6.4 Cloning and biotechnology Flashcards
define clones
genetically identical organisms or cells
how are clones produced
by asexual reproduction in which the nucleus is divided by mitosis where is creates two identical copies of DNA. These cells may not always be physically or chemically identical as, after division, they may differentiate to form two different types of cell
What is an example of cloning
yeasts reprudce by budding
bacteria reproduce by binary fission
what are advanatages of natual cloning
-if the conditions for growth are good for the parent, they will also be good for the offspring
-cloning is relatively rapid so the population can increase quickly to take advanatage of the suitable environmental conditions
-reproduction can be carried out even if there is only one parent
what are the disadvantages of natural cloning
-the offspring may become overcrowded
-there will be no genetic diversity (except for when theres mutation)
-the population shows little variation
-selection is not possible
-if the environment changes to be less advanatgeous as the whole population is susceptible
what is vegetative propagation
the process of reproduction through vegetative parts of the plant, rather than through specialised reproductive structures
What are runners, stolen and rhizomes?
-horizontal stems that can form roots at certain points
-they are called stolens of they grow on the surface of the ground
-they are called rhizomes if they are underground
How are some rhizomes adapted?
-they are thickened over-wintering organs from which one of more new stems will grow in the spring
what are suckers?
-new stems that grow from the roots of a plant
-these may be close to the base of an older stem or could be some distance away
-in all cases, the original horizontal branch may die, leaving the new stem as a separate individual
Describe Bulbs
-an over-wintering mechanism for many perennial monocotyledonous plants
-they consist of an underground stem from which grow a series of flesh leaf bases
-they have an apical bud which will grow into a new plant in the spring
-often a bulb contains more that one apical bud and each will grow into a new plant
Describe Corms
-solid rather than fleshy like a bulb
-they are an underground stem with scaly leaves and buds
-they remain in the ground over winter
-in the spring, the buds grow to produce one or more new plants
-croci and gladioli reproduce using corms
Describe leaves
-Kalanchoe plant reproduces asexually as clones grow on the leaf margins
-the immature plants drop off the leaf and take root
Describe Tubers
-another type of underground stem
-potatoes are tubers
-one potato will grow into one or more plants
-each new plant can then produce many new tubers
Describe cloning in animals
-do not clone as often as plants
-mammals clone when identical twins are formed
-occurs when a zygote divides as normal but two daughter cells then split to become two separate cells
-each cell grows and develops into a new individual
-the water flea and greenfly are examples of animals that commonly reproduce asexually to produce clones
Define micropropagation
growing large numbers of new plants from meristem tissue taken from a sample plant
Define tissue culture
growing new tissues, organs or plants from certain tissues cut from a sample plant
What is the easiest way to create clones?
-through making cuttings
-to make a cutting, a stem is cut between two leaf joints (nodes)
-the cut end of the stem is then placed in moist soil
-new roots will grow from the tissues in the stem (usually from the node) but they may grow from other parts of the buried stem
What are cuttings used for?
To produce large numbers of plants very quickly
What other treatment may some plants need as well as making cuttings?
-dipping the cut stem in rooting hormone to help stimulate root growth
-may be helpful to wound or remove the bark from the cut end of the stem as this encourages the plant to produce a callus
What parts of the plant can be used to make cuttings?
-Root cuttings, a section of root is buried just below the soil surface and produces new shoots
-scion cuttings, which are dormant woody twigs
-leaf cuttings, a leaf is placed on moist soil. the leaves develop new stems and new roots. some leaves may produce many new plants from one cutting
Why is cloning by taking cuttings not alway appropriate?
-can be time consuming
-needs a lot of space
-some plants don’t respond well to taking cuttings
How is tissue culture carried out?
-carried out on a nutrient medium under sterile conditions
-application of plant growth substances at the correct time can encourage the cells in the growing tissue to differentiate
-it is widely used commercially to increase the number of new plants
What does micropropagation involve?
- Suitable plant material is selected and cut into small pieces (explants) Meristem tissue is often used as it is always free virus infection
2.They are sterilised using dilute bleach or alcohol- essential to kill any bacteria and fungi, as these would thrive in the conditions supplied to help the plant grow well - They are placed on a sterile growth medium containing suitable nutrients such as glucose, amino acids and phosphates. The gel contains high concentrations of he plant growth substances auxin and cytokinin which stimulates the cells of each explant to divide by mitosis to form a callus.
