Cloning and Biotechnology

• Reproductive cloning is used to make a complete organism that's genetically identical to another organism.
• Scientists use cloned animals for research purposes. They're all genetically identical, so the variables which come from genetic differences are removed. This makes results more reliable.
• Reproductive cloning can also be used to save endangered animals from extinction by clonign new individuals.
• It can also be used by farmers to increase the number of animals with desireable characteristics to breed from.

• Non - Reproductive cloning is used to make embryonic stem cells that are genetically identical to another organism. Also called therapeutic cloning.
• Embryonic stem cells are harvested from young embryos.
• They have the potential to become any cell in an organism, so scientists think they could be used to replace damaged tissue, or help cure degenerative conditions.
• If replacement tissue is made from cloned embryonic cells thar are genetically identical to the patient's own cells, then the tissue won't be rejected by their immune system.

• A body cell is taken from sheep A. The nucleus is extracted and kept.
• An egg cell is taken from sheep B. It's nucleus is removed to form an enucleated egg.
• The nucleus from sheep A is inserted into the enucleated egg - the egg cell from sheep B contains the genetic information from sheep A.
• The implanted nucleus creates a diploid cell which grows like a zygote.
• The egg cell is stimulated to divide and an embryo is formed.
• The embryo is implanted into a surrogate mother. A lamb is produced that's genetically identical to sheep A.
• In non-repreoductive cloning, stem cells identical to sheep A are harvested from the embryo.

• Blunt pipette holds cells in place - sharp pipette sucks out and reinserts nucleus.
• Chemicals as well as electric shock can be used to stimulate division.

Advantages

• Desireable genetic characteristics are always passed onto the clones - removes risk factor of genetic variation
• Infertile organsims can reproduce.
• Animals can be cloned at any time of the year - do not need to wait for breeding season.
• Animals which are critically endangered/extinct can be brought back.

Disadvantages

• Undesireable genetics always passed on to clones.
• Difficult, time consuming, and expensive.
• Some evidence suggests that clones have a reduced life span.

• A small piece of tissue is taken from the plant to be cloned, usually from the shoot tip. This is called an explant.
• The cells are sterilised to kill any microorganisms which may compete for nutrients with the plant cells, decreasing their growth rate.
• The explant is placed on a nutrient growth medium.
• Cells in the tissue divide, but they do not differentiate. Instead, they form a  mass of undifferentiate cells called a callus.
• After a few weeks, single callus cells can be removed from the mass and placed on a growth medium containing plant hormones to encourage shoot growth.
• The growing shoots are transferred onto medium containing hormones to encourage root growth.
• The growing plants are transferred to a greenhouse to be acclimatised and grown further before they are grown outside.
• Each plant is gentically identical the the original plant.

• Elm trees produce clones from structures called suckers/basal sprouts.
• A sucker grows from the shallow roots of an elm tree.
• Suckers grow from sucker buds scattered around the tree's root system. The buds are normally dormant.
• During times of stress, or when the tree is dying, the buds are activated and suckers begin to form.
• Suckers can pop up many metres away from the parent tree, which can help them to avoid the stress that triggered their growth.
• They eventually form completely separate trees - clones of the tree from which they grew.

Advantages

• Desireable genetic characteristics are always passed on to the clones.
• Plants can be produced in any season because tissue culture is carried out indoors.
• Sterile plants can be reproduced.
• Plants that take a long time to reproduce can be reproduced quickly.

Disadvantages

• Undesireable genetic characteristics are always passed on to clones.
• Clined plant populations have no genetic variability, so a single disease could kill them all.
• Production costs are very high due to high energy use and the training of skilled workers.

• Biotechnology is the industrial use of living organisms to produce food, drugs, and other products.
• The living organisms used are mostly microorganisms;
  • Their ideal growth conditions can easily be created.
  • They grow rapidly under the right conditions, so products can be made quickly.
  • They can grow on a range of inexpensive materials.
  • They can be grown at any time of the year.
• Biotechnology also uses parts of living organisms, such as enzymes, to make products.
• Enzymes used in industry can be contained within the cells of organisms - these are intracellular enzymes.
• Enzymes can also be isolated, not contained within cells.
• Extracellular enzymes are secreted by organisms, but others have to be extracted.
• Naturally secreted enzymes are cheaper to use because it can be expensive to extract enzymes from cells.

• Adsorption - Enzyme molecules are mixed with the immobilising support and bind to it due to a combination of hydrophobic interactions and ionic links. Adsorbing agents used include porous carbon, glass beads, clays, and resins. Because the bonding forces are not that strong, enzymes can become detached. However, provided the enzyme molecules are held so their active site is not changed, adsorption can give high reaction rates.
• Covalent Bonding - Enzyme molecules are covalently bonded to a support, often by covalently linking enzymes together and to an insoluble material using a cross-linking agent like gluteraldehyde and sepharose. This method does not immobilise a large quantity of enzyme but binding is very strong so there is little leakage of enzyme from the support.
• Entrapment - Enzymes may be trapped in a gel bead or a network of cellulose fibres. The enzymes are trapped in their natural state. However, reaction rates can be reduced because substrate molecules need to get through the trapping barrier. this means the active site is less easily available than with adsorbed or covalently bonded enzymes.
• Membrane Separation - Enzymes may be physically separated from the substrate mixture by a partially permeable membrane. substrate molecules are small enough to pass through the membrane for the reaction to take place, and product molecules are small enough to pass back through the membrane.

