Biotechnology & gene technologies (Pt 1), Bio

• Genes, cells or whole organisms that carry identical genetic material because they are derived from the same original DNA.
• Distinction should be made between cloned genes, cloned cells, and cloned organisms.

• Twins (when zygote splits into two)
• Bacteria/prokaryotes divide asexually 
• Plants asexual reproduction (some cells produced by mitosis can grow into new organisms)
• Vegetative propagation in elm trees (the one we need to know about).

• Ad: Quick, allow organism to reproduce rapidly and take advantage of resources in environment
• Can be completed if sexual reproduction fails (plants may not have to rely on agents of pollination such as insects)
• All offspring have the genetic info to enable them to survive in their environment.
• Disad: Does not produce genetic variety, so any genetic parental weakness will be in offspring. Also all will be equally susceptible to environmental changes.

• Production of structures in an organism from non-reproductive tissue (I think - like leaves, roots,stems) that can grow into new individual organisms. These offspring contain the same genetic info as the parent and so are clones of the parent.
• Eg. elm tree produce clones from structures called suckers.

• Root suckers grow from sucker buds that are scattered around the tree's root system. The suckers are from meristem tissue from shallow roots of tree. They are normally dormant.
• During times of stress (eg. disease or some damage to tree in form of felling, or when tree is dying), the buds are activated and suckers begin to form.
• They begin to grow up into separate trees (clones), metres away from parent tree, which can help avoid the stress that triggered their growth.

• Ad: Allow elm to spread. The new circle of clones will in turn put out suckers which grow when they die. 
• The suckers grow metres (sometimes many metres) away from dying trunk so to help avoid the stress that triggered the suckers in the first place.
• Disad: The new trees are clones of the parent tree so they won't have resistance to disease (in case of Dutch elm disease), so they are just as vulnerable as the original tree.

• Taking cuttings - section of stem cut. Cut end often treated with plant hormones ton encourage root growth, then planted.
• Grafting - shoot section of a woody plant you want to propagate, is joined to an already growing root and stem (rootstock), making sure vascular tissue lined up. Graft grows and is genetically identical to parent plant, but rootstock is genetically different.
• [However, this is slow and sometime plants do not reproduce well from either cuttings or grafts - in these cases tissue culture is better]

• Cells/small pice of tissue is taken from the plant to be cloned, usually shoot tip (because there're meristem cells there). Called the explant.
• Cells sterilised to kill any microorganisms (to stop disease, and also increases growth rate)
• Explant placed on a nutrient growth medium.
• Cells divide, but do not differentiate and form a mass of undifferentiated cells called a callus.
• After few weeks, single callus cells removed and placed on growing medium containing plant hormones encouraging shoot growth.
• Growing shoots transferred to soil in greenhouse.
• [called micropopagation and can produce many clones in short time]

The separation of cells of any tissue type and their growth in or on a nutrient medium. In plants, the undifferentiated callus tissue is grown in nutrient medium containing plant hormones that stimulate development of the complete plant.

• Desirable genetic characteristics (eg. high fruit production) are always passed on to clones. This doesn't always happen in sexual reproduction.
• Plants can be reproduced in any season if tissue culture is used - carried out indoors.
• Sterile plants can be reproduced.
• Plants that take a long time to produce seeds can be reproduced quickly.
• Might be cost efficient because all the crop is ready for harvest at same time.

• Again, similar to disadvantages of asexual reproduction...
• Undesirable characteristics always passed on
• Cloned plant population have no genetic variability so a single disease could kill them all.
• (Maybe, CHECK, production costs high because of high energy use and training skilled workers..)

• Embryonic stem cells
• They are totipotent - capable of differentiating into any type of adult cell found in organism.
• These cells are able to "switch on" any of the genes present in the genome.

• Used to make embryonic stem cells that are genetically identical to another organism. It is also called therapeutic cloning. 
• Usually these embryonic stem cells are harvested from young embyos.