4.Once a callus has formed, it is divided to produce a larger number of small clumps of undifferentiated cells
5.These clumps of cells are stimulated to grow, divide and differentiate into different plant tissues. This is achieved by moving the cells to different growth media. each medium contains different ratios of auxin and cytokinin. The first medium contains the ratio 100 auxin: 1 cytokinin, and this stimulates roots to form. The second medium contains the ratio of 4 auxin: 1 cytokinin which stimulates the shoots to form. - Once tiny platelets have been formed, these are transferred to a greenhouse to be grown in compost or soil and acclimatised to normal growing conditions
What are explants?
-can be tiny pieces of leaf, stem, root or bud
What are advantages of artificial cloning?
-rapid method of producing new plants rather than growing them from seed
-can be carried out where sexual reproduction is not possible e.g commercially grown bananas
-plants selected will be genetically identical to the parent plant- display same characteristics eg. high yield, resistance to disease and colour
-new plants are uniform in their phenotype which makes them easier to grow and harvest
-using the apical bud as an explant for tissue culture ensures the new plants are free from viruses
What are disadvantages of artificial cloning?
-tissue culture is labour intensive
-expensive to set up the facilities to perform tissue culture
-cloned offspring are genetically identical so are susceptible to same pests/disease
-crops grown in monocultures allow rapid spread or a disease
-no genetic variation, exceptions of mutation
define embryo twinning
splitting an embryo to create two genetically identical embryos
define enucleation
removal of the cell nucleus
define somatic cell nuclear transfer
a technique that involves transferring the nucleus from a somatic cell to an egg cell
What may cloning of animals be useful for?
-elite farm animals produced by selective breeding or genetic modification eg. a bull whose value is as a stud, supplying sperm for artificial insemination
-genetically-modified animals developed with unusual characteristics eg. cows that produce less methane
What are the 2 main techniques to achieve reproductive cloning?
-embryo twinning (produce identical offspring)
-somatic cell nuclear transfer (only way to clone an adult)
Describe how Embryo splitting is done
- a zygote is created by IVF
- The zygotę is allowed to divide by mitosis to form a small ball of cells
- The cells are separated and allowed to continue dividing
- Each small mass of cells is placed into the uterus of a surrogate mother
What has Embryo splitting been used for?
-to clone elite farm animals or animals for scientific research.
-precise genotype and phenotype of offspring is unknown until animals are born as they depend on the sperm and egg used
Describe Somatic cell nuclear transfer
- An egg cell is obtained and its nucleus is removed (enucleation)
- A normal body cell (somatic cell) from the adult to be cloned is isolated and may have the nucleus removed
- The complete adult somatic cell or its nucleus is fused with the empty egg cell by applying an electric shock
- The shock also triggers the egg cell to start developing, as though it had just been fertilised
- The cell undergoes mitosis to produce a small ball of cells
- The young embryo is placed into the uterus of a surrogate mother
What is non-reproductive cloning?
The production of cloned cells and tissues for purposes other than reproduction
What is therapeutic cloning?
When new tissues can be grown as replacement parts for people who are not well:
-Skin can be grown in vitro to act as a graft over burned areas
-cloned cells have been used to repair damage to the spinal cord of a mouse and to restore the capability to produce insulin in the pancreas
-There is potential to grow whole new organs to replace diseased organs
-Tissues grown from the patients own cells will be genetically identical and so avoid rejection, which battles the problem when transplanting donated organs
How can cloning be used for scientific research?
-research into the action of genes that control development and differentiation
-used to grow specific tissues or organs for use in tests on the effects of medicinal drugs
What are the arguments for artificial cloning in animals?
-can produce a herd of animals with a high yield or showing an unusual combination of characteristics
-produces genetically identical copies of very high value individuals containing the same characteristics
-using genetically identical embryo and tissues allows the effects of genes and hormones to be assessed with no interference of other genotype
-testing medicinal drugs on clones cells avoids using animals for testing
-can produce cells genetically identical to the donor to repair damage from accidents
-individuals of endangered species can be cloned to increase numbers
What are the arguments against artificial cloning in animals?
