Biotechnology and gene technology Flashcards
What is the basis of asexual reproduction in eukaryotes?
What about in prokaryotes?
How do genetic differences arise in organisms that reproduce asexually?
Mitosis
Binary fission
Via mutations
Define vegetative propagation
The production of structures in an organism that can grow into new individual organisms, they are genetically identical to their parents and are therefore clones
Define clone
Genes, cells or whole organisms that carry genetically identical material as they are derived from the same original DNA
How are identical twins produced?
They are produced when a zygote splits in 2, they are natural clones
Finish the sentence…
When organisms reproduce asexually all of the offspring are…
…genetically identical and are clones with cloned DNA
Can cloning occur naturally? Explain
Yes because it’s essential for growth and reproduction
There is a distinction between cloned genes, cloned cells and cloned organisms. Give examples of when each of the occur.
Cloned genes: DNA replication, protein synthesis
Cloned cells: Mitosis for growth of any multicellular organism
Cloned organisms: Identical twins, offspring from asexual reproduction
Briefly describe binary fission. Are the offspring genetically identical or not?
It occurs in prokaryotes, DNA replicates and then the cell divides into 2.
The offspring are genetically identical to each other and their parents
Briefly describe mitosis
When DNA replicates then separates to form 2 new nuclei and then 2 genetically identical daughter cells are formed
In multicellular organisms, especially plants, what can happen to the cells produced via mitosis?
They can grow into new separate organisms which are genetically identical to the parent and they are therefore clones. This is vegetative propagation.
Give three advantages of asexual reproduction
- It’s quick so organisms can reproduce rapidly, allowing them to take advantage of their environment
- It can be completed if sexual reproduction fails or isn’t possible
- All of the offspring have the genetic information in order to survive in their environment
Give 1 disadvantage of asexual reproduction
There is no genetic variation so all the offspring will have the genetic parental weaknesses. This means that if the environment changes, all of the organisms will be equally susceptible to death.
What happens when English Elm is damaged due to disease or burning?
Vegetative propagation: It reproduces asexually allowing the species to survive catastrophes. Root suckers/basal sprouts appear within 2 months of the destruction, they grow from the meristem tissue in the trunk close to the ground because this is where the least damaged is likely to have occurred.
Give 1 detailed advantage of vegetative propagation in English Elm trees
The root suckers can help the elm spread because they form a clonal patch (a circle of root suckers) after the Elm dies due to stress or felling. These clones will then produce more root suckers and the Elm clones will spread.
Give 1 detailed disadvantage of vegetative propagation in English Elm trees.
Dutch Elm disease, which has spread through Europe, kills the leaves of an Elm, then the branches, then the trunks. The fungal disease is spread via a beetle. When an English Elm gets infected, it grows root suckers but because there is no genetic variation, the root suckers get infected also. This means that natural selection doesn’t occur.
Define tissue culture
What type of tissue can be grown?
What type of tissue is usually grown?
Why?
The separation of cells from a tissue and their growth in or on a nutrient medium.
Any type
Undifferentiated callus tissue because it can, with the addition of hormones, stimulate the development of a complete plant.
Describe the 2 main methods of artificial vegetative propagation
- Taking cuttings: A section of stem is cut between nodes, the cut end is then treated with root growth promoting hormones. You then plant this cutting and it grows into an individual new organism. Large numbers of plants can be produced quickly from this method
- Grafting: A shoot section from a woody plant is joined to an already growing rootstock. This grows an individual organism. The graft is genetically identical to the parent but not to the rootstock. Side grafting is when you cut a slit in the plant and slide the graft into the slit.
Describe 2 disadvantages of grafting and cuttings
They don’t always reproduce well
They can’t produce huge numbers of cloned plants
Name a modern method of artificial vegetative propagation and list 3 advantages of it.
Tissue culture
It can produce huge numbers of genetically identical plants from a very small amount of plant material, it can generate large stocks very quickly and the plants are known to be disease free
What is the most common method for large scale cloning in plants?
What plant is usually cloned?
