Cloning and Biotechnology Flashcards
What are clones?
Genetically identical organisms or cells
By what process are clones produced?
Clones are produced by asexual reproduction which involves the process of mitosis (for cell division).
2 Identical DNA formed –> 2 Identical Nuclei formed –> 2 Identical cells formed
*If stem cells these may then differentiate into different cells.
Examples of cloning in nature?
When organisms reproduce asexually they produce clones:
- Single celled yeast reproduce asexually via budding
- Bacteria reproduce asexually via binary fission
Advantages of Natural Cloning/Asexual reproduction?
- Good conditions for parent means good condition for the offspring - this is because the parent will already have the genes that allows it to survive and this will be passed onto the offspring directly without change in their genome.
- Rapid reproduction = produce large numbers of offspring to take advantage if suitable environmental conditions
- Requires only one parent
- Can be a backup to sexual reproduction (for organisms that can do both)
Disadvantages of Natural cloning/Asexual reproduction?
- Offspring may be overcrowded
- No genetic diversity = little variation
- Selection is not possible - like no natural selection
- If the environment changes to be less advantageous, the whole population is susceptible.
What is plant cloning by natural vegetative propagation?
The natural asexual reproduction of plant clones from vegetative parts of a plant i.e. from non-reproductive tissues such as roots leaves + stems.
(Not specialised reproductive structures)
Why can plants clone by vegetative propagation?
Many plant cells retain the ability to divide and differentiate (meristem tissues) into a range of different plant cells. This means the plants are able to reproduce by cloning. An adult body cannot do this as they don’t contain many stem cells. This is why a lot more plants reproduce asexually.
What are runners/stolens?
Horizontal stems that run above the ground and produce new clone plants at their nodes (plants form at the nodes of the runner - the roots and the main stem of the cloned plant forms)
What are rhizomes?
Horizontal stems that run below the ground and produce new clone plants at their nodes (new plants form at the nodes of the rhizome - the roots and the main stem of the cloned plant forms)
What are nodes?
The points on a stem where the buds, leaves, branches and roots originate. A small growth zone.
What are suckers?
New stems that grow from the roots or the lower main stem of a plant.
Why are suckers bad sometimes?
Suckers are fast growing, so require lots of nutrients and water. This may cause the original stem to to not recieve enough of the nutrients and water it requires, so may starve and die. This may cause the stem formed as a sucker to become a seperate individual
Why do many plants grow suckers after the main stem has been damaged?
This is because it allows the plant to continue living and growing, even when the parent plant is damaged or dies. The genetic line of that plant is conserved as the stem formed as a sucker will have the same genetic information as the original plant stem.
What are bulbs?
Bulbs consist of an underground stem surrounded by a series of fleshy leaves. They also contain one or more apical bud, that will each grow into plant. New bulbs can develop from the original bulb.
*Look at a diagram on Google images by searching “apical bud of a bulb”
Bulbs are an over-wintering mechanism for perennial monocotyledonous.
What is an overwintering mechanism?
Why are bulbs an overwintering mechanism? FIND ANSWER
An overwintering mechanism is a mechanism by which some organisms survive the winter season, when activity is low and survival would otherwise be difficult.
What are corms?
Corms are swollen underground plant stem with scaly leaves and buds that serve as a storage organ.
This is used by some plants as an overwintering mechanism - corms stay underground in the winter (how is the an overwitnering maechanism) and then in the spring, their buds grow to produce one or more new stems.
When are leaves used for asexual reproduction of plants?
On some plants, their clones grow on teh leaf margins. These immature plant clones then frop off the leaf and take root.
What are tubers?
Tubers are another type of large underground stem/structure that act as a food store for the plant. These tubers can grow into one or more plant and then each plant can produce more tubers.
Name all the methods of natural vegetative propagation and give an example of which plant uses it.
- Runners/Stolons: The Strawberry
- Rhizomes: Bamboo
- Suckers: The Elm Tree
- Bulbs: The onion
- Corms: Root vegetable Taro
- Leaves: The Kalanchoe plant
- Tubers: Potatoes
Examples of natural cloning in animals?
Mammals clone when identical twins are formed - zygote splits to produce two daughter cells which become two separate cell. Each cells grows and develops into a new individual.
- Other animals that naturally clone (asexually reproduce) = the water flea + the greenfly
What is plant cloning by artificial vegetative propagation?