-lack of genetic variation may expose herd to certain diseases or pests
-animals may be produced with little regard for their welfare (eg chickens used for meat unable to walk)
-success rate of adult cell cloning is very poor and method is more expensive than breeding
-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
-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
How are microorganisms in food used in biotechnology?
-ethanol in beer and wine
-co2 used to make bread rise
-lactic acid used to make yoghurt and cheese
-soya beans are fermented to produce soy sauce
How are microorganisms in pharmaceutical drugs used in biotechnology?
-penicillin
-other antibiotics
-insulin, other therapeutic human proteins
How are microorganisms in enzymes used in biotechnology?
-protease and lipase used in washing powders
-pectinase used to extract juice from fruit
-sucrase used to digest sugar to make food sweeter
-amylase to digest starch into sugar to produce syrup used as a sweetener
-protease used to tenderise meat
-lactase to make lactose-free milk
How are microorganisms in other products used in biotechnology?
-biogas, a combination of co2 and methane
-citric acid, a food preservative
-bioremediation- cleaning waste water
What are advantages of using microorganisms in biotechnology?
-relatively cheap and easy to grow
-production process takes place at lower temp than would be required by chemical engineering which means it saves fuel and reduces costs
-can take place at normal atmospheric pressure, safer than using chemical reactions that may require high pressure
-microorganism can be fed by products from other food industries
-have a short life cycle and reproduce quickly so large population can grow very quickly inside the reaction vessel
-they can be genetically modified relatively easily
-there are fewer ethical considerations to worry about
-products are released from the microorganism into the surrounding medium which makes the product easy to harvest
-product is more pure/easier to isolate than in conventional chemical engineering which means lower downstream processesing costs
What does biotechnology also encompass?
-gene technology
-genetic modification and gene therapy
-selective breeding
-cloning by embryo-splitting and micropropagation
-the use of enzymes in industrial processes
-immunology
How is biotechnology used to make yoghurt?
-yoghurt is milk that has undergone fermentation
-bacteria convert lactose to lactic acid
-the acidity denatures the milk protein causing it to coagulate
-bacteria partially digest the milk making it easy to digest
-fermentation also produces the flavours of yoghurt
How is biotechnology used to make cheese?
-bacteria convert lactose to lactic acid
-the acidity denatures the milk protein causing it to coagulate
-once it is acidified, milk is mixed with rennet which contains the enzyme rennin
-rennin coagulates the milk protein (casein) in the presence of calcium ions
-kappa-caesin, keeps the casein in solution, is broken down which makes casein insoluble
-casein is precipitated by the action of calcium ions, which binds the molecules together
-resulting solid, curd, is separated from the liquid component by cutting, stirring and heating
-the bacteria continue to grow, producing more lactic acid and the curd is pressed into moulds
How is biotechnology used to make bread?
1.mixing- ingredients are mixed through kneading
2. proving/fermenting- dough is left in warm place so yeast respires anaerobically which produces co2 bubbles, causing the dough to rise
3. cooking- the risen dough is baked and any alcohol evaporated during the cooking process
How is biotechnology used to make alcoholic beverages ?
-wine is made using grapes that have yeasts on their skin. They contain fructose and glucose so when they are crushed, the yeast uses these sugars to produce co2 and alcohol
-beer is brewed using barley grains which are begininning to germinate which is called malting. as the grain germinates, it converts stored starch to maltose, which is respired by yeast. Anaerobic respiration produces co2 and alcohol.
Describe how biotechnology contributes to single-cell protein
-microorganisms have been used to manufacture protein that is directly used as food
-The fungal protein or mycoprotein is known as single-cell protein eg Quorn which is marketed as a meat substitute for veggies and a healthy option for non veggies
-microorganisms such as candida can produce protein with a similar amino acid profile to animal and plant protein, can grow on almost any organic substrate including waste materials such as paper and whey
What are the advantages of using microorganisms in biotechnology?
-production of protein can be faster
-biomass produced has high protein content
-production can be increases and decreased based on demand
-no animal welfare issues
-microorganisms provide good source of protein
-protein contains no animal fat or cholesterol
-it can be easily genetically modified to adjust the amino acid content of the protein
-SCP production could be combined with removal of waste products
-production is independent of seasonal variations
-not much land is required
What are the disadvantages of using microorganisms in biotechnology?
