Cloning Flashcards
cloning
the production of genetically identical individuals
vegetative propagation - strawberry plants
produce runners that grow sideways along the ground - new shoots and roots grow along the runner, runners eventually die off between plants
vegetative propagation - daffodil bulbs
leaf bases swell with stored food from photosynthesis, buds form internally to grow in next season
vegetative propagation - marram grass
rhizome - specialised horizontal stem running underground, often swollen with stored food, buds develop and form new vertical shoots which become independent plants
vegatative propagation - potato
stem tubers - tip of underground stem becomes swollen with stored food to form tuber or storage organ, buds grow on it to produce new shoots (eyes)
examples of natural cloning uses in horticulture
- splitting bulbs, removing plants from runners and rhizomes - increases plant numbers cheaply
- taking cuttings from plants and adding growth hormone powder to base to encourage growth of new roots
- this guarantees quality of the plant and is quicker
advantages natural cloning in horticulture
- guarantees quality of crop
- planting cuttings is quicker than growing from seed
disadvantages of natural cloning in horticulture
lack of genetic variation - vulnerable to disease
micropropagation uses
- making large numbers of genetically identical offspring from a single parent plant using tissue culture techniques
- used when a desirable plant : doesn’t produce seeds, doesn’t respond well to natural cloning, is rare, required to be pathogen free
process of micropropagation
- cut a small tissue sample from stem of plant to be cloned using a scalpel in sterile conditions - meristem tissue
- sterilise sample (eg. by immersing it in ethanol)
- explant is placed in sterile culture medium containing plant hormones (rooting powder) to induce root formation
- plant the cutting in a suitable growth medium
- grow the cutting in a warm, moist environment until roots are formed then the plant can be transferred elsewhere
advantages of micropropagation
- rapid production of large numbers of plants with a known genetic makeup
- disease-free plants as they’re from meristem tissue
- large numbers of seedless fruit
- growing rare plants
disadvantages of micropropagation
- monocultures - genetically identical so susceptible to same diseases
- expensive, requires skilled workers
tissue culture
- the starting step of micropropagation
1. cells removed from stem/root tip to be cloned
2. cells sterilised and grown on suitable culture medium
3. cells grow and divide into small plant
4. small plant moved into soil to grow
natural animal cloning
- invertebrates - starfish regenerate entire animals from fragments, hydra get small buds on side of body which develop into clones
- vertebrates - monozygotic twins
artificial twinning of animals (cows)
- cow with desirable trait treated with hormones so she produces more mature ova than normal
- ova fertilised naturally or by artificial insemination from a bull with desirable traits
- when cells are still totipotent, cells of the earlier embryo are split to produce several smaller embryos
- embryos are grown in the lab for a few days then implanted into different surrogate mothers
- embryos develop into foetuses and are born normally
somatic cell nuclear transfer
- nucleus removed from somatic cell of adult animal
- nucleus removed from mature ovum of a different female of same species (enucleated)
- nucleus from adult somatic cell placed in enucleated ovum and given small electric shock so they fuse and begin to divide
- embryo develops and is transferred into uterus of third animal
- new animal is clone of animal where somatic cell came from, but mitochondrial DNA came from ovum
advantages of artificial animal cloning
artificial twinning:
- high yield farming - more offspring
- enables success of male passing on desirable genes to be determined
SCNT:
- enables GM embryos to be replicated and develop to give many embryos from one procedure
- enables scientists to clone animals with desirable traits
- rare or endangered animals can be reproduced
disadvantages of artificial animal cloning
- SCNT is inefficient - can taje many eggs to produce single cloned offspring
- many cloned animals fail to develop and miscarry or produced malformed offspring
- many animals produced in cloning have short lifespans
biotechnology
- applying biological organisms or enzymes to the synthesis, breakdown or transformation of materials in the service of people
- eg. cheese, bread, wine making
- eg. genetic engineering
why are microorganisms used in biotechnology?
