Cloning and biotechnology

Natural clones - vegetative propagation

• Plants can reproduce by cloning because they have many cell meristems that retain the ability to differentiate.

• The new plant may be propagated from the stem, leaf, bud or root of the parents depending on the type of plant.

How can plants produce clones naturally?

• Runners - Horizontal stems that grow above ground, developing roots at nodes to form new plants (e.g., strawberries, spider plants)

• Tubers - Large underground plant structure that act as a food store for the plant, they are covered in ‘eyes’ which can sprout and form a new plant. (e.g. potatoes)

• Bulbs - Underground food store, new bulbs are able to develop from the original bulb and form new plants. (e.g. onions)

• Rhizomes - Underground stems that grow horizontally, they have nodes which new shoots and roots develop. (e.g. bamboo)

Cuttings to make a clone

• Take a cutting from the end of a stem, at an angle

• Remove all excess leaves ( to stop transpiration)

• Dip the end in rooting powder (hormones)

• Plant in optimal conditions

Tissue culture

• An artificial cloning technique

• Meristem cells are taken from the shoot or root tip of a plant - this is called the explant

• The cells are sterilised to kill any microorganisms

• The cells are placed on a culture medium containing nutrients such as glucose for respiration and growth hormones e.g. auxins

• The explant cells divide to form a callus culture

• When the cells have divided by mitosis into a small plant they can be transferred and planted in soil, they will grow to be a clone of the original plant.

purpose of sterile conditions

• To remove competition from any other microorganisms e.g. bacteria and fungi which would compete for nutrients and decrease the growth rate of the plant.

Micropropagation

• When tissue culture is used to produce lots of cloned plants very quickly.

• Cells are taken from developing cloned plants and subcultured (grown on another fresh growth medium)

• This is repeated with large numbers of clones

• This technique is used in horticulture and agriculture to produce fields full of a crop that has been genetically engineered the be insect resistant.

Advantages of micropropagation and tissue culture

• rapid

• produces large numbers of disease resistant crops

• New plants will have desired characteristics

  • Can produce large numbers of seedless fruit plants

• Can be used to reproduce rare or endangered plants

• Less space is required by tissue culture.

Disadvantages of micropropagation/tissue culture

• Monoculture, loss of genetic diversity. Means that plants will be susceptible to the same disease - could kill them all.

• Expensive - requires skilled workers and high energy

• Risk of contamination could result in loss of plants, if source materials are infected with a virus all the clones would be too.

Natural clones in animals

Monozygotic twins

When a single zygote split in half and develops into 2 separate embryos with the same DNA.

This produces 2 genetically identical clones.

Artificial embryo twinning e.g. in a cow

1. An egg cell is extracted and fertilised in a petri dish

2. The zygote divides by mitosis to form a ball of totipotent cells (embryo)

3. The cells are separated then divide further to form blastocysts

4. Each mass of cells is placed into the uterus of a different surrogate mother

5. The 4 surrogate mothers will give birth to 4 genetically identical offspring

Somatic Cell Nuclear Transfer (SCNT)

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

2. An immature egg cell is taken from sheep B it is enucleated.

3. The nucleus from sheep A is inserted into the enucleated egg cell from sheep B.

4. These are fused together and stimulated to divide by an electric shock. This produces an embryo.

5. Then the embryo is implanted into a surrogate mother, eventually a lamb is born that is a clone of sheep A.

Uses of cloned animals

• Scientific research. E.g. when testing drugs having genetically identical animals removes variables caused by genetic differences.

• Save endangered animals

• In agriculture it can be used to ensure desirable characteristics e.g. high milk yield or body mass

• Genetically modified animals used for ‘pharming’ could be cloned

Arguments for animal cloning

• desirable characteristics can be passed on

• Infertile animals can be reproduced

• Increasing population of endangered species preserves biodiversity.

• No need to wait for breeding season

• pharming

Arguments against animal cloning

• very difficult, time consuming and expensive.

• very few embryos survive to birth

• no genetic variation, all susceptible to the same disease

• Ambiguity around differing life expectancy of clones, if it is less this is unethical

• undesirable characteristics passed on

Biotechnology

The industrial use of living organisms or enzymes to produce food, drugs and other products

Benefits of using microorganisms in manufacturing processes

• Short life cycle and rapid growth rate

• No ethical issues

• They require little nutrients - genetic manipulation means we can modify them so micro-organisms can utilise waste materials for growth

• Allows us to artificially manipulate microorganisms to carry out synthesis reactions which they would not naturally do

Difference between direct and indirect food production

• Direct = eating the microorganism and what they produce e.g. Quorn

• Indirect = eating just the product of the microorganisms

Role of Microorganisms in brewing beer

• yeast is added to barley

• It respires anaerobically using glucose from the grain and produces ethanol and CO2 (fermentation)

Role of microorganisms in bread

• yeast is added and respires anaerobically

• The fermentation produces CO2 which makes the bread rise

Role of microorganisms in cheese

• Chymosin enzyme clots the milk, this is produced by GM yeast

• Lactobacillus turns it sour and solidifies it

Yoghurt production

• Lactobacillus and streptococcus

• produce lactic acid by anaerobic respiration, this makes the proteins in milk denature and coagulate.

