiration of yeast uses glucose from the grain

- Fermentation produces ethanol and CO2

How are microorganisms used in the baking industry? (2)

- CO2 is produced during the fermentation of sugars in dough

- This makes the bread rise

How are microorganisms used in cheese production? (4)

- Used to rely on rennet (enzyme chymosin) to clot milk

- Chymosin can now be obtained from genetically modified yeast

- Lactic acid bacteria (e.g. Lactobacillus, Streptococcus) convert lactose in milk to lactic acid, souring the milk and helping it solidify

- Fungi are used in the production of blue cheese

How are microorganisms used in yoghurt production? (2)

- Lactic acid bacteria are used to convert lactose in milk to lactic acid

- Turning the milk sour and solidifying it

How are microorganisms used to produce penicillin? (2)

- Penicillium fungi produce penicillin when under stress to inhibit bacterial growth

- The fungus is grown in industrial fermenters and the penicillin is collected and processed for medicine

How are microorganisms used to produce insulin? (2)

- Genetically modified bacteria are used to produce human insulin by inserting the insulin gene into their DNA

- The bacteria are grown in industrial fermenters, and the insulin is collected and purified

How are microorganisms used in bioremediation? (2)

- Bacteria that can remove pollutants from contaminated sites (e.g. oil spills) are provided with extra nutrients and enhanced conditions

- The bacteria break down pollutants into less harmful products.

What is Single-Cell Protein (SCP)? (3)

- Microorganisms grown as a source of protein for human consumption

- Fusarium venenatum (fungus used to make Quorn)

- Methylophilus methylotrophus (bacteria used for SCP

What are the advantages of using microorganisms to make food for human consumption? (5)

- Microorganisms can grow on waste products (e.g. molasses), reducing waste

- Quick, easy, and cheap growth requirements

- Can be grown with less land than crops/livestock

- Can be cultured anywhere (helpful in regions where food production is difficult)

- Considered a healthier alternative to animal protein

What are the disadvantages of using microorganisms to make food for human consumption? (4)

- Conditions for microorganism growth can also grow contaminating microbes

- People may dislike the idea of food grown from waste

- Texture and taste may not match that of meat

- High consumption of SCP can lead to uric acid buildup from amino acid breakdown

How can microorganisms be cultured in a lab? (3)

- Use a sterile inoculation loop to transfer microorganisms in a zig-zag streak across an agar plate

- Loosely tape the Petri dish lid to allow oxygen in, preventing anaerobic bacteria growth

- Incubate plates at a suitable temperature and add nutrients to the agar to improve growth

What aseptic techniques are used to prevent contamination in microorganism cultures? (6)

1. Close windows/doors to prevent draughts

2. Regularly disinfect work surfaces

3. Avoid placing utensils on work surfaces

4. Use sterile equipment (autoclave glassware or use pre-sterilised tools)

5. Work near a Bunsen flame to draw microbes away from the plate

6. Flame the neck of broth containers after opening/closing to prevent contamination

What is Batch Fermentation? (2)

- Microorganisms are grown in individual batches in a fermentation vessel

- Closed culture: once a culture ends, it's removed, and a new batch is grown

What is Continuous Fermentation? (2)

- Microorganisms are grown continuously without stopping

- Nutrients are added and waste products removed at a constant rate

What conditions are needed for optimal growth in fermentation? (5)

- pH: Monitored by a pH probe for efficient enzyme activity

- Temperature: Controlled by a water jacket to maintain enzyme activity

- Nutrients: Paddles circulate fresh nutrient medium for constant access

- Oxygen: Sterile air is pumped into the vessel for respiration

- Sterility: Superheated steam sterilises the vessel to prevent competition from other organisms

What is the Lag Phase in the standard growth curve of microorganisms? (2)

- Population increases slowly

- Microorganisms need time to produce enzymes and prepare for reproduction

What is the Exponential Phase in the standard growth curve of microorganisms? (2)

- Population size doubles at regular intervals

- Conditions are most favorable: lots of nutrients, little competition

What is the Stationary Phase in the standard growth curve of microorganisms? (2)

- Death rate equals reproductive rate, so population size stays level

- Nutrients are running out and waste products are building up

What is the Decline Phase in the standard growth curve of microorganisms? (2)

- Death rate exceeds reproductive rate

- Population size decreases as food becomes limited and waste reaches toxic levels

Draw and label a growth curve (4)

What is the formula to calculate the number of bacterial cells after a certain number of divisions? (3)

- N= N0​× 2^n

- N0​ = initial number of bacteria

- n = number of divisions/generations

A bacterial cell divides every 20 minutes. How many cells will there be after 4 hours? (3)

- 4 hours ÷ 20 minutes = 12 divisions

- Initial bacteria N0 = 1

- Therefore, using equation, there will be 4096 bacterial cell

How do you investigate the effect of temperature on bacterial growth? (7)

1. Add a set volume of bacteria sample to an agar plate using a sterile pipette.

2. Spread the broth across the surface using a sterile spreader

3. Tape the lid shut and repeat for 6 plates

4. Place 3 plates in a fridge (4°C) and 3 plates in an incubator (25°C)

5. Incubate the plates upside down for 48 hours

6. Count and record the number of colonies formed

7. Use a negative control: an agar plate with no bacteria in both temperature conditions

What are other factors that can affect bacterial growth, and how can you investigate them? (2)