Micropropagation
Orcids
Describe the 6 stages of micropropagation.
- An explant (the shoot tip) is taken from a plant to be cloned
- The explant is placed in a nutrient medium
- The cells divide but don’t differentiate, which means that they form a callus (a mass of undifferentiated cells)
- After a few weeks, single callus cells can be removed from the mass and placed on a growing medium containing hormones that promote shoot growth
- After a few more weeks, the growing shoots can be placed onto a different growing medium with different concentrations of hormones that promote root growth.
- The plants are then transferred to a greenhouse where they then acclimatise before being planted outside.
Is micropropagation a form of genetic engineering?
No because genes aren’t added or removed
Define cloned animal
One that has been produced using the same genetic information as another animal. It has the same genotype as the donor.
Define totipotent stem cells
Cells that are naturally capable of going through the stages of development to generate a new individual. In animals, only embryonic cells are capable of this. They’re able to switch on any of the genes present in the genome.
List the two methods of artificial cloning in terms of animals
Splitting embryos and nuclear transfer.
Describe the procedure for splitting an embryo
An egg and sperm goes through in vitro fertilisation, and the embryo is grown in vitro also. When the embryo gets to 16 cells, the cells from the developing embryo can be separated out and implanted in a surrogate mother. This produces separate genetically identical organisms. It has been used on sheep, cattle, rabbits and toads
Describe in detail the procedure for nuclear transfer
A differentiated cell from an adult can be taken, and its nucleus can be placed in an enucleated egg cell via electro-fusion. The egg is then cultured in an oviduct. After this, it’s implanted into a surrogate mother and it then goes through the stages of development using the genetic information from the inserted nucleus. This method has been used on sheep.
List 3 advantages of cloning animals
- High value animals (high yields) can be cloned in large numbers
- Rare animals can be cloned to preserve the species
- Advantageous genetically modified animals (sheep that produce pharmaceutical chemicals) can be quickly reproduced
List 3 disadvantages of cloning animals
- High value animals aren’t always produced with animal welfare in mind, e.g. meat producing chickens that are unable to walk.
- Genetic uniformity reduces variation so plants wouldn’t be able to cope with or adapt to changes in the environment
- Cloning animals via nuclear material might affect the health of the animals’ cells long term. E.g. the first cloned sheep had premature ageing and had to be put down.
What can cloned cells be used for?
They can be used to generate cells, tissues and organs to replace those damaged by diseases or accidents
List 4 advantages of cloning cells
- They will be genetically identical to the individuals own cells and won’t be rejected by the immune system
- It could end problems like waiting for donor organs
- They are totipotent so they can be used to generate any type of cell which is good because currently some damage can’t repaired via transplantation.
- It’s less dangerous than major operations/transplantations
Define non reproductive cloning
Cloning that’s used to produce some parts of the body rather than creating a whole organism which is what reproductive cloning does.
Give 3 examples of what non-reproductive cloning can be used for
- The regeneration of heart muscles after a heart attack
- The repair of nervous tissue that’s destroyed by multiple sclerosis
- The repairing of the spinal cord that’s been paralysed due to an accident
What is another term for non-reproductive cloning?
Give 2 disadvantages for non-reproductive cloning
Therapeutic cloning
Ethical issues when using embryonic material
Lack of understanding about how cloned cells will behave over time.
Define biotechnology
It says it in the name. It exploits living organisms/biological processes to improve agriculture, food science, animal husbandry, medicine and industry.
List 4 reasons for using biotechnological processes for commercial uses.
To produce foods
To produce drugs/pharmaceutical chemicals
To produce enzymes/commercial chemicals
For the bioremediation of waste products.
Give 3 examples of foods that are produced via biotechnological processes
Describe the process and the organisms involved
- Cheese and yoghurt - Lactobacillus is grown in milk which changes the texture and flavour of milk. It also prevents other bacteria from growing, which preserves the food
- Mycoprotein/Quorn - Fusarium fungus is grown in a culture which produces mycelium fungus. This is then separated and processed as food.
