CLONING + BIOTECHNOLOGY

What are natural clones used for?

Horticulture

Examples of natural plant clones

Bulbs, runners, rhizomes, stem tubers

Other phrase for natural cloning

Vegetative propagation

How bulbs do natural cloning

Leaf base swells with stored food, buds form internally, develop into new shoots and plants in the next season

How runners do natural cloning

Lateral stem grows away from the parent plant, roots develop where the runner touches the ground, new plant develops, runner withers away

How rhizomes do natural cloning

Specialised horizontal stem swollen with food running underground, buds develop, form new vertical shoots, become plants

How stem tubers do natural cloning

Tip of underground stem becomes swollen with stored food, forms tuber, buds on tuber produce new shoots

How are natural clones used in horticulture?

Splitting of bulbs, removing young plants from runners, cutting up rhizomes, all increase plant numbers cheaply

How to take a plant cutting

Select a non-flowering stem, angled cut across the stem below a node, reduce the leaves, dip in rooting powder, put in watered soil, cover with a plastic bag for a few days, water

Example of a simple cloning technique

Cuttings

Way of making artificial clones

Tissue culture

Process of tissue culture

Small samples of meristematic tissue tissue taken from virus free plant from shoot tips or axial buds, sterile conditions, sterilised in bleach or sodium dichloroisocyanurate, removed material now an explant, explant placed on sterile culture medium, callus forms, callus divided into smaller clumps of cells, placed on new culture medium, platelets grow, placed in compost

What's in the sterile nutrient medium for tissue culture?

Auxins, cytokinins, glucose, amino acids

Callus

Group of undifferentiated genetically identical cells

Difference between micropropagation and tissue culture

Micropropagation is a type of tissue culture

Advantages of artificial plant cloning

Can grow plants which have low levels of fertility, large number of plants with a known genetic code produced rapidly, can grow rare or endangered plant species, can produce large numbers of seedless plants, disease free plants because meristem is used

Disadvantages of artificial plant cloning

Skilled workers required, have to make sure tissue isn't infected with a virus, infection possible during production, all plants at risk when environment changes or when exposed to a disease, limits evolution, large numbers of plants lost during production

Example of use of plant cloning in horticulture and agriculture

Sugar cane

How is sugar cane cloned?

Short lengths of cane with three nodes are cut, buried in clear field in shallow trenches, covered with thin layer of soil

Examples of natural animal clones

Starfish, flatworms, sponges, Hydra, monozygotic twins

How are starfish natural animal clones?

Entire animals can be regenerated from fragments of the original

How are flatworms and sponges natural animal clones?

Fragment and form identical animals as part of the natural reproductive cycle

How are Hydra natural animal clones

Produce buds on the side of the body, develop into genetically identical clones

Process by which monozygotic twins occur

Embryo splitting

How can artificial clones in animals be made?

Artificial embryo twinning, somatic cell nuclear transfer

How does artificial embryo twinning occur?

Cow treated with hormones so she super-ovulates, ova fertilised naturally or by artificial insemination, early embryos gently flushed out of the uterus, at day six the early embryo is split into smaller embryos, split embryos grown in lab for a few days, implanted into a surrogate mother

Process of somatic cell nuclear transfer

Nucleus removed from somatic cell of an adult animal, mature ovum enucleated, nucleus from somatic cell put into enucleated ovum, given a mild electric sock to get it to divide, embryo that develops is transferred into the uterus of a surrogate mother

Electrofusion

When the nucleus from a somatic cell is placed next to the enucleated ovum and fuses with it when the electric current is introduced

Uses of artificial clones of animals in agriculture

Pharming

Pharming

Production of animals which have been genetically engineered to produce therapeutic human proteins

Uses of artificial clones of animals in medicine

Production of GM animals which grow organs for transplants

Arguments for animal cloning

High-yielding animals, GM embryos can be replicated and develop to give many embryos from the original procedure, cloning specific animals, enabling rare or extinct animals to be reproduced

How is artificial twinning used in agriculture?

Identical animals can be reared from frozen clones of the original animal which has proven to be successful

How is SCNT used in agriculture?

Replication of GM embryos is used in it

Arguments against animal cloning

Very inefficient process, many fail to develop, miscarriage is common, malformed offspring, shortened lifespans

Biotechnology

Using biological organisms or enzymes in the production of materials for humans

Most commonly used organisms in biotechnology

Fungi, bacteria

Benefits of using microorganisms in biotechnology

No welfare issues, wide variety which can carry out different chemical reactions, can be genetically engineered, very short life cycle, rapid growth rate, nutrient requirements are simple and cheap, can use waste resources

Microorganism involved in baking

Yeast

How is yeast used in baking?

Active yeast mixture added to flour and other ingredients, mixed, left to rise as yeast produces carbon dioxide, dough is knocked off, cooked, yeast killed in cooking

Microorganism involved in brewing

Yeast

How is yeast used in brewing?