-people may not want to eat fungal protein or food that was grown on waste
-microorganisms are grown in huge fermenters and need to be isolated from the material on which they grow
-protein has to be purified to ensure it is uncontaminated
-microbial biomass can have high proportion of nucleic acids which must be removed
-amino acid profile may be different from traditional animal protein, can be deficient in methionine
-conditions needed for microorganisms to grow are ideal for pathogenic organisms so there is risk of infection and extra care must be taken to ensure culture is not infected with wrong organisms
-the protein does not have the taste or texture of traditional protein sources which affects the palatability
what does commercial drug production use?
-large containers called fermenters
-the growing conditions can be controlled to ensure the best possible yield of the product
what are the conditions that must controlled in a fermenter?
-temperature: too hot, enzymes denature, too cold, growth limited
-nutrients available: sources of carbon, nitrogen, minerals and vitamins are needed for microorganisms to grow and synthesise the product
-oxygen availability: microorganisms respire aerobically
-PH: enzyme activity and growth are affected by extremes of pH
-concentration of product: if product is allowed to build up it may affect the synthesis process
What is the first step of a fermenter?
-must be sterilised using superheated steam
-then can be filled with all the components required for growth and supplied with a starter culture of the microorganism to be used
-culture will be left to grow and synthesise the products
Describe the structure of a industrial fermenter
-on top left there is pressure vent to prevent gas build up
-next on top left is air inlet which provides sterile air to enter to provide oxygen in aerobic fermenters
-bottom left there is water jacket inlet which allows circulation of water around the fermenter to regulate temperature
-top middle is motor which rotates the blades to mix culture evenly
-under blades there are air outlets which release air bubbles to mix with the culture (known as sparging)
-top right is inlet for the addition of nutrients
-below that is water jacket outlet
-right bottom are electronic probes for measuring oxygen, ph and temp
-bottom middle is outlet tap for draining fermenter
what are all inlets and outlets fitted with in a fermenter?
filters to prevent contamination
Define primary metabolites
products that are synthesised by the microorganism during normal metabolism when they are actively growing
describe continuous culture
-keeps the microorganism growing at a specific growth rate
-products are continually releases from the cells and can be extracted continuously from the fermenting broth
-broth is topped up with nutrients as they are used by microorganisms
-some broth is removed regularly to extract the product and remove cells from the broth as otherwise, the population will become too dense
Define secondary metabolites
-products that are produced only when the cells are placed under stress, such as high population density or limited nutrient availability
-produced mostly during the stationary phase of growth
describe batch culture
-culture is set up with a limited quantity of nutrients and is allowed to ferment for a specific time
-after this time, the fermenter is emptied and the product can be extracted from the culture
describe asepsis
-it is ensuring that sterile conditions are maintained
The nutrient medium also supports the growth of unwanted microorganisms which would reduce production as the unwanted microorganisms:
-compete with the cultured micro-organisms for nutrients and space
-reduce the yield of useful products
-spoil the product
-may produce toxic chemicals
-may destroy the cultured micro-organism and their products
What must be done if unwanted microorganisms contaminate the cultured microorganisms?
All products must be discarded in any processes where foods or medicinal chemicals are produced
What is Penicillin?
-a secondary metabolite as it is only produced once the population has reached a certain size
-therefore manufactured through batch culture
Describe the production of Penicillin
- fermenter is run for 6-8 days then culture is filtered to remove the cells
- Antibiotic is precipitated as crystals by the addition of potassium compounds, may be modified by the action of other microorganisms or by chemical means
3.Antibiotic is mixed with inert substances and prepared for administration in tablet, syrup or form suitable for injection
What does microorganisms using enzymes to carry out required processes mean?
any condition suitable for enzyme activity is likely to be suitable for bacterial and fungal growth
Describe the production of Insulin
-widely used to treat type 1 diabetes
-previosuly extracted from the pancreas of animals but this wasn’t identical to human insulin so it was less effective than human insulin and expensive to extract
-gene for human insulin was combined with a plasmid to act as vector so it could be inserted into the bacterium e-coli
-this enabled the production of large quantities of human insulin at low cost
-it is manufactured through continuous culture
What is Bioremediation
-the use of microorganisms to clean the soil and underground water on polluted sites
-the organisms convert the toxic pollutants to less harmful substances
-can be used for treating oil spills, solvents and pesticides
What does Bioremediation involve
-stimulating the growth of suitable microbes that use the contaminants as a source of food
-it requires the right conditions for the growth of microorganisms which are: available water, a suitable temperature, suitable pH
What happens when the conditions are not suitable for stimulating growth for microbes therefore bioremediation?