The artificial asexual reproduction of plant clones from vegetative parts of a plant i.e. from non-reproductive tissues such as roots, leaves + stems. This requires human intervention.
Methods of artificial vegetative propagation?
- Cuttings
- Callus Tissue Culture/Micropropagation
*Unsure about grafting
How to take a cutting (simple method)?
1) To make a cutting, a stem (or root, scion or leaf) is cut between two leaf joints (nodes).
2) The cut end of the stem is then dipped in rooting hormone (if required to stimulate growth) before placing that end in moist soil.
3) New roots will grow from the tissues in the stem or around the buried stem, usually from the node to produce a cloned plant.
watch a video or find an aimge to see exaclty whic bit is cut etc.
What is a scion cutting?
Cuttings using a scion rather than a stem - scions are dormant woody twigs.
What is different about leaf cuttings?
- You remove a complete leaf and scoring large veins on the lower leaf surface, and placing that in a growth medium with a warm and moist environment. Once roots have formed, you can plant in soil.
What is tissue culture?
- A series of techniques used to grow cells, tissue or organs from a small sample of cells or tissue.
- Carried out on a nutrient medium under sterile conditions.
- Used commercially to produce large numbers of new plants in micropropagation.
What is micropropagation?
Growing large numbers of plants from meristem tissue taken from a sample plant, using tissue culture.
*Tissue culture usually gets cells/tissues from a plant, places them in culture to multiply + differentiate, allowing a new plant to grow, which is then rooted in soil to produce one plant. Micropropagation is a technique which uses tissue culture, to produce lots of cloned plants. When the cells divide in the culture to one large ball of cells, they are seperated into small clusters of cells (subcultured), each which form a new plant. In tissue culture and micropropagation, meristem cells are usually used.
Stages of micropropagation?
1) Leaf / shoot/ meristem tissue, known as explants, are cut into small pieces. Meristem tissue is usually used as it free from viral infection.
2) Explants are then sterilised using bleach/alcohol to kill any bacteria or fungi which would thrive in the conditions supplied to help the plant grow well.
3) Place sterilised explants in a sterile aseptic growth medium containing nutrients like glucose, amino acids + phosphates. They also contain varying concentrations of auxin and cytokinin which stimulate explants to divide by mitosis to form a callus.
4) The callus is then divided into a large number of small clusters, each made of undifferentiated cells.
5) These small clusters are encouraged to grow divide and differentiate into different plant tissues. This is done by moving the cells for different growth media. The concentration ratio of auxin to cytokinin is used to control which plant tissues will develop.
6) 100 auxin: 1 cytokinin ratio stimulate root formation. 4 auxin: 1 cytokinin ratio stimulate shoot formation.
7) When plantlets, transfer to sterile soil (in greenhouse) to grow.
What is grafting?
A form of asexual reproduction which involves joining the shoot/scion of one plant to the growing stem of another plant.
*How is grafting a form of asexual reproduction? He said it wasn’t and to take it as a seperate concept, not part of artificial vegetative reproduction.
Advantages of artificial cloning?
- Relatively rapid reproduction (faster than from a seed) = tissue culture is much faster than cuttings, but cuttings in general is a fast method of reproduction.
- Backup to sexual reproduction (plant may have lost ability to reproduce sexually/few individuals of plant specie)
- Clones are genetically identical to parent plant = ∴ will display same desirable characteristics.
- The unusual combination of characteristics a plant has due to selective breeding or genetic modification can be retained without the risk of losing that combination in sexual reproduction.
- Uniform phenotype/characteristics means easier to grow and harvest - e.g. same height so can be cut at a specific height for all the genetically identical plants
- If an apical bud is used as an explant and aseptic technique is used, new cloned plant formed from tissue culture will be free from viruses as it contains meristem.
What is the apical bud?
This is the location where shoot growth occurs, usually found at the end of the a shoot.
Disadvantages of artificial cloning?
- Labour intensive
- Expensive to set up facilities to perform successful tissue culture.
- Can fail due to microbial contamination
- Cloned offspring genetically identical = little or no variation (mutation is the only way for variation to occur) susceptible to same pests or disease which would spread rapidly.
Stages of Grafting?
