Cloning and biotechnology Flashcards
What are clones and how produced
genetically identical organisms or cells
formed from mitosis
Advantages of natural cloning in plants
-conditions good for parent, then good for offspring
-quick
-only need one parent
Disadvantages of natural cloning in plants
-offspring may become overcrowded
-no genetic diversity
-little variation so if the environment changes, the whole population is susceptible
Examples of natural clones in plants
-runners/stolons- horizontal stems that form roots on the surface
-rhizomes- same as runners but grow underground
-suckers- new stems that grow from the root of the plant
-bulbs- contain multiple apical buds which grow into new plants
-corms-underground stem with scaly leaves and bulbs
-leaves
-tubers
Using natural clones (cuttings)
-farmers and gardeners take advantage of vegetative propagation
-where cut the stem at the nodes and place in new soil to grow
-also take cuttings from root, scion and leaf
Tissue culture
-growing new tissues, organs or plants from certain tissues cut from a sample plant
-used in micropropagation
Micropropagation steps
-plant tissue is cut into small pieces= explant
-explant sterilised in bleach or alcohol
-placed on growth medium eg agar with auxin and cytokinins
-this stimulates the explant to divide forming a callus
-callus is divided into smaller clumps and stimulated to grow by moving them through different mediums of auxin and cytokinins
-when tiny plantlets form they are transferred to a greenhouse
Advantages of artificial cloning in plants
-quick
-only needs one parent
-genetically identical so will show same desired characteristics
-uniform phenotype so easy to grow and harvest
Disadvantages of artificial cloning in plants
-labour intensive
-expensive to set up and perform tissue culture successfully
-all genetically identical and susceptible to same diseases
-no genetic variation
Examples of natural clones in animals
-identical twins
-water flea and greenfly divide asexually
What cells used in artificial cloning in animals
totipotent cells that can differentiate into any cell
Examples of reproductive cloning in animals
Embryo splitting
Somatic cell nuclear transfer
Embryo splitting
-zygote is created by in vitro fertilisation (IVF)
-zygote allowed to divide by mitosis to form a small ball of cells
-cells are separated and allowed to continue dividing
-each small mass of cells is placed into the uterus of a surrogate mother
Somatic cell nuclear transfer
-egg obtained and nucleus removed= enucleation
-normal body cell from the adult to be cloned is isolated
-complete adult somatic cell is fused with empty egg cell by electric shock
-shock triggers egg to develop as if it’s fertilised
-cell divides by mitosis producing ball of cells
-embryo places into the uterus of a surrogate
Non reproductive cloning
Therapeutic cloning
-skin grown in vitro to graft over burnt areas
-repair damage to spinal cord of mice that are used to help pancreas produce insulin
Cloning for scientific research
-research action of genes that control development and differentiation
-grow tissues to test effects of drugs
Advantages artificial cloning in animals
-produce high yield of animals with desired characteristics eg. cows that produce lots milk
-genetically identical copies with same characteristics
-endangered species can be cloned to increase numbers
Disadvantages of artificial cloning in animals
-lack genetic variation, susceptible to disease
-poor success rate of cloning and expensive
-ethical issues as use of embryo
-coming endangered species won’t increase genetic diversity
Biotechnology
-the use of living organisms or parts of living organisms in industrial processes
-nowadays it has come to mean the use of organisms in production processes eg. gene technology, immunology, selective breeding
4 ways that biotechnology is used
-food eg. alcohol, bread rising, cheese production
-pharmaceutical drugs eg. penicillin, insulin
-Enzymes eg. protease and lipase used in washing powders, lactase to make lactose free milk
-other products eg. biogas= combo CO2 and methane, citric acid
Advantages of using microorganisms in biotechnology
-cheap and easy to grow
-production take place at lower temperatures, saving energy
-not dependent on climate
-reproduce quickly
-fewer ethical considerations
Yoghurt production
-milk that has been fermented by bacteria
-bacteria convert lactose to lactic acid
-causes milk to coagulate= clump
Cheese production
-milk treated bacteria to produce lactic acid from lactose
-mixed rennet that contains enzyme rennin
-coagulates the milk protein (casein) in presence Ca2+
-kappa casein broken down making casein insoluble
-casein ppt by Ca2+ which bind together
-forms curd which is pressed into moulds
Bread production
-mix ingredients to produce dough
-proving/ fermenting- left in warm place while yeast respires anaerobically producing CO2
-dough baked and alcohol evaporates
Alcohol production
-product anaerobic respiration of yeast
-use grapes with yeast on skin that when crushed, sugars produce CO2 and alcohol
Single cell protein production eg quorn
-use fungus which produces mycoproteins eg quorn
Advantages of using microorganisms in food
-faster than animal or plant protein production
-high protein content in biomass
-production altered according to demand
-no animal welfare issues
Disadvantages of using microorganisms in food
-people may not want to eat fungal protein
-protein has to be purified to make sure it’s not contaminated
-can have large proportion of nucleic acids that need to be removed
Commercial drug productions
-uses a fermenter which controls conditions for max yield of product