Advantages

• Enzymes are not present with products so purifications/downstream costs are low.
• Enzymes are immediately available for reuse. This is useful in allowing a continuous process.
• Immobilised enzymes are more stable because the immobilising matrix protects the enzyme molecules.

Disadvantages

• Immobilisation requires additional time, equipment, and materials, so is more expensive to set up.
• Immobilised enzymes can be less active because they do not mix freely with the substrate.
• Any contamination is costly to deal with because the whole system would need to be stopped.

• In a closed culture, a population of microorganisms follows a standard growth curve.
• Lag Phase - The population size increases slowly because the microorganisms have to make enzymes and other molecules before they can reproduce. reproduction rate is low.
• Exponential Phase - The population size increases quickly because the culture conditions are at their most favourable for reproduction. The nnumber of microorganisms doubles at regular intervals.
• Stationary Phase - The population size stays level because the death rate of the microorganism equals their reproductive rate. Microorganisms die because there's not enough food and poisonous waste products build up.
• Decline Phase - The population size falls because the death rate is greater than the reproductive rate. food is very scarce and waste products are at toxis levels.
• When growing conditions are favourable, microorganisms produce primary metabolites - small molecules that are essential to growth.
• When growing conditions are less favourable, some microorganisms produce secondary metabolites - molecules that aren't essential to growth but are useful in other ways.
• Secondary metabolistes help the microorganisms to survive, and some are desireable to biotechnology industries.

• Cultures of microorganisms are grown in fermentation vessels.
• The conditions are kept at the optimum for growth to maximise the yield of desireable products.
• The pH is monitored by a probe and kept at the optimum level. this increases the product yield as enzymes are working efficiently, so the rate of reaction is high as possible.
• The temperature is kept at the optimum level by a water jacket which surrounds the vessel. This increases the yield as the enzymes work efficiently.
• Vessels are sterilised with superheated steam to kill any unwanted organisms. This increases the product yield because the microorganisms aren't competing with other organisms.
• The volume of oxygen is kept at the optimum level for respiration by pumping sterile air in when needed. This increases the product yield because microorganisms can always respire to provide the energy for growth.
• Microorganisms are kept in contact with the fresh medium by paddles that circulate the medium around the vessel. This increases the product yield because microorganisms can always access the nutrient needed for growth.

• Batch culture is where microorganisms are grown in individual batches in a fermentation vessel.
• When one culture ends it's removed and then a different batch of microorganisms are grown in the vessel.
• a fixed volume of growth medium is added to the fermentation vessel at the start of the culture and no more is added. This is a closed culture.
• Each culture goes through the lag, stationary, and exponential growth phases.
• The product is harvested once, during the stationary phase.
• The product yield is relatively low - stopping the reaction and sterilising the vessel between fermentations means there's a period where no product is harvested.
• If contamination occurs it only affects one batch. It's not very expensive to discard the contaminated batch and start a new one.
• Used when you want to produce secondary metabolites. 

• Continuous culture is where microorganisms are continuously grown in a fermentation vessel without stopping.
• Growth medium flows through the vessel at a stead rate so there's a constant supply of fresh medium. The culture is an open system.
• The culture goes throguh the lag phase but is then kept at the exponential growth phase.
• The product is continuously taken out of the fermentation vessel at a steady rate.
• The product yield is relatively high - microorganisms are constantly growing at an exponential rate.
• If the culture is contaminated the whole lot has to be discarded - this is very expensive when the cultures are grown on an industrial scale.
• Usually used when you want primary metabolites or the microorganisms themselves are the desired product.

• Asepsis is the practise of preventing contamination of cultures by unwanted microorganisms.
• It's important when culturing microorganisms because contamination can affect the growth of the microorganism you're interested in.
• Contaminated cultures in laboratory experiments give inaccurate results.
• Contamination on an industrial scale can be very costly because entire cultures may have to be thrown away.
• A number of aspetic techniques can be used when working with microorganisms;
  • Work surfaces are regularly disinfected to minimise contamination.
  • Gloves should be worn and long hair is tied back to prevent it from falling into anything.
  • The instruments used to transfer cultures are sterilised before and after each use.
  • In laboratories, the necks of culture containers are briefly flamed before they're opened or closed - this causes air to move out of the container, preventing unwanted microorganisms from falling in.
  • Lids are held over open containers after they're removed, instead of putting them on a work surface. This prevents unwanted mircroorganisms from falling into the culture.