- A stem section of a woody plant (scion) is cut into a wedge
- The root stock is cut to match the scion
- The vascular tissue is lined up
- Binding tape is wrapped around the graft area to hold it in place
Similarities between tissue culture and take cuttings
Tissue culture:
- Requires warm temperatures
- Genetically identical cloned plants are produced
- it’s Thousands of clones can be produced from one plant
- More expensive
- More advanced technology
- Slower method to make cloned plants
- Only require a few cells
Take cuttings: - Requires warm temperatures - Genetically identical cloned plants are produced - Cheaper - Less advanced technology required - Older plants can be used Faster method to make cloned plants - Needs hormones for root growth to speed up process - Requires leaves
Differences between tissue culture and take cuttings
Tissue Culture
- Thousands of clones can be produced from one plant
- More expensive
- More advanced technology
- Slower method to make cloned plants
- Only require a few cells
Take cuttings: - Cheaper - Less advanced technology required - Older plants can be used Faster method to make cloned plants - Needs hormones for root growth to speed up process - Requires leaves
When taking a stem cutting, why are most of the leaves removed?
Most of the leaves are removed from the cutting to reduce water loss. Otherwise, the cutting could dry out very quickly and not grow.
Why is it important to leave some leaves on stem cuttings?
It is important to leave some leaves on cuttings so that photosynthesis occurs to make glucose so new roots can grow.
Suggest reasons why the cuttings should be kept out of direct sunlight.
The cuttings could overheat and lose water; damages very young leaves.
ANIMAL CLONING
What is reproductive cloning?
Reproductive cloning is used to produce genetically identical clone from a donor animal using embryo splitting/twinning or somatic cell nuclear transfer.
Why is cloning useful?
- To increase the number of farm animals with a desirable characteristic (produced from artificial selection/selective breeding or genetic modification)
- Scientists can use cloned animals for research purposes - testing drugs etc.
- Save endangered animals from extinction by cloning new individuals.
Two methods of reproductive cloning?
- Embryo twinning/splitting
- Somatic cell nuclear transfer(SCNT)
Steps of embryo twinning?
1) A zygote (a fertilized egg) is created by in vitro fertilization (fertilization outside a living organism, i.e. in a petri dish)
2) The zygote is allowed to divide by mitosis to form a small ball of cells.
3) The cells are separated and allowed to continue dividing.
4) Each small mass of cells formed is placed into the uterus of a surrogate mother.
5) The embryos continue to develop inside the surrogate mothers and eventually the offspring are born.
6) The offspring are genetically identical to each other. The precise genotype and phenotype of the offspring produced will depend upon the sperm and egg used so they will not be known until the offspring is born
Steps of SCNT in sheep?
1) Differentiated somatic cell is taken from an adult sheep (e.g. udder cell)
2) A ewe is superovulated (using FSH) and egg cells are collected from the ovary / washed out of the oviduct
3) The nucleus is removed from the somatic cell and placed in an enucleated egg cell – an egg cell which has had its nucleus removed.
4) An electric shock is given to fuse the nucleus and the empty egg cell
5) This cell is then placed in a tied oviduct of a ewe to develop into an embryo (in culture)
5) The embryo is then recovered from that ewe and treated with hormones.
6) It is then placed into a surrogate mother’s uterus where it will develop and eventually an offspring will be born.
7) The offspring will be genetically identical to the sheep, which provided the nucleus to be placed in an enucleate egg cell.
Advantage of SCNT over embryo splitting?
The phenotype of the offspring is known before cloning starts. This is not possible with embryo twinning.
- This is the only method that allows cloning of an adult - embryo twinning does not clone adults because the cells of the embryo are formed from random fertilisation which results in the mixing of genetic information from the egg and the sperm - it allows clones of the offspring to be made.
Suggest why an offspring produced through SCNT is not a 100% genetically identical?
- Mitochondrial DNA remains in the enucleated egg cell
- Cloned individual has DNA from the nucleus donor and mitochondrial DNA from the egg donor
What is non-reproductive cloning?
Non-reproductive cloning is the production of cloned cells and tissues for purposes other than reproduction.
Methods of non-reproductive cloning?
Therapeutic Cloning
What is the potential use of therapeutic cloning/reproductive cloning?
- New tissues can be grown to replace dead or damaged tissues - cloned cells have been used to repair damage to nervous tissue in mice (i.e. repair to spinal cord after paralysis) and to regenerate tissue such as that of the heart muscle (after heart attack)
nervous tissue in mice (i.e. spinal cord after paralysis). Skin can be grown in vitro to act as a graft over burned areas - Whole new organs can be grownto replace damaged or diseased ones.