Malting, mashing, fermentation, maturation, finishing

Malting

Barley germinates to produce enzymes which breaks down starch molecules, yeast can use produced sugar molecules, malt produced

Mashing

Malt mixed with hot water, enzymes break down starch to produce wort, hops added, wort sterilised and cooled

Fermentation

Wort inoculated with yeast, temperature for anaerobic respiration maintained, yeast inhibited by falling pH

Maturation

Beer conditioned in tanks at low temperatures

Finishing

Beer filtered, pasteurised and bottled

Microorganism used in cheese making

Bacteria

Process of cheese making

Milk pasteurised and homogenised, mixed with bacterial cultures, bacteria feed on lactose to change the texture and taste, separates into curds and whey, curds cut and cooked in the whey, curds put into drums and pressed, left to mature

Microorganism used in yoghurt making

Lactobacillus bulgaricus, Streptococcus thermophilus

Process of yoghurt making

Skimmed milk powder added to milk, mixture is pasteurised and homogenised and cooled, milk mixed with bacteria, incubated, bacteria produce polymers that give the yoghurt its texture, yoghurt put into cartons

Product of Lactobacillus bulgaricus

Ethanal

Product of Streptococcus thermophilus

Lactic acid

Uses of microorganisms in biotechnology

Brewing, baking, cheese making, yoghurt production, penicillin production, insulin production, bioremediation

Process of penicillin production

Fungus grows from Pencillium chrysogenum, penicillin then produced, drug extracted and purified, uses a semi-continuous batch process

Conditions for penicillin production

Relatively small fermenters, continuously stored to keep it oxygenated, rich nutrient medium, contains a buffer to maintain pH, temperature maintained at 25 degrees celsius

Why are relatively small fermenters used in the production of penicillin?

Difficult to maintain high levels of oxygenation in very large bioreactors

Process of insulin production

Genetically modified bacteria grown in a fermenter, downstream processing gives a constant supply of pure human insulin

Bioremediation

The use of microorganisms to break down pollutants and contaminants in soil or in water

Example of fungal source being used to make food for human consumption

Single celled Fusarium venetatum grown in fermenters with glucose syrup, combined with albumen, compressed, makes Quorn

Advantages of using microorganisms to produce human food

Reproduce fast, produce proteins faster than animals and plants, high protein content with little fat, wide variety of waste materials used, genetic modification, not dependent on weather or breeding cycles, no welfare issues, can taste like anything

Disadvantages of using microorganisms to produce human food

Can produce toxins if not optimum conditions, have to be separated from the nutrient broth, needs sterile conditions which is expensive, concerns over GM, protein has to be purified, people don't like eating food grown on waste, needs additives

How to culture microorganisms effectively

Temperature, oxygen concentration, pH, sterile enriched nutrient media containing glucose and amino acids, use aseptic conditions

Why aseptic conditions are important

To prevent contamination with pathogenic microorganisms, to prevent the environment becoming contaminated if a mutation occurs

Primary metabolite

Substances which are formed as an essential part of the normal functioning of a microorganism

Secondary metabolites

Substances which are not essential for normal growth of a microorganism

Process of batch fermentation

Microorganisms inoculated into a fixed volume of medium, nutrients used up, biomass and waste products build up, stationary phase reached, products still made, process stopped before the death phase, products harvested

Process of continuous fermentation

Microorganisms inoculated into sterile nutrient medium, nutrient medium added continuously once exponential point of growth reached, culture broth continuously removed, volume in bioreactor is constant

Culture broth

Medium, waste products, microorganisms, product

Conditions to maintain in bioreactors

Temperature, nutrients, oxygen, mixing, asepsis

Why is it important to regulate temperature in bioreactors?

Enzymes

How to regulate temperature in bioreactors

Heating or cooling system, temperature sensors, negative feedback system

How to regulate oxygen and nutrient conditions

Added in controlled amounts, probes or sample tests to test levels

Type of metabolite formed from continuous culture

Primary

Type of metabolite formed from batch culture

Secondary

Why do you have to mix things in a bioreactor?

Diffusion not sufficient, to keep temperature constant

How to do asepsis in bioreactors

Seal the bioreactor

Stages of a standard growth curve for a microorganism

Lag, exponential, stationary, death

Lag phase

When bacteria are adapting to their new environment

Things happening in the lag phase

Growth, synthesis of enzymes, not reproducing at maximum rate

Log/Exponential phase

Rate of bacterial reproduction is close to the theoretical maximum

Stationary phase

Total growth rate is zero, number of new cells formed by binary fission is the same as the number of cells dying

Death stage

Reproduction has pretty much stopped, death rate of cells increases

Limiting factors in a culture of bacteria

Nutrients, oxygen levels, temperature, build-up of waste, change in pH

How to investigate the effect of conditions on the growth of microorganisms

Serial dilutions of the original broth in aseptic conditions, multiply number of visible colonies by the dilution factor to find number of original bacteria

Advantages of using immobilised enzymes

Can be reused, can be separated from products, reduces downstream costs, more reliable, greater temperature tolerance

Disadvantages of using immobilised enzymes

Reduced efficiency, higher initial cost, higher initial cost of bioreactor, more technical issues

Why are immobilised enzymes cheaper to use?

Can be reused, can be separated from reactants and products

Why are immobilised enzymes more reliable?

Insoluble support provides stable microenvironment for the immobilised enzymes

Why do immobilised enzymes have a greater temperature tolerance?

Less easily denatured

Methods of enzyme immobilisation

Surface immobilisation by adsorption to inorganic carriers or covalent bonds to inorganic carriers, entrapment in a matrix, membrane entrapment, semi-permeable membrane entrapment

Examples of inorganic carriers

Cellulose, silica, carbon nanotubes, polyacrylamide gel

Examples of organic carriers

Polysaccharides

Advantages of surface immobilisation by adsorption

Simple, cheap, used with many different processes, enzymes accessible to substrate

Disadvantages of surface immobilisation by adsorption

Enzyme easily lost

Advantages of surface immobilisation by covalent or ionic bonding

Strongly bound so less likely to be lost, accessible to substrate, pH and substrate concentrations don't affect rate

Disadvantages of surface immobilisation by covalent or ionic bonding

Active site may be modified in the process

Advantages of entrapment in matrix

Widely applicable

Examples of matrices in entrapment

Polysaccharides, gelatine, activated carbon

Disadvantages of entrapment in matrix

Expensive, difficult to entrap, substrate has to diffuse which can slow down rate