Unit 6 - Cloning and biotechnology Flashcards
Clones
Carry identical genetic material because they are derived from the same orig. DNA
Cloning
Process of producing genetically identical cells or organisms from the cells of existing organisms through nonsexual means
Examples of processes that form genetically identical organisms
Mitosis
Binary fission (bacteria)
Budding (yeast)
Natural plant cloning
Vegetative propagation through runners or suckering
Artificial plant cloning
Artificial vegetative propagation through cuttings or micropropagation (tissue culture)
Vegetative propagation
Ability of plants to reproduce w/out sexual reproduction by producing new plants from existing vegetative structures
Vegetative structures
Non-reproductive tissues e.g. roots, leaves and stems
Cuttings
Cut stem 1/4 “ below internode at 45-60 degrees
Treat cut end w/ rooting hormones
Cover in clear plastic bag
Transfer to another growing medium
Why do you cover cuttings w/ a clear plastic bag
To keep it moist and warm
Explant
A small piece of tissue
Callus
Undifferentiated mass of tissue containing totipotent cells
Micropropagation
Cut out explant from vegetative structures (leaf)
Sterilise explant w. alcohol
Place explant in sterile agar w/ glucose, cytokinins and auxins
Subdivide callus and place on growth medium to induce root growth (prepares plant for transplanting)
Transferred to greenhouse to acclimatise before being planted outside
Advantages of artificial plant cloning
Can produce large no. v. quickly
Can grow plants that dont reproduce easily
No need to wait for seed production
Reproduce sterile plant
Disadvantages of artificial plant cloning
Labour intensive and requires skilled workers
Trial and error to find ideal conditions for growth
Undesirable traits also passed on
Can fail to microbial contamination
Runners
Side stem grows out from bud at the base of the main stem
Creates new bud and grows a vertical stem
Suckering
Grow from shallow roots from buds that are normally dormant
Duing times of stress, buds are activated and suckers form many metres away from parents tree (to avoid stress that triggered growth)
Eventually form clonal patch of new trees
Trees in clonal patch put out new sucker buds
Advantages of natural plant cloning
Large colonies can form quickly
Allows species to survive catastrophic events
Disadvantages of natural plant coning
No natural selection
Susceptible to gentic disease; no variation
Runners vs. suckers
Runners are overground and suckers are underground
Why must the agar used in micropropagation be sterile
Prevents infection
Competition of resources e.g. oxygen/nutrients if other organisms e.g bacteria and fungi are present
Somatic cell
Biological cell forming the body of an organism
Germline cell
Biological cell that gives rise to the gametes of an organism that reproduces sexually
Natural animal reproductive cloning
Some animals can regenerate entire animals from fragments of the orig. (starfish)
Others fragment and form new identical animals as part of their normal reproductive process (flatworms and sponges)
MZ twins form when an early embryo splits and and two foetuses develop from two halves
Artificial animal reproductive cloning
Somatic cell nuclear transfer
Artificial twinning
Somatic cell nuclear transfer
Extract nucleus from somatic cell from Sheep A
Remove nucleus from egg cell from Sheep B
Insert nucleus from A into innoculated egg(electrofusion)
Stimulates to divide in vitro and implant embryo into sheep C
Artificial embryo twinning
Get fertilised egg and allow to divide until 16 cell stage
Harvest embryo and split into smaller ones manually
Implant into surrogate mothers which give birth to identical high quality animals
Advantages of artifical animal cloning
Produce identical clones w/ desirable traits
Stem cell research
Clones so offspring can be produced all year round
Disadvantages of artificial animal cloning
Difficult and time consuming
Destruction of embryos unethical
Clones have shorter life expectancies
No genetic identity so selection pressures affects all
Similarities between AT and SCNT
Produce clones Both have surrogates Divides by mitosis Unnateral Expensive
Differences between AT and SCNT
AT forms several clones at once
Gametes meet outside the body is AT
SCNT involves only maternal DNA
No fertilisation in SCNT
Biotechnology
Industrial exploitation of living micro-organisms (or parts of them) and biological process to produce useful substances for human use
Why are microorganisms used in biotech
Easy/ not labour intensive
Obtain pure products if aseptic technique is followed
Can be easily genetically enginerred to produce spp products
Short life cycle
Simple requirements for growth - can be left w/ little intervention
Can be grown v. quickly
Can be grown on waste material from other processes
Use of microorganism in biological processes