Arguments for artificial cloning in animals?
- We can increase the number of farm animals with a desirable characteristic, as clones retain the same genotype and phenotype of the animal that is cloned.
- Using genetically identical embryos and tissue for research allows the effect of genes and hormones to be assessed with no interference from different genotypes.
- Testing medicinal drugs on cloned cells and tissues avoids using animals or people for testing
- Cells and tissues grown from the patient’s own cells are genetically identical to the donor so can be used in repairing damage caused by disease or injury, without rejection*
- Individuals from an endangered species can be cloned to increase numbers
*e.g. taking stem cells, cloning them by therapeutic cloning, and allowing them to diffferentiate into the correct cells and tissues)
Arguments against artificial cloning in animals?
- Lack of genetic variation, means all members of the herd are susceptible to certain pests or diseases.
- Animals may be produced with little regard for their welfare, which may have undesirable side affects such as meat-producing chickens that cannot walk
- The success rate of adult cell cloning is very poor and the method is a lot more expensive than conventional breeding. (DOES THE TERM ‘ADULT CELL CLONING’ REFER TO BOTH EMBRYO TWINNING AND SCNT?)
- Cloned animals may be less healthy and have shorter life spans.
- There are ethical issues regarding how long the embryo survives and whether it is right to create a life simply to destroy it.
- This does not help increase genetic diversity.
Define biotechnology?
The use of living organisms or parts of living organisms in industrial processes. this could be to produce food drugs or other products
What are the 4 main areas in which microorganisms are used in biotechnology?
- Healthcare and medical processes – e.g. producing drugs (e.g. insulin) with microorganisms and gene therapy
- Agriculture – micropropagation of plants (tissue culture) and GM crops (e.g. with drought tolerance)
- Industry – using microorganisms to produce enzymes
- Food science – developing foods with better taste, nutrition, texture or appearance
What are the advantages of using microorganisms (M) in biotechnology? (in place of chemical processes)
- M = cheap + easy to grow
- Production process of molecules using M can occur at lower temperatures = saves fuel + reduces costs.
- Production process of molecules using M can occur at normal atmospheric pressures = safer
- Production process not dependent on climate - so can occur anywhere in the world as long as the required resources and suitable equipment are available.
- M have short life cycle + reproduce quickly = large population can grow quickly inside the reaction vessel/fermenter.
- M can be genetically modified easily = specific production process is can be achieved
- fewer ethical considerations when using M
- Product made by M are released into the surrounding medium = easy to harvest
- Product made by M are often more pure or easier to isolate = lower downstream processing costs
- M can be fed using by-products from other food industries e.g. starch, waste water or molasses (easy to maintain M, low economic cost)
Examples of when microorganisms are used for food production? Give 4 example.
- Beer production + Making bread rise
- Production of cheese and yoghurt
How are microorganisms used to produce yoghurt?
- Milk which has undergone fermentation by L. bulgaricus + S. thermophilus.
- These convert lactose –> lactic acid.
- Aciditiy of lactic acid denatures milk protein = causing it to clot/coagulate
- Bacteria partially digest milk also = making it easy to digest
- Bacteria like L. acidophilus may be added as probiotics (bacteria which may benefit human health, by improving digestion of lactose, improving gastrointestinal health).
How are microorganisms used to produce cheese?
- Milk which has undergone fermentation by L. bulgaricus + S. thermophilus.
- These convert lactose –> lactic acid.
- Once acidified, milk is mixed with rennet, which contains the enzyme chymosin.
- Rennin coagulates the milk protein (casein) in the presence of calcium ions.
- Kappa-casein, which keeps the casein in solution, is broken down by chymosin, making casein insoluble.
- The casein is precipitated by the action of calcium ions, which bind the molecules together.
- Resulting solid, known as curd, Is separated from the liquid component (by by cutting stiring and heating)
- the bacteria continue to grow producing more lactic acid
- the Curd is then pressed into mould treatment while making impressing the curd determines the characteristics of the cheese.
- Bacteria like L. acidophilus may be added as probiotics (bacteria which may benefit human health, by improving digestion of lactose, improving gastrointestinal health).
How are microorganisms used to produce single-cell proteins (SCP)?