• Won't be rejected because it's identical to individual's own cells
• Cloning and cell culture could mean an end to current problems of waiting for donor organs
• Cells totipotent so can be used to generate any cell type
• Can be used for: regeneration of heart muscle cells following heart attack
• repair of nervous tissue

• Used to make a complete organism that's genetically identical to another organism.
• Can be used for: research purposes - genetic makeup is a variable that can be controlled by using clones in drug testing etc.
• Can save endangered animals
• Used by farmers

• Splitting embryos: Cells from developing embryo (produced maybe in vitro by sperm and egg) can be separated out, with each one then going on to produce a separate, genetically identical organism (by implanting into surrogate mothers). As the cloned organism is genetically identical to the zygote (made by male and female), you can't tell what characteristics it will have.
• Nuclear transfer: Differentiated cell from adult to be cloned taken, and nucleus placed in an egg cell which has its nucleus removed (an enucleated egg cell).
• Electro-fusion (some UV) I think, is used here to fuse the egg (ovum) and nucleus together.
• Developing embryo implanted in surrogate mother. [This is how Dolly was created]

• Desirable genetic characteristics are always passed on, allowing cloning of high-value animals in large numbers.
• Infertile animals can be reproduced.
• Rare animals can be cloned to preserve species.
• Animals can be cloned at any time.

• Undesirable genetic characteristics are alwyas passed on.
• (As with animals) excessive genetic uniformity in a species makes it unlikely to be able to adapt to changes in environment.
• Reproductive cloning very difficult, time-consuming and expensive (Dolly was created after 277 attempts!)
• Some evidence suggests that clones may not live as long as natural offspring.
• Ethical issues surrounding use of human embryonic cells.

• The industrial use of living organisms (or parts of them, or biological processes) to produce food, drugs or other products.
• used in...
• medicine (drugs and microorganisms in gene therapy)
• agriculture (includes micropropagation)
• industry (enzymes)
• food science

• Bacteria (eg. for insulin)
• Fungi (eg. antibiotic penicillin is produced by these)

• Grow rapidly under the right conditions, so products made quickly.
• Can be genetically engineered to produce specific products.
• Their ideal growth conditions can be easily created (relatively low temps, grown anywhere).
• Can be grown at any time of the year.
• Can often be grown on a range of inexpensive materials, or some that are otherwise useless or even harmful to humans.
• Often produce proteins/chemicals that are given out into surrounding medium so it can be collected - and often in a more pure form than those generated via chemical processes.

Growth/Population of microorganisms in an environment where all conditions are fixed and contained. No new materials are added and no waste products or organisms removed.

• 1. Lag phase: population remains fairly constant with population size increasing v. slowly. Because organisms are adjusting to new environment. Could be cell expansion, activating specific genes, synthesising specific enzymes or taking in water.
• 2. Exponential (Log) Phase: Population size increases very quickly, number doubling with each generation. Because it is most favourable condition for reproduction, with enough space and nutrients and little competition.
• 3. Stationary phase: Population size stays level. Because death rate equals their reproduction rate. Nutrients decrease and waste products like CO2 and other metabolites build up. [note - in open system, this is carrying capacity of environment.]
• Decline (death) phase: Population size falls until eventually all organisms die. Because nutrients are exhausted and increased levels of toxic waste products and metabolites; death rate higher than reproduction rate.

• process
• metabolites

• Primary: 
• are substances produced by organism as part of its normal growth
• production of primary metabolites matches the growth in population of the organism, because products associated with growth of organism itself.
• eg. amino acids, proteins, enzymes, nucleic acids, ethanol and lactate.
• Secondary:
• are substances produced by organism that are not part of its normal growth
• production of secondary metabolites usually begins after the main growth period of organisms and so does not match the growth in population of the organism.
• eg. antibiotic chemicals produced by a number of microorganisms are almost all secondary metabolites.
• [Another difference is that all microorganisms produce primary metabolites, but only a relatively small number of them produce secondary metabolites.]