Brewing - anaerobic respiration of yeast Baking - yeast Cheese making - bacteria and rennin Penicillin production - fermentation by fungus Insulin production - GM bacteria Bioremediation
Bioremediation
Using microorganisms to clean up pollution
Convert toxic pollutants to less harmful substances
Advantages of bioremediation
Uses natural systems Less labour and equipment required Treatment can be carried out on site Few waste products produced Less risk of harmful exposure to clean-up personnel
Culturing microorganisms
Sterilisation - Sterilising equipment in an autoclave (15 mins at 121 degrees)
Inoculation - Introducing a sample of microorganisms to the growth medium
Incubation - Place in a warm environment and place agar plate upside down (condensation)
Types of growth medium
Agar jelly in a petri dish
Nutrient broth in a bijoux bottle
Standard procedure of aseptic techniques
Wash hands thoroughly
Disinfect working area
Light and keep Bunsen burner on
Flame neck of bottle before and after taking sample
Lift lid of petri dish slightly to introduce microbe by streaking
Close petri dish and tape (not completely)
Flame all equipment after use
Wash hands again
Why do you keep the Bunsen burner on during aseptic procedures
Heats the air, causing it to rise so air-borne microbes don’t settle
Why cant you seal the petri dish completely
Introduces O2 for aerobic respiration
Doesn’t introduce harmful anaerobically respiration organism
Continuous culture
Culture is set up and nutrients are added and products removed from the culture at intervals - done at same rate to keep vol constant
Culture is maintained at exponential growth phase - grows and produces metabolites faster
Metabolites
Substances produced by living organisms in order to survive e.g ATP synthase
Batch culture
A starter population of the microorganism is given a fixed amount of nutrients and at the end of the time period products are extracted
Examples of continuous culture
Insulin
Single-cell protein (from Fusarium)
Examples of batch culture
Wine
Beer
Yogurt
Advantages of continuous culture
Fermenter is always in use - increases efficiency
High growth rate as nutrient levels maintained
Useful for primary metabolites
Disadvantages of continuous culture
Contamination is more likely
Difficult to maintain and control product consistency
In cases of contamination losses are great and all production halts
Advantages of batch culture
Less likelihood of contamination
Can be left for a set time period
Useful for secondary metabolites
In the event of contamination, only one batch is lost
Disadvantage of batch culture
Fermenter isn’t in use constantly - less efficient
Time spent cleaning
Primary metabolites
Produced in the course of normal metabolism e.g. proteins, enzymes, alcohol
Produced in lag and log phase
Secondary metabolites
Produced after main population growth has occurred, nutrients are in short supply and population isn’t growing rapidly
Produced in stationary and death phase
How do fermenters maximise the yield
Tube for sterile air to provide oxygen for aerobic reactions
Sparger - diffuses air through culture medium
Powerful motors - mixes contents ensuring equal distribution of nutrients and so microbes don’t settle at base of fermenter
Acid-base injection site - controls pH
Culture broth - contains sources of carbon and nitrogen (NH3) and vitamins/minerals
Jacket - filled with hot/cold water to provide optimum temp as respiration releases heat (denaturing)
Asepsis in fermenters
Washing. disinfecting and steam cleaning all equipment
Using fermenter made of polished stainless steel so microbes cant stick
Sterilising all nutrients w/ steam or heat
Only bubbling in sterile air- v. fine filters
Phases in microorganim growth curves (bacteria/fungi)
Lag
Exponential
Stationary
Death
Lag phase
Reproduction v. slow as cells acclimitase, absorb nutrients
Gene expresion for spp enzymes
Synthesis of enzymes and organelles
Exponential phase
Reproduction is rapid
No limiting conditions
Few cells die
Stationary phase
Population remains constant as death and reproduction rates are the same
Death phase
Lack of resources
Build up of CO2 - fatal
Culture
A method of multiplying microbial organisms by letting them reproduce in predetermined culture medium under controlled lab conditions
Isolated enzymes
Taking enzymes out of microorganism
Issue w/ isolated enzymes
Product must be seperated from enzymes and extraction is v. expensive
Immobilised enzyme
Enzymes fixed to a surface and do not freely mix w/ the substrate
Ways to immobilise enzymes
Covalent bonding
Encapsulation
Adsorption
Entrapment
Covalent bonding to immobilise enzymes
Covalently bonded to a supporting surface
Enzymes are also covalently bonded together using a cross-linking agent
Adsorption
Bound to supporting surface by a combination of hydrophobic