SCPs (aka mycoprotein) are proteins manufactured by microorganisms that can be directly used as food. A microorganism which produces a type SCP is the fungus Fusarium venetatum. An example of an SCP or mycoprotein is Quorn. They can produce proteins with a similar amino acid progile to animal and plant protein and they have no animal fat or cholesterol = suitable substitute for meat/a healthier option than meat.
How are microorganisms used to produce bread?
Anaerobic respiration of yeast (S. cervisae, a microorganism) in the presence of glucose, produces CO2 and ethanol. Fermentation of glucose by yeast is used in used in making bread, by placing the dough in a warm place for 3 hours, so when the yeast respires anaerobically, CO2 is produced to make bread rise. When dough is cooked/baked, any alcohol produced in fermentation evaporates.
How are microorganisms used for brewing of beer?
Anaerobic respiration of yeast (S. cervisae, a microorganism) in the presence of glucose, produces CO2 and ethanol. Fermentation of glucose by yeast is used in beer production, as it produces the ethanol needed. The yeast is added to dried germinating barley grains (malting). The grain converts starch to maltose, which is respired by the yeast anaerobically to produce ethanol and CO2
How are microorganisms used in medicine/for the production of pharmaceutical drugs? Give 2 examples.
- Penicillin production = the fungus, Penicillium notatum, produce an antibiotic called penicillin to stop bacteria from growing + competing for resources. The fungus is now grown on a large scale, in stressful to produce this antibiotic, which is collected and purifies to be used as drug against bacteria in the body.
- Insulin production = Bacteria are genetically modified to produce insulin. The bacteria is then grown in an industrial fermenter on a large scale, to produce insulin which is then collected and purified for use in the body.
How are microorganisms used as enzymes? Give 3 examples.
- Protease and lipase produced by the bacteria, B. licheniformis, is used in washing powders
- Pectinase produced by A. niger is used to extract juice from fruit.
- A. niger (and A . oryzae) produces lactase which breaks down lactose so that lactose-free milk is available
Other uses of microorganisms in biotechnology?
- Biogas (mix of CO2 and methane) produced by anaerobic bacteria (decomposers)
- Bioremediation = a variety of bacteria and fungi remove pollutants, like oil and pesticide from contaminated sites like waste water, by using them as a food source and breaking them down into less harmful products. May be provided with extra nutrients so they grow and multiply quicker.
Advantages of using microorganisms in biotechnology to produce food:
- Protein production by M is faster than by animals/plants.
- Biomass has very high protein content = good source of protein
- Production can be increased and decreased according to demand
- No animal welfare issues
- Protein contains no animal faster or cholesterol
- M can be easily genetically modified to adjust the amino acid content of the protein
- M can be fed using waste/by-products
- Production independent of seasonal variation
- Does not require a lot of land
Disadvantages over using microorganisms in biotechnology to produce food:
- Some people may not want to eat fungal protein all food that has been grown on waste
- Protein needs to be isolated from the fermenter which contains the materials in which they grew and microorganisms.
- protein has to be purified to ensure it is uncontaminated (biomass contains lots of nucleic acids that need to be removed)
- Amino acid profile may differ from traditional animal/plant protein e.g. deficient in certain amino acids
- Conditions for growth of M are also ideal for growth of pathogenic organisms = Need to insure culture is not infected/contaminated.
- Palatability - protein does not have taste or texture of traditional protein sources
Which other processes would come under the term biotechnology?
- Selective breeding
- Genetic modification
- Cloning through embryo-splitting + micropropagation
- Using enzymes in industrial processes
- Immunology
What are fermenters?
Vessels used for commercial drug production. The growing conditions for microbes inside of these vessels are controlled to ensure the best possible yield of product.
What conditions are controlled inside the fermenter?
- Temperature (too hot = enzyme denaturation, too slow = limited microbe growth)
- Nutrient availability (needed for growth of microorganisms)
- Oxygen availability (most microorganisms respire aerobically)
- pH (extreme pH restricts enzyme activity = denaturation)
- Concentration of product – (if product builds up = may affect synthesis process)
Before a fermenter is filled with nutrients + a starter culture, what must be done to it and how?
- It must be sterilised using a superheated steam.
Have a look at the diagram on pg253 of an industrial fermenter, what aspects does it have?