- no welfare issues
- large range of microorganisms which carry out different chemical syntheses
- genetic engineering allows us to artificially manipulate microorganisms to carry out specific synthesis reactions
- short life cycle, rapid growth rate - huge quantities in short period of time
- nutrient requirements are simple and cheap
disadvantages of using microorganisms for indirect food production (has an effect on the food eaten rather than eating the microorganism itself)
- conditions must be ideal to grow properly and avoid contamination by unwanted bacteria
- some people have ethical issues with eating genetically engineered organisms
how are microorganisms used to directly produce food?
eg. Quorn (a single-celled protein)
- single-celled fungus grown in large fermenters using glucose syrup as food source
- microorganisms combined with egg white then compressed into meat substitutes
advantages of using microorganisms to directly produce food
- microorganisms reproduce fast
- high protein low fat
- can use waste materials, reducing costs
- can be genetically modifies to produce required protein
- not dependent on weather or breeding cycles etc. , takes place constantly, can be increased or decreased to match demand
- can be made to taste like anything
disadvantages of using microorganisms to directly produce food
- can produce toxins if not kept at correct temp
- have to be separated from nutrient broth and processed to make the food
- sterile conditions - expensive
- often involve GM organisms - ethical issues
- protein has to be purified
- little natural flavour
- consumption of high quantities of single-celled protein can cause health problems by releasing uric acid when its broken down
producing penicillin
- P. chrysogenum - allowed for commercial production of penicillin
- needs high oxygen levels and rich nutrient medium to grow
- uses small fermenters - hard to maintain high levels of oxygen in large ones
- continuously stirred to keep it oxygenated
- rich nutrient medium
- growth medium contains buffer to maintain pH 6.5
- maintained at 25-27 degrees
culturing microorganisms - inoculating the broth
adding bacteria to nutrient broth
1. make a suspension of the bacteria to be grown
2. mix a known volume with the sterile nutrient broth in the flask
3. use cotton wool as a stopper for the flask to prevent contamination with the air
4. incubate in suitable temp, shake regularly to provide oxygen for the growing bacteria
culturing microorganisms - inoculating the agar
- the wire loop is sterilised by flaming it in a bunsen flame until it glows
- dip the sterilised loop in the bacterial suspension, remove lid of petri dish and make zig-zag streak across surface
- put lid back on petri dish and hold down with tape
stages on growth curve of bacteria
- lag phase - bacteria adapting to new environment - growing, synthesising enzymes
- log phase - rate of bacterial reproduction at theoretical maximum
- stationary phase - growth rate is 0
- death phase - reproduction slows
limiting factors preventing exponential growth in culture of bacteria
- nutrient availability - will be used up, can’t support growth and reproduction
- oxygen levels - population rises, oxygen demands rise
- temperature - enzymes need optimum temps
- build up of waste - toxic material may inhibit further growth or poison the culture
- change in pH - CO2 produced from respiration decreases pH of culture affecting enzyme activity
primary metabolites
- substances produced as part of normal functioning of an organism
- eg. ethanol, amino acids, enzymes
secondary metabolites
- substances produced by organisms which aren’t essential for normal growth
- eg. antibiotic chemicals produced by some fungi (penicillin)
describe the graph for no. of cells produced and ethanol produced for primary metabolites
- both increase and decrease together, cells produced slightly above the ethanol produced line
- flat S shape
describe the graph for no. of cells produced and penicillin produced for secondary metabolites
- cells increase, penicillin increases after a lag period
- flat S shape
batch culture
- microorganisms inoculated for a fixed time in a fixed amount of nutrients
- growth takes place, nutrients used up, new biomass and waste products built up
- the culture reaches stationary phase - overall growth stops, biochemical changes may be carried out to form desired end products
- process stopped before death phase, products harvested
- system is sterilised and new batch started up
continuous culture
- microorganisms inoculated into sterile nutrient medium and start to grow
- sterile nutrient medium added continually to the culture once it reaches exponential growth
- culture broth continually removed - medium, product, waste products, microorganisms - keeps culture volume in bioreactor constant
downsteam processing
- products separated for further processing
- purifies product
asepsis during industrial production
- prevents contamination with unwanted microbes
- most bioreactors are sealed units
- GM organisms are legally required to be contained
why use isolated enzymes in biotech rather than whole microorganisms?