- pH: Add buffers at different pH levels to the broth

- Nutrient availability: Use agar preparations with different nutrients

What does a spectrophotometer measure in microbial broth and how can it be used? (3)

- Measures turbidity (cloudiness) of the broth

- High turbidity means more cells are present, indicating more replication

- It can be used to investigate microbial growth directly without needing to plate onto agar

If too many colonies form on agar, how can you make them more manageable to count? (2)

- Produce serial dilutions of the bacterial broth

- Plate diluted samples onto agar to get a more manageable number of colonies

How do you make serial dilutions starting from a 2M solution and diluting by a factor of 2? (5)

1. Line up the required number of test tubes

2. Add 10cm³ of the 2M solution to the first tube

3. Add 5cm³ of distilled water** to the rest

4. Transfer 5cm³ of the 2M solution to the second test tube (now 1M solution)

5. Repeat until the desired dilutions are made

How do you dilute a solution by a factor of 10? (1)

Add 1cm³ of the initial solution to 9cm³ of distilled water

How do you prepare 15cm³ of a 0.2 mol dm⁻³ sucrose solution from a stock solution of 0.8 mol dm⁻³ sucrose? (2)

What are isolated and immobilised enzymes? (3)

- Isolated enzymes = Enzymes that are not contained within cells

- Immobilised enzymes = Enzymes attached to an inert support system

- So the substrate can pass over them and convert to product without mixing the enzymes with the product

What are the advantages of enzyme immobilisation by adsorption onto an inorganic carrier? (4)

- Simple

- Cheap

- Can be used with many processes

- Enzymes are very accessible to the substrate; activity remains unchanged.

What are the disadvantages of enzyme immobilisation by adsorption onto an inorganic carrier? (1)

Enzymes can be lost from the matrix quite easily

What are the advantages of enzyme immobilisation by covalent or ionic bonds? (4)

- Cost varies

- Enzymes strongly bound, unlikely to be lost

- Enzymes accessible to the substrate

- pH and substrate concentration have little effect on activity

What are the disadvantages of enzyme immobilisation by covalent or ionic bonds? (2)

- Cost varies.

- Active site of enzyme may be modified in the process, making it less effective

What are the advantages of enzyme entrapment in a matrix (e.g. polysaccharides, gelatin)? (1)

Can be used with many different processes

What are the disadvantages of enzyme entrapment in a matrix (e.g. polysaccharides, gelatin)? (3)

- May be expensive

- Difficult to entrap

- Diffusion of substrate/product to/from the active site can be slow, reducing reaction rate

What are the advantages of membrane entrapment in microcapsules or semi-permeable membranes? (3)

- Relatively simple to do

- Small effect on enzyme activity

- Can be used in various processes

What are the disadvantages of membrane entrapment in microcapsules or semi-permeable membranes? (2)

- Relatively expensive

- Diffusion to/from active site can be slow, reducing reaction rate

What are the advantages of using immobilised enzymes over free enzymes in industry? (3)

- Immobilised enzyme columns can be washed and reused, reducing cost

- Product is not mixed with the enzyme, saving time and money on separation

- Immobilised enzymes are more stable, less likely to denature under extreme pH/temperature conditions

What are the disadvantages of using immobilised enzymes over free enzymes in industry? (3)

- Extra equipment needed, which is expensive

- Immobilised enzymes are costlier to buy compared to free enzymes, not ideal for small-scale use

- Immobilisation can reduce enzyme activity as enzymes cannot mix freely with the substrate

What are the advantages of using immobilised enzymes over whole microorganisms in industrial processes? (4)

- Less wasteful: isolated enzymes don't use substrate for growth/reproduction

- More efficient: enzymes work at higher concentrations than whole microorganisms

- More specific: conditions can be optimised for a specific enzyme

- Less downstream processing: isolated enzymes produce a pure product, unlike microorganisms that produce multiple by-products

How can immobilisation increase the effectiveness of an enzyme? (4)

- Enzymes are accessible to substrates

- Allows continuous production by a continuous flow of substrate over the enzyme

- Conditions can be tightly controlled over enzyme columns

- Immobilised enzymes are less affected by changes in pH and temperature

How can immobilisation decrease the effectiveness of an enzyme? (3)

- Immobilisation may affect the enzyme's ability to catalyse a reaction

- Diffusion of substrate to and from the active site may be inhibited, slowing the reaction

- Surface-immobilised enzymes may be lost from the matrix easily

What are the advantages of using extracellular enzymes over intracellular enzymes? (3)

- Extracellular enzymes are secreted, making them easier to isolate

- Microorganisms produce fewer extracellular enzymes, simplifying isolation

- Extracellular enzymes are more robust, as they are adapted to cope with greater variations in temperature and pH

What are some industrial uses of immobilised enzymes? (5)

- Conversion of lactose to glucose and galactose

- Production of semi-synthetic penicillins

- Conversion of dextrins to glucose

- Conversion of glucose to fructose

- Production of pure L-amino acids