- Water jacket outlet
- Water jacket inlet
- Air Inlet
- Air Outlets
- Outlet tap
- Mixing blades/Impellers
- Motor
- Electronic probes
- Inlet
- Pressure vent
What is the role of pressure vents?
Prevents any gas build-up.
What is the role of air inlets and air outlets ?
Provides sterile air needed for aerobic fermenters, which contain microorganisms that respire aerobically using oxygen in oxidative phosphorylation. Once the air enters the air inlets, it is released into the mixture as air bubbles through the air outlet.
What is the role of mixing blades?
Mixes the contents of the fermenter evenly (i.e. Mixes microorganism with substrate)
What is the role of the water jacket inlet and the water jacket outlet?
The water jacket inlet and outlet allows the circulation of water around the fermenter to regulate temperature. Allows cooling (as respiration produces heat) To maintain optimum temperature for enzyme action.
What is the role of the pressure vent?
Prevents buildup of pressure due to the release of gaseous products.
What is the role of motors?
Rotates the mixing blades.
What is the role of the outlet tap?
Drains the fermenter when necessary.
What is the role of the inlet?
Necessary for the addition of nutrients to synthesise amino acids the microorganism needs.
What is the role of electronic probes?
Needed for measuring oxygen pH and temperature levels in the fermenter. For example, if pH changes, indicates the need for a buffer.
Why are all inlets and outlets fitted with filters?
To prevent contamination.
There are two main methods for culturing microorganisms. What are they?
Batch fermentation
Continuous fermentation
What is continuous fermentation?
- Nutrients are added and products are removed at regular intervals (I.e. continuously) = causes a high microbe growth rate. Volume in the fermenter is kept constant.
- The growing conditions can be difficult to maintain/kept constant (temp, PH, oxygen, nutrients).
- Very inefficient as fermenter is in constant use but if contaminated huge volumes will be lost.
- Useful in producing primary metabolites such as proteins like human insulin from GM E. coli.
What is batch fermentation/culture?
- The microorganism starter population (so population has to be established) is mixed with a set quantity of nutrients, grown for a set time and then the products are removed.
- Produces secondary metabolites (e.g. antibiotics like penicillin) – but not very efficient as fermenter not in use all of the time.
- Short log phase, then the metabolite is produced during stationary phase (after the growth phase).
- Nutrients are only added at start and decrease over time (so growth rate of microbe is slower).
- Build-up of waste products causes reproduction to drop.
- If batch contaminated it can be discarded, without huge losses.
In the case of batch fermentation, the cells must be under _____. How does batch culture achieve this?
- Stress
- This is done by using a high population density of microorganisms with limited nutrient availability in the fermenter.
What is the difference between primary metabolites and secondary metabolites?
- Primary metabolites are the products synthesised by the microorganism during their normal metabolism, when they are actively growing and hence primary metabolites are produced in the growth phase (when the population of microorganisms are being established i.e. log and lag phase). It is initiated when the nutrients required are present in the medium for the microorganism to grow.
- Secondary metabolites are the product synthesised by the microorganism that are not essential for their growth, development and reproduction. These metabolites help protect the nutrient supply from being used by competitors. Secondary metabolites are produced in the stationary phase (so once the population of microorganisms have been established).
Examples of primary and secondary metabolites?
- P: AAs, enzymes, proteins nucleic acids
- S: Antibiotics like penicillin
What is the importance of the aseptic technique?
Ensures sterile conditions are maintained.
This is important because, otherwise the nutrient medium would support the growth of unwanted microorganisms which would:
- compete with the culture microorganisms for nutrients and space
- reduce the yield of useful products
- spoil the product
- -produce toxic chemicals
- destroy the cultured microorganisms and their products.
What happens if the culture becomes contaminated?
It need to be discarded
On a graph of “Production of product vs. time”, there are two graph lines: fungal growth + mass of penicillin (pg254). Using the graph indicate if penicillin is a primary or secondary metabolite and by which method penicillin is made?
- There is a lag (log + lag phase i.e. growth phase) in penicillin production. This indicates the fungus was in the growth phase during this lag and as no penicillin was produced, this tells us that the fungus population has to be established first - the fungus has to be in the stationary phase for penicillin to be produced. Therefore as secondary metabolites are only produced in the stationary phase, penicillin must be a secondary metabolite.
- Secondary metabolites are produced in batch fermentation/culture, so this was the method used.