• a) Unlikely to have any effect. During this phase, organisms are adjusting to the new environment and synthesising enzymes they need. Nutrient supply is probably not a limiting factor during this period.
• b) Would most likely lead to increase in growth. Because in this stage, nutrient supply is likely to be limiting (the carrying capacity has been reached.) However, additional nutrients would not have any effect if reason for entering stationary phase was accumulation of waste products or lack of space, with plenty of nutrients remaining.

When conditions are favourable in the lag and exponential phases, metabolic processes are dedicated to growth (ie primary metabolites). But when conditions worsen and competition arises, secondary metabolites, such as antibiotics, are required to enable the organism to survive.

• Temperature: too hot and enzymes denatured; too cold and growth will be slowed. So reaction rate kept high.
• pH: optimum pH essential for enzymes to work efficiently, so rate of reaction high.
• Type and time of addition of nutrient: growth requires nutrient supply, including sources of carbon, nitrogen and essential minerals/vitamins. Timing of addition can be manipulated, depending on whether the process is designed to produce primary or secondary metabolite.
• Oxygen conc: most commercial applications use growth of organisms under aerobic conditions, so sufficient oxygen must be made available. Lack of oxygen would lead to unwanted products as a result of anaerobic respiration.

• Conditions are monitored by electronic probes (measuring oxygen, pH and temp levels)
• Temp: water jacket inlet allows circulation of water around fermenter vessel to regulate temp.
• Oxygen: air inlet provides sterile oxygen in aerobic fermenters. (Often released in small bubbles, so that oxygen is mixed and increase access. [NB. All inlets & outlets fitted with filter to prevent contamination].
• Pressure vents: prevent any gas build-up
• Rotating blades: so microorganisms are kept in contact with fresh medium. This increases product yield because they can always access the nutrients needed for growth.

• Vessels are sterilised between uses with superheated steam to kill any unwanted organisms. 
• This obviously prevents unwanted products from forming and contamination that ruins the products.
• Also, it increases product yeild because the microorganisms aren't competing.

• Nutrition will always be kept at sufficient levels in the primary metabolite culture, because that will increase the growth of the microorganisms and hence increase yield of products that are directly associated with the growth. (eg. enzymes)
• Nutrition, however, will be restricted to an effective level in the secondary metabolite culture, because secondary metabolites will only begin to be produced when conditions are unfavourable and there is a lot of competition for  nutrients.

• Batch: fixed volume of nutrients added at start, and no more added. Closed system. Continuous: Constant supply of fresh nutrients. Open system. 
• B: Each culture goes through lag, exponential and stationary growth phases. C: lag, but is then kept at exponential growth phase.
• B: Product harvest once, during stationary phase. C: product continuously taken out at steady rate.
• B: Product yield relatively low (less efficient because fermenter not in use all of time, esp when sterilised at intervals). C: Product yield high (more efficient, fermenter operates continuously).
• B: Growth rate smaller because nutrient level declines with time. C: Growth rate higher, nutrients continuously added to vessel.
• B: If contamination happens, only one batch is lost C: If culture contaminated, huge volumes of product may be lost.
• B: Used for secondary metabolites. C: Used for primary metabolites or microorganisms themselves. (usually).

• Compete with culture microorganisms for nutrients and space.
• Reduce yield of useful products
• May cause spoilage of product
• May produce toxic chemicals
• May destroy culture microorganism and their products.

• aseptic technique
• asepsis

• Lab: all aparatus used is sterilised before and after use, by heating in flame, glowing under UV light, or steam-sterilise.
• Work carried out in fume cupboard, where air circulation carries any airborne contaminants away from bench space.
• At large-scale culture level: Steam-cleaning fermenter and associated pipes.
• Fermenter surface polished stainless steel to prevent microbes sticking to surfaces.
• Sterilising all  nutrients and oxygen before adding.
• Fine filters on inlet and outlet pipies.