- less wasteful - whole microorganisms use substrate growing and reproducing
- more efficient - isolated enzymes work at higher conc than is possible in microorganisms
- more specific - no unwanted side reactions from unwanted enzymes
- maximise efficiency - can be given ideal conditions for max product formation
- less downstream processing - pure product is produced, microorgansisms give variety of products in final broth
why use extracellular enzymes in biotech rather than intracellular?
- extracellular are secreted - easier to isolate
- microorganisms produce few extracellular enzymes - easy tp identify and isolate
- extracellular are more robust bc conditions outside cell are less controlled
reasons some may choose to use intracellular and examples
- bigger range of intracellular - produce ideal enzyme for a process
- eg. glucose oxidase - food preservation, asparaginase - cancer treatment, penicillin acylase - converts natural penicillin into drugs which are more effective
immobilised enzyme meaning
- an enzymes is attachned to an inert support system that the substrate passes over and is converted into product
- enzymes can be recovered from the reaction mixture and reused
advantages of using immobilised enzymes
- can be reused - cheaper
- easily separated from reactants and products if the reaction so less downstream processing - cheaper
- more reliable - control over the process as the support provides a stable microenvironment for the immobilised enzymes
- better temperature tolerance - less easily denatured by heat, work at optimum levels at range of temperatures - bioreactor less expensive
- ease of manipulation - properties can be altered to fit particular process easier
disadvantages of using immobilised enzymes
- process of immobilising it could reduce its activity rate
- immobilised enzymes more expensive than free enzymes or microorganisms but they don’t need to be replaced frequently
- bioreactor to use immobilised enzymes is expensive
- reactors which use immobilised enzymes are more complex than simple fermenters more can go wrong
entrapment - immobilising an enzyme and pros and cons
- enzyme trapped in a matrix so it can’t move
- remains fully active
- substrate diffuses into matrix
- product diffuses out
advantages: - widely applicable to different processes
disadvantages: - expensive
- hard to entrap
- diffusion of substrate to and from active site can hold up reaction
- effect on the enzymes activity varies depending on the matrix
adsorption - immobilising an enzyme and pros and cons
- enzymes bound to supporting surface eg. clay, porous carbon, resins
- hydrophobic interactions and ionic links hold them in place
- enzymes stay exposed to substrates
pros: - simple and cheap
- used with many diff processes
- enzymes v accessible to substrate
cons: - enzymes can be lost from matrix easily - bonding forces not always strong
- can distort active site - reduces activity
covalent bonding - immobilising enzymes and pros and cons
- enzymes bonded to supporting surface such as clay using strong covalent bonds
pros - unlikely to become detached
- enzymes v accessible to substrate
- pH and substrate conc often have little effect on enzyme activity
cons - expensive
- active site can be distorted
using immobilised enzymes - penicillin acylase
- used to make semi-synthetic penicillins
- many penicillin resistant microorganisms aren’t resistant to these
using immobilised enzymes - glucose isomerase
- converts glucose to fructose
- fructose - much sweeter than glucose so used in food industries
- cheaper than sucrose
using immobilised enzymes - lactase
- converts lactose to glucose and galactose
- used to produce lactose-free milk
using immoblised enzymes - aminoacylase
- used to produce pure samples of L-amino acids used for pharmaceuticals, organic chemicals, cosmetics, food
using immobilised enzymes - glucoamylase
- (amylase breaks down starch to short chain polymers - dextrins)
- breaks down dextrins to glucose
using immoblised enzymes - nitrile hydratase
- converts nitriles to amides
- acrylamide can be polymerised to form polyacryamide
- can be used to treat water - sticks small contaminants togther so they can be easily filtered
- also used in paper making and gel for electrphoresis