How is insulin produced? (Brief)
Gene for human insulin combined with plasmid of E.coli (acts as a vector) before inserting the E.coli. The E.coli are then grown in culture to produce insulin.
We have already looked at bioremediation. (These are basically the conditions to control in a fermenter). What conditions are needed?
- Water availability
- Suitable temperature
- Suitable pH
- Oxygen availability
- Nutrient availability
Advantages of bioremediation:
- Uses natural systems
- Less labour/equipment required
- Treatment in situ
- Few waste products
- Less risk of exposure
Disadvantages of bioremediation:
- Treatment time is typically longer than that of other remediation technologies.
- If the process is not controlled it is possible the organic contaminants may not be broken down fully resulting in toxic by-products that could be more mobile than the initial contamination.
What is a closed culture?
A closed culture is a culture which has no exchange of nutrients or gases with the eternal environment (batch cultures have a closed culture). No waste products are removed either.
In a closed culture, like batch fermentation, how does a population of microorganisms grow?
The population of microorganisms follow a standard growth curve. (pg.260 in book, pg.243 in CGP)
*Doesn’t both continuous culture and batch culture produce primary metabolites because they both have a growth phase. Continuous culture will not produce secondary metabolites because it will not reach a stationary phase as nutrients are always being added. If this is right, read through notes and make changes (only a few places).
What are the 4 key phases of the standard growth curve?
- Lag phase
- Log (exponential) phase
- Stationary phase
- Decline or death phase (in closed population)
What is the lag phase?
- Only a few individuals
- Low rate of reproduction/growth as individuals are acclimatising to the environment
What is the log phase?
- Plenty of resources for all microorganisms
- Good environmental conditions as organisms have adjusted to it.
- Rate of reproduction is higher than mortality, so population size grows rapidly
What is the stationary phase?
- The population size has levelled out at the carrying capacity
- Increasing number of microorganisms means lack of nutrients available and waste products start to build up, so number of individuals dieing increases until rate of reproduction and mortality are equal (no population growth)
What is the decline phase?
- Fewer resources available i.e. becomes very scarce (increased competition)
- Waste products build up (high concentrations may be lethal)
- The rate of mortality is greater than reproduction rate, so population size decreases.
What does the length of the lag phase depend on?
Adjusting to growing conditions (e.g. O2 levels, availability of nutrients, pH levels) and synthesis of enzymes is required which takes tim.
What does the length of the log phase depend on?
Competition with other bacteria (if not a closed pure culture), nutrient, O2 & space avail., pH
How would adding nutrients in the lag phase affect the population growth of bacteria?
Adding nutrients would not affect growth at this stage as the cells are active but not reproducing
How would adding nutrients to the stationary phase affect the population growth of bacteria?
The graph line would curve upwards as population size would increase.
Suggest how high temperature, pH and oxygen would affect the growth of a closed culture?
Temperature & pH affects enzyme activity (denature enzymes; cell metabolism ceases). Oxygen is required for aerobic respiration (to release energy for culture/microbe growth)
What are immobilised enzymes in biotechnology?
an enzyme that is held in place in not free to diffuse through the solution.
Some biotechnological process use enzymes that have been taken out of microorganisms. What happens to the enzymes when they take part in these bio technological processes?
They catalyse the reaction by binding to substrates and converting it into the product. The enzyme is not used up in the reaction.
What is the disadvantage of enzymes not being used up in a reaction?
Enzymes remain in suspension/in the final solution when the reaction is completed therefore the product must be isolated from the enzymes before use - this could be expensive.
What are the advantages of enzymes in catalysing industrial processes?
- Enzymes produce fewer by-products
- Lower optimum temp compares to chemical catalysts (less expensive)
- No need to grow whole organisms in order to make a product – requires sometimes only one isolated enzymes.
How could we prevent the enzymes being mixed with the products at the end?
Use immobilised enzymes - these are held in position so that they do not mix freely with the substrate so will not be present in the final solution.
What is the benefit of immobilised enzymes?
- Enzymes do not mix with product = extraction/purification costs lower
- Enzymes can easily be reused
- Easier to carry out continuous processes as there are no cells requiring nutrients or producing waste products that would otherwise need to be removed, only enzymes present.
- Enzymes are surrounded by immobilising matrix, which protects it from extreme conditions - so higher temperatures or wide pH range can be used without causing denaturing.