Advantages of using microorganisms in food production

• Reproduce very quickly and produce proteins faster than animals and plants

• can be grown on waste products

• Production costs are low

• can be genetically modified

• no ethical issues

Disadvantages of using microorganisms in food production

• need sterile conditions that are carefully controlled to prevent contamination

• Some microorganisms can produce toxins if the conditions are not maintained at the optimum

• People can be concerned about eating GM food or single-celled protein e.g. quorn if it has been grown using waste products

Penicillin production

• Produced from a mould called Penicillin Chrysogenum

• in times of stress it produces antibiotics to stop bacteria growing as competition

• produced in an industrial fermenter

Bioremediation

process of using microorganisms to remove pollutants from contaminated sites

pollutant-removing bacteria occur naturally but are provided with nutrients so they an thrive.
Bacteria breakdown pollutants into less harmful products, cleaning up the area. It has been used to clean up oil spills at sea.

culture

A population of one type of microorganism that has been grown under controlled conditions.

where are cultures grown

In fermentation vessels, to obtain lots of the microorganism or to collect lots of useful product that the microorganism makes.

2 methods of culturing

• Batch fermentation → This is where microorganisms are grown in individual batches in a fermentation vessel, when one culture ends its removed and then a different batch is grown. This is known as closed culture.

• Continuous fermentation →This is where microorganisms are continually grown in a fermentation vessel without stopping. Nutrients are put in and waste products taken out at a continuous rate

How are temperature and pH kept at optimum in fermentation vessels? +why

• Temperature: Kept constant by a water jacket that surrounds the whole vessel, metabolic reactions are exothermic.

• pH: Constantly monitored by a pH probe and kept at the optimum level

• optimum for enzymes

How is access to nutrients regulated? benefits?

Paddle constantly circulates fresh nutrient medium around the vessel.

This ensures the microorganism always have access to their required nutrients.

How is the volume of O₂ regulated and why?

• Sterile air is pumped into the vessel when needed

• O2 for respiration

How is the vessel kept sterile and why?

• Superheated steam sterilises the vessel after each use.

• Kills any unwanted organisms that may compete with the ones being cultured.

Closed culture of microorganisms follow a standard growth curve

Lag phase

• The population size increases slowly

• Because the microorganisms have to synthesise enzymes and other molecules

• The reproduction rate is low

The Log/exponential phase

When the rate of bacterial reproduction is close to or at its theoretical maximum

Because the culture conditions are at their optimum.

The stationary phase

• population stays level

• the death rate and reproduction rate are equal

• Microorganisms die because there is not enough food and toxic waste product build up

The death phase

The death rate is greater than the reproductive rate

scarce food and toxic waste.

The aseptic technique

• regularly disinfecting surfaces

• Work near a bunsen flame (hot air rises so airborne microorganisms will be drawn away from the culture)

• sterilise instruments before and after each use

• pass necks of containers through the flame

• minimise time that agar plate is open

• wear lab coat, gloves and tie hair back.

Culturing bacteria

• Can be done in a nutrient broth or a nutrient agar

• A sterile inoculating loop is used to transfer bacteria from the original stock culture to the nutrient medium.

• The medium is then incubated at a suitable temperature

Immobilised enzyme

An enzyme attached to an insoluble material to prevent mixing with the product

Methods for immobilising enzymes

1. Encapsulated in alginate beads / a capsule which act as a semi-permeable membrane

2. Trapped in a silica gel matrix

3. Covalently bonded to an inert substances, collagen or cellulose fibres

Process of using immobilised enzymes

• The immobilised enzymes are contained within a column through which the substrate solution is filtered

• As the substrate runs through the column, enzyme-substrate complexes are formed and products are produced

• The products then flow out of the column, leaving the enzymes behind. The enzymes can then be reused.

Advantages of using immobilised enzymes in industry

• columns of immobilised enzymes can be washed and reused - this reduces the cost of running a reaction on an industrial scale because you don’t have to keep buying new enzymes

• The product isn’t mixed with the enzymes - no money or time is spent separating them out.

• Immobilised enzymes are more stable than free enzymes, they are less likely to denature in extreme pHs or temperatures

Disadvantages of using immobilised enzymes in industry

• Specialist equipment is required - which can be expensive to buy

• Expensive to buy

• The rate of reaction is sometimes lower due to immobilised enzymes not being able to freely mix with the substrate

Example - Lactose free milk

• some people are unable to digest lactose due to producing insufficient lactase enzyme

• Lactase breaks down lactose into glucose and galactose via hydrolysis reaction

• Fresh milk can be passed over immobilised lactase to produce lactose-free milk for use in the production of lactose-free dairy products.

Production of Semi-Synthetic Penicillins

• can be effective against penicillin-resistant organisms

• Immobilised enzyme penicillin acylase enzyme is used in their production

Conversion of glucose to fructose

• fructose is sweeter then glucose so it can be used in smaller quantities as a sweetener

• Immobilised glucose isomerase is used to convert glucose to fructose on an industrial scale