-the conditions may be modified by the addition of suitable substances
-in some cases, additional nutrients such as molasses may be needed to ensure the microorganisms can grow effectively
-may be necessary to pump in oxygen for aerobic bacteria
-where conditions cannot be made suitable in situ, the soil may be dug up and moved to be treated ex situ
What are the advantages of bioremediation?
-uses natural systems
-less labour/equipment is required
-treatment in situ
-few waste products
-less risk of exposure to clean-up personnel
What are the disadvantages of bioremediation?
only suitable for certain products; heavy metals such as cadmium and lead cannot be treated
define aseptic technique
sterile techniques used in culturing and manipulating microorganisms
Where can microorganisms grow?
on almost any material that provides carbon compounds for respiration and a source of nitrogen for protein synthesis
Where are microorganisms grown in the laboratory?
one of two types of growth medium:
-soup-like liquid called a broth, kept in bottles or tubes
-set jelly-like substance called agar, which is melted and poured into Petri dishes
What have aseptic techniques been developed to do?
-reduce the likelihood of contaminating the medium with unwanted bacteria or fungi
Describe the standard procedure of aseptic techniques
- wash hands
2.disinfect working area - have Bunsen burner operating to heat air so the air rises and prevents air-borne microorganisms settling
4.do not lift lid of Petri dish completely, only open enough for introduction of desired microorganism
5.as you open a vessel, pass the neck of the bottle over the flame to prevent bacteria in the air entering the bottle, bottle should be flamed when closed
6.glassware or metal equipment should be passed through flame before and after contact with the desired microorganisms
What are the main steps of growing microorganisms on agar plates?
1.sterilisation
2.inoculation
3.incubation
Describe sterilisation
-medium is sterilised by heating in an autoclave at 121*c for 15 mins
-this kills all living organisms including bacterial and fungal spores
-when medium has cooled, it is poured into a sterile Petri dish and left to sit: important lid is kept on to prevent infection
-everything must be sterilised through flame before and after use
Define inoculation
the introduction of microorganisms to a sterile medium
What are the 4 ways of inoculating
-streaking
-seeding
-spreading
-small cotton swap moistened with distilled water used to collect microorganisms from a surface and then wiped over surface of agar medium
describe streaking
wire inoculating loop used to transfer a drop of liquid medium onto the surface of the agar
drop is drawn out into a streak by dragging the loop across the surface
describe seeding
sterile pipette used to transfer a small drop of liquid medium to the surface of the agar or to the Petri dish before agar is poured in
describe spreading
sterile glass spreader used to spread the inoculated drop over the surface of the agar
Describe incubation
-Petri dish must be labelled and the top taped to the bottom
-do not seal dish completely as this can lead to selection of anaerobic bacteria which may be pathogenic
-dish must then be placed in a warm environment eg incubator
-must be placed upside down so drops of condensation do not fall onto the surface of the agar and so the agar medium doesn’t dry out too quickly
-cultures can be examined after 24-36hrs
-Do not open the Petri dish- bacteria grow into visible colonies
-filamentous fungi grow into a mass of hyphae: mass is not shiny and looks like cotton wool with fluffy aerial hyphae
-single celled fungi grow as circular colonies
-all Petri dishes must be completely sterilised after use and before disposal
what will you see if you grow microorganisms in a liquid medium?
-liquid broth is initially clear but will turn cloudy when bacteria have grown
-can be useful to increase the numbers of microorganisms before transferring to agar plates for counting or identification
-aspectic technique must also be applied
What can a liquid broth be used for?
-can be used to measure the growth rate of a microorganism population
-sterile broth is inoculated and population size is measured at regular intervals during incubation
-population size can be measured by transferring a small sample to an agar plate and incubating the agar culture
What may a serial dilution be used for?