- Soya sauce - Roasted soya beans are fermented with fungus or yeast, e.g. Aspergillus
Give 2 examples of drugs that are produced via biotechnological processes
Describe the process and the organisms involved
- Penicillin, an antibiotic - Penicillium fungus is grown in a culture and it produces penicillin as a by-product of its metabolism
- Insulin - E. coli bacteria are genetically modified to carry the human insulin gene and the bacteria produces insulin as it grows.
Give 3 examples of enzymes/commercial chemicals that are produced via biotechnological processes
Describe the process and the organisms involved
- Pectinase, used in fruit juice extraction - A. niger fungus is grown in certain conditions to produce and secrete pectinase
- Calcium citrate, for detergents - A. niger fungus produces citric acid as a by-product of its normal metabolism
- Bio-gas fuel production - Methanogenic bacteria is grown on concentrated sewage. It respires anaerobically and generates gases that can be used for fuel.
Give 1 example of the bioremediation of waste products that uses a biotechnological process.
Describe the process and the organism involved
- Waste water treatment - A variety of bacteria/fungi use the organic waste in water as a source of nutrients, this makes the waste harmless. For example, Fusarium is a waste product of the corn milling industry
What type of organism is often used for biotechnological processes?
Microorganisms like bacteria and fungus
Give 5 out of 7 reasons as to why microorganisms are most commonly used in biotechnological processes
- They grow rapidly in favourable conditions with generation times as little as 30 minutes
- They often produce proteins and chemicals that can be harvested
- They can be genetically engineered to produce specific products
- They grow well at relatively low temperatures, much lower than those needed for chemical engineering
- They aren’t dependent on climate so can be grown anywhere in the world
- They generate more pure products in comparison to chemical engineering
- They can be grown using materials that are useless or toxic to humans.
Define culture
A growth of microorganisms. It can be 1 species aka pure culture or it can be a variety aka mixed culture. It can be grown in a nutrient broth or on a nutrient agar gel.
Define closed culture
The growth of microorganisms in an environment where all conditions are fixed and contained. No new materials are added and no waste products/organisms are removed
What is the standard growth curve?
The series of events that occur in a closed culture of microorganisms. This can be plotted on a graph
What are the 4 phases of a standard growth curve in terms of a closed culture?
Describe each phase.
Lag phase: Organisms are adjusting to their environment, they are taking in water, expanding cells, activating genes, synthesising enzymes etc. They are active but not reproducing so the population remains fairly constant.
Log phase: Also called the exponential phase. The population size doubles each generation as there’s enough space and nutrients to reproduce. The population (in some bacteria) can double every 20-30 minutes. Just after this phase, there are less resources so secondary metabolites are produced, these are antibiotics (e.g. penicillin) and they kill off surrounding bacteria to reduce competition.
Stationary phase: Nutrient levels decrease and waste products like CO2/other metabolites increase. Organisms die at the same rate that they are being produced. In an open culture, this would be the carrying capacity.
Death/decline phase: Nutrient exhaustion and increased levels of toxic waste products/metabolites lead to the death rate being higher than the production rate. Eventually all organisms die.
Define the 2 definitions for fermentation
- The use of anaerobic respiration to produce substances, e.g. ethanol from yeast which is a natural by-product of anaerobic respiration.
- The culturing of microorganisms in aerobic or anaerobic fermentation tanks. The useful substances that are generated are separated and treated.
List 3 things that are produced from metabolic processes
- New cells/cellular components
- Chemicals like hormones and enzymes
- Waste products which vary depending on the organism
Define primary metabolites and describe how its production would be represented on a graph
Substances produced by an organism as part of its normal growth. E.g. Enzymes, amino acids, proteins, ethanol, lactate and nucleic acids. On a graph, the production of primary metabolites should match the growth of a population as every organism needs these molecules to survive.
Define secondary metabolites and describe how its production would be represented on a graph
Substances produced by an organism that aren’t part of its normal growth, e.g. penicillium producing the secondary metabolite penicillin. On a graph, it doesn’t match the growth rate because they are only produced after the main growth period.