-temperature to prevent enzymes denaturing by having a water jacket and temperature probe
-nutrients available by having an inlet
-oxygen availability for respiration by having an air inlet
-pH- prevent enzyme denature and measured using probe
-concentration of product
Batch and continuous culture
Batch
-cells are put under stress by low nutrients or high population density
-this produces secondary metabolites from the stationary phase of growth
-set up with limited nutrient and allowed to ferment for a certain time before all product is collected
Continuous
-products synthesised during normal metabolism= primary metabolites
-products continuously released from cells and extracted from the fermenting broth
-nutrients are then continuously topped up
Production of penicillin
-secondary metabolite therefore produced by batch culture
- fermenter run for 6-8 days. Culture filtered to remove cells
- antibiotic precipitated as crystals by addition of potassium compounds
- antibiotic mixed with inert substances and prepared for administration
Growth curve graph
-shows growth of microorganisms in a closed culture= batch
Lag phase- slow growth in population as acclimatising to new environment
Log phase/exponential- acclimatised and have sufficient nutrients so pop grows rapidly
Stationary phase- using up nutrients and build up of waste causing birth rate to equal death rate
Death/ decline phase- nutrients run out and the concentration of waste products is high. This kills organisms, causing pop to decrease
Bioremediation
-use of microorganisms to clean soil and water on polluted sites
-organisms convert toxic pollutants to less harmful substances
-bacteria can be used to breakdown crude oil to treat oil spills
-involves stimulating the growth of suitable microbes that use contaminants as a source of food
Advantages of bioremediation
-uses natural systems
-less labour and equipment required
-treatment in situ
-few waste products
-less risk of exposure to clean up personnel
2 growth mediums and what they contain
-agar jelly
-broth
contain peptones, yeast extract, salts, water
carbon compounds for respiration
nitrogen compounds for growth
Typical aseptic procedure
-wash hands
-disinfect working area
-bunsen burner to sterilise air nearby
-as you open a vessel pass the neck of the bottle through the flame
-don’t fully open petri dish lid, just enough to introduce microorganism
-metal/ glassware should be flamed
3 steps to grow microorganisms on agar plates
sterilisation
inoculation
incubation
Sterilisation
-heated in autoclave at 121°
-this kills any microorganisms that could contaminate
Inoculation
-streaking- wire inoculating loop spread across the agar and the plate rotated
-seeding- sterile pipette to drop liquid onto agar
-spreading- use a sterile glass spreader to wipe over agar
-cotton bud wiped on surface to collect microorganisms then wiped on agar
Incubation
-tape dish with 2 bits of tape so oxygen can get in
-put upside down so condensation doesn’t drop onto jelly to contaminate
-place in warm environment
Why we use a liquid growth medium
-use a broth to increase the number of microorganisms before transferring to the agar
Why we do serial dilutions
-to reduce population density making the microorganisms easier to count
How serial dilutions are performed
-1cm3 of solution diluted with 9cm3 of water. Forms a solution diluted by 1 in 10 = x no. by 10
-then add 1cm3 of that solution to another test tube with 9cm3 of water. Forms a solution diluted by 1 in 100 = x no. by 1000
How to calculate CFU/Cm3
-CFU- colony forming unit
-first divide by the number of cultures counted by the volume used
-the times by the amount they’re diluted by
eg. 20 colonies in 0.1 diluted by 0.01
20/0.1=200
200x100= 20,000
What is culturing microorganisms
-when microorganisms are grown on agar plate/ nutrient broth
-do this using aseptic techniques
Immobilised enzymes
an enzyme that is held in place and not free to diffuse through the solution
Advantages of immobilised enzymes
-don’t mix with product so extraction costs are less
-enzymes easily removed
-continuous process easier as no cells requiring nutrients, reproducing and producing waste
-enzymes surrounded by immobilising matrix which protects enzymes from extreme conditions which could cause them to denature
4 methods immobilised enzymes
Adsorption:
-enzymes bound to supporting surface by hydrophobic reactions and ionic links
-suitable surfaces= clay, porous carbon, glass beads, resins
Covalent bonding
-bonding to surface eg clay by covalent bonds. More expensive but less likely to leak into reaction mixture
Entrapment
-enzyme trapped in matrix eg. alginate beads. The substrate then move past beads
Membrane separation
-enzymes separated from substrate by membrane
-substrate passes through membrane then products leave membrane
Industrial enzyme examples
Glucose isomerase
Penicillin acylase
Lactase
Aminoacylase
Glucoamylase
Nitrile hydratase
Glucose isomerase
-convert glucose to fructose
-fructose sweeter than glucose so used for diabetics as only need small amount
Penicillin acylase
-forms semi synthetic penicillin
-microorganisms aren’t resistant to it
Lactase
-convert lactose to glucose and galactose
-good for people lactose intolerant
Aminoacylase
-produces pure sample L-amino acids by removing acyl group from N-acyl-amino acid
-L amino acids used in synthesis pharmaceutical compounds
Glucoamylase
-Convert dextrins to glucose
-as sometimes in break down of starch dextrins are produced
Nitrile hydratase
-covert nitrile to amides
-polymerised to form polyacrylamide which is used in water treatment