Disadvantages of immobilised enzymes?
- If contamination occurs, current reaction mixture must be discarded – expensive + loss of product.
- Setting up the immobilised enzyme process is more expensive.
- Immobilised enzymes are usually less active than free enzymes (not mixed together as well) = making the process slower.
What are the methods to immobilise enzymes?
- Adsorption
- Covalent bonding
- Entrapment
- Membrane separation
What is adsorption?
Enzyme molecules bound to a supporting surface by hydrophobic interactions + ionic links, with their access site exposed and accessible to substrate.
Suitable surfaces for adsorption?
Clay, porous carbon, glass beads and resin.
Disadvantages of adsorption?
- Active site of enzymes may be slightly distorted by the additional interactions to the supporting surface = this may affect enzyme activity.
- Interactions and forces between enzyme and supporting surface are not strong so enzyme may become detached and leak into the reaction mixture.
What is covalent bonding in the production of immobilised enzymes?
Enzymes are bonded to the supporting surface using strong covalent bonds. They are bonded using a cross linking agent which may also link them in the chain.
Disadvantages of covalent bonding?
Production of covalent bonding could be expensive and can distort the enzyme active site = reducing enzyme activity, however the enzymes are much less likely to become detached and leak into the reaction mixture .
What is entrapment?
Enzymes trapped in a matrix by a network of e.g. cellulose fibers or gel bead that does not allow free movement of enzymes. These enzymes are unaffected by entrapment and remain fully active. However, the substrate molecules must be small enough to diffuse into the entrapment matrix and the product molecules must be small enough to diffuse out.
Disadvantages of entrapment?
Access to enzymes may limit the reaction.
What is membrane separation?
Enzyme molecules separated from the reaction mixture by a partially permeable membrane. The substrate and product molecules are small enough to pass through the partially permeable membrane by diffusion.
Disadvantage of membrane separation?
Access to enzymes may limit the reaction
Industrial use of immobilised enzymes?
Glucose isomerase Glucoamylase Penicillin acylase Lactase Aminoacylase Nitrile Hydratase
How does penicillin acylase work? (immobilised enzymes)
Method of immobilisation: Entrapment, adsorption and covalent bonding
Substrate: Penicillin
Product: Amino penicillanic acid
Use:: Amino penicillanic acid is the base molecule used to produce a range of penicillin–type antibiotics (including amoxicillin and ampicillin). Useful because some penicillin-resistant bacteria are not resistant to these synthetic types
How does glucose isomerise (aka xylose isomerase) work? (immobilised enzymes)
Method of immobilisation: Adsorption or covalent bonding
Substrate: Glucose + xylose
Product: Fructose + xylulose
Use: To produce high fructose corn syrup
How does lactase work?
Method of immobilisation: entrapment (alginate beads)
Substrate: Lactose
Product: Glucose and Galactose (by hydrolysis)
Use: To produce lactose-free milk. Milk provides calcium to prevent the development of weak bones and osteoporosis therefore it is important in a diet. This allows lactose intolerant people to still be able to get access to the calcium in milk.
What is the use of aminocyclase? (immobilised enzymes)
Method of immobilisation: adsorption
Substrate: N-acyl-amino acid
Product: L-amino acid by removing acyl group from the nitrogen on the N-acyl-amino acid.
Use: L-amino acids are the base molecules for the synthesis of pharmaceutical and agrochemical compounds like animal feed, pesticides and herbicides
What is the use of glucoamylase? (immobilised enzymes)
Method of immobilisation: Covalent bonding
Substrate: Dextrins or maltose
Product: Glucose (When starch hydrolysed to glucose, dextrins, which are short polymers of glucose, may be formed. Glucoamylase further hydrolyses dextrins to glucose).
Use: Digest sources of starch like corn, used in fermentation processes including conversion of starch pulp to alcohol used to produce gasahol – an alternative fuel for motor vehicles. Also used to make high fructose corn syrup.
What is the use of nitrile hydratase? (immobilised enzymes)
Method of immobilisation: Entrapment
Substrate: Nitriles
Product: Amides
Used for: Conversion of acrylamide to polyacrylamide, which is a plastic used as a thickener. Polyacrylamide is mainly used in the treatment of water as it helps stick contaminants together so they are precipitated out and easy to filter out of the water. Used to make gel for electrophoresis + involved in paper-making.