If a broth is used to inoculate an agar place, there may be too many colonies which merge together making a count impossible
To investigate the rate of growth of the population of microorganisms, it will be essential to reduce the population density
Define closed culture
a culture which has no exchange of nutrients or gases with the external environment
define serial dilution
a sequence of dilutions used to reduce the concentration of a solution or suspension
What factor is broth diluted by? and what occurs after?
-At each step the broth is diluted by a factor of 10. 1cm3 of broth and 9cm3 of distilled water to make 10-1 and repeat step to achieve 10-2 etc.
-drop of each dilution can be used to inoculate an agar plate, one of them will produce a culture plate in which the number of colonies can be counted
-when recording the population density, do not forget to multiply your count by the dilution factor and also by the volume added to the plate
Describe the growth curve
-a small population of microorganisms in a closed culture that contains all the nutrients required for growth will undergo population growth
1.lag phase- beginning of curve
2. log/exponential phase- when curve goes up
3.stationary phase- curve plateaus
4.death/decline phase- curve drops
Describe the lag phase of a growth curve
-population does not grow quickly bc population is small and organisms are adjusting to their new environment
This may involve: taking up water, cell growth, switching on certain genes, synthesising specific proteins
Describe the Log phase of the growth curve
-organisms have adjusted to their environment
-each have enzymes needed to survive, sufficient nutrients, space to grow rapidly and reproduce
-population doubles in size with each generation
Describe the stationary phase of the growth curve
-there is no population growth
-increasing numbers of organisms use up the nutrients and produce increasing amounts of waste such as CO2 and other metabolites
-rate of population growth declines and number of individuals dying increases until the reproduction rate equals the death rate
Describe the Death phase of the growth curve
- nutrients run out and concentration of waste products may become lethal
-more individuals die than are produced and population begins to fall
-eventually all organisms die
When will primary metabolites be collected from a fermenter?
-during the log phase
-population is not kept in a closed culture, but conditions are maintained for optimal growth
When will secondary metabolites be collected from a fermenter?
-end of the stationary phase
-population must be kept in a closed culture and the metabolites can be collected at the end of the stationary phase of during decline phase
define immobilised enzyme
an enzyme that is held in place and not free to diffuse through the solution
What are the advantages of immobilised enzymes?
-enzymes do not mix with the product, so extraction costs are lower
-enzymes can easily be reused
-a continuous process is made easier as there are no cells requiring nutrients, reproducing and releasing waste products
-enzymes are surrounded by the immobilising matrix which protects them from extreme conditions so higher temp or wider pH range can be used w/o causing denaturing
What are the disadvantages of immobilised enzymes?
-setting up immobilised enzyme process is more expensive
-immobilised enzymes are usually less active than free enzymes making the process slower
What are the methods of immobilising enzymes?
-Adsorption
-covalent bonding
-Entrapment
-Membrane seperation
Describe adsorption for immobilised enzymes
-enzymes are bound to a supporting surface by a combination of hydrophobic interactions and ionic links
-suitable surfaces include clay, porous carbon, glass beads and resin
-enzymes are bound with the active site exposed and accessible to the substrate
-active site may be slightly distorted by the additional interactions affecting enzyme activity
-bonding forces are not always strain so enzymes can become detached and leak into the reaction mixture
Describe covalent bonding for immobilised enzymes
-enzymes are bonded to a supporting surface such as clay using strong covalent bonds
-they are bonded using a cross-linking agent which may link them in a chain
-can be expensive and distort enzyme active site reducing activity
-enzymes are less likely to become detached and leak into the reaction mixture
Describe Entrapment for immobilised enzymes
-enzymes are trapped in a matrix that doesn’t allow free movement
-they are unaffected by entrapment and remain fully active
-substrate molecules must diffuse into the entrapment matrix and the product molecules must be able to diffuse out
-only suitable for processes where the substrate and product molecules are relatively small
-calcium alginate beads are often used and cellulose mesh may be used in industrial processes
Describe membrane separation for immobilised enzymes
-enzymes are separated from the reaction mixture by a partially permeable membrane
-substrate and product molecules must be small enough to pass through the partially permeable membrane by diffusion
-access to enzymes may limit the reaction rate