Do a lot of or not many microorganisms produce secondary metabolites?
Not many microorganisms produce secondary metabolites but all of the produce primary ones.
Define an aseptic technique
Any measure taken at any point in a biotechnological process to ensure that unwanted microogranisms don’t contaminate the culture/products.
Define asepsis
The absence of unwanted microorganisms
What is usually used in order to produce the growth of a particular microorganism on an enormous scale
A industrial-scale fermenter.
What is an industrial scale fermenter?
A huge tank that has a capacity of tens of thousands of litres. The growing conditions in it can be manipulated and controlled to ensure that the best possible yield is obtained.
What are the specific growing conditions of a microorganism?
It depends on the microorganism and whether the process is designed to produce primary or secondary metabolites.
What conditions need to be controlled when growing microorganisms? 4 things
- Temperature - too hot the enzymes will be denatured, too cold the growth will be slowed
- Type/time of addition of nutrient - A nutrient supply is required (e.g. carbon, nitrogen etc.) but the timing depends on whether you want primary or secondary metabolites.
- Oxygen concentration - Most commercial applications grow organisms in aerobic conditions. A lack of oxygen will produce unwanted products from anaerobic respiration and also growth rates will be slowed
- pH - Changes in pH can reduce the activity of enzymes which will reduce the growth rates.
How are starter populations obtained? These are needed for large cultures
By taking a pure culture and growing it in a sterile nutrient broth
List eleven features of a large scale industrial fermenter
- Pressure vent- prevents gas build up
- Sterile air inlet- provides oxygen for aerobic respiration
- Mixing blades- to stir the culture
- Water jacket inlet- allows a circulation of water around the tank to regulate temperature
- Outlet tap- to drain the culture
- Motor- to rotate the mixing blades
- Inlet- for the addition of nutrients
- Water jacket outlet- to remove water
- Electronic probes- to measure oxygen, pH and temperature
- Air outlets- release air into the culture, which mixes it, this is called sparging
- Filters in all the inlets and outlets- this prevents contamination and ensures everything is sterile
What are the two types of cultures/ fermentation operations?
Bath cultures and continuous cultures
Describe a batch culture
The microorganism starter population is mixed with a specific quantity of nutrient solution and is then allowed to grow for a fixed period of time. No additional nutrients are added and this produces secondary metabolites. The fermentation tank is emptied after a set amount of time in order to get the secondary metabolites, which is usually penicillin.
Describe a continuous culture
When nutrients are added to the fermentation tank and products are removed at regular intervals. This process produces primary metabolites like insulin from genetically modified E. Coli
What are the advantages and disadvantages of a batch culture?
Advantages: easy to set up and maintain, if contamination occurs only one batch is lost, useful for producing secondary metabolites.
Disadvantages: growth rate is slower than in continuous cultures as nutrient levels decline, less efficient as it isn’t in operation all if the time.
What are the advantages and disadvantages of a continuous culture?
Advantages: growth rate is higher than batch culture as there’s a constant supply of nutrients, more efficient because the fermenter is always operating, useful for producing primary metabolites.
Disadvantages: set up is difficult and maintenance is hard as optimum growing conditions are constantly required, if contamination occurs huge volumes of product are lost.
What is a contaminant in terms of fermentations?
The growth of unwanted microorganisms in a culture
Give 5 reasons as to why a contaminant is bad in a culture?
- They compete for space and nutrients with the culture microorganisms
- They reduce the yield of useful products from the culture microorganisms
- They can cause spoilage of the product
- They can produce toxic chemicals
- They could destroy the culture microorganisms and their products
List 3 aseptic techniques when growing a starter population
- All apparatus are sterilised before and after use via steam sterilisation, UV light or heat from a flame.
- Work is carried out in a fume cupboard/laminar flow cabinet to provide air circulation which carries the contaminants away from the work space
- The culture is kept closed and away from the mech space whenever possible
List 4 aseptic techniques when using a large scale culture
- Washing, disinfecting and steam cleaning the fermenter and pipes when not in use
- The fermenter surfaces are made from polished stainless steal to prevent microbes sticking to surfaces
- All nutrient media is sterilised before being added to the culture
- Filters on all inlet and outlet pipes to avoid microorganisms entering the fermentation tank
Define immobilisation
Any technique where enzyme molecules are held, separated from the reaction mixture. The substrate molecules can still bind to the enzyme and the products formed go back into the reaction mixture
Name and describe two features that enzymes have which makes them useful in industrial processes
- Specificity- they catalyse reactions between specific chemicals even when other chemicals are present. This means that there are fewer by-products and therefore less purification of the products is needed
- Temperature of enzyme action- they function at relatively low temperatures in comparison to many industrial chemical processes which saves money on fuel costs.
Give an example of an organism that has enzymes which need a high temperature to function. These enzymes are sometimes used in industrial processes.
Thermophilic bacteria which thrive at high temperatures
How many products are usually required when carrying out clinical research/diagnosis/industrial processes?
Only one product is required from a single chemical reaction
Why is it more efficient to use isolated enzymes instead of growing whole organisms or using inorganic catalysts during clinical processes?
Because it would be a waste of time, energy and money to grow a whole organism for just one chemical reaction to produce one product.
Name a process that enables you to isolated an enzyme from a fermentation mixture. Can isolated enzymes be produced in large quantities from this process?
Downstream processing
Yes
What is downstream processing?
A process that involves the separation and purification of any product from large scale fermentations
How can you easily achieve making enzyme-substrate complexes?
By mixing quantities of substrate and isolated enzymes together under suitable conditions
The product generated from enzyme substrate complexes needs to be extracted, is this a cheap or expensive process?
Expensive
Because extracting products from a mixture is expensive, what can you do instead which is cheaper?
You can immobilise enzymes so that they still catalyse enzyme-controlled reactions but they don’t mix freely with the substrate and in turn the product. This means that extraction is cheaper.
When do enzymes and catalysts mix freely with each other?
When don’t they?
In cells or isolated systems
When the enzymes have been immobilised
List 3 advantages of using immobilised enzymes
- Enzymes don’t mix with the products so purification/downstream processing costs are low.
- Enzymes are immediately available for reuse which is useful in continuous processes
- Immobilised enzymes are more stable because the immobilising matrix protects the enzymes
List 3 disadvantages of using immobilised enzymes
- Immobilisation requires additional time, equipment and materials making it more expensive
- Immobilised enzymes can be less active as they aren’t mixing freely with the substrate
- Contamination is costly to deal with as the whole process needs to be stopped
List 4 methods of immobilising enzymes
Adsorption, covalent bonding, entrapment and membrane separation
How do you know which method of immobilising enzymes to use?
It depends on the ease of preparation, cost, relative importance of enzyme leakage/discharge and efficiency
Describe the adsorption method when immobilising enzymes
The enzyme molecules are mixed with the immobilising support and they bind to the support via hydrophobic reactions and ionic links. The bonds are weak and enzymes can become detached (leakage). Nevertheless, adsorption can still give very high reaction rates. Adsorbing agents/support: porous carbon, glass beads, clays and resins
Describe the method of covalent bonding when immobilising enzymes
The enzymes covalently bond to a support and they covalently link with each other and with the insoluble molecule (support). They covalently link using a cross-linking agent like gluteraldehyde or sepharose. This method doesn’t immobilise a large quantity of enzymes but the binding is strong so there is very little leakage. Insoluble material: clay particles
Describe the method of entrapment when immobilising enzymes
Enzymes are trapped in a gel bead or a network of cellulose fibres. They are trapped in their natural state as they don’t bind to any other molecules which could affect their active site (adsorption and covalent bonding). Reaction rates are reduced because the substrates need to get through the trapping barrier which makes the active site less easily available compared to absorption and covalent bonding.
Describe the method of membrane separation when immobilising enzymes
Enzymes are physically separated from the substrate mixture via a partially permeable membrane. The enzymes are on one side and the substrate solution is passed along the other. The substrates are small enough to pass through the membrane and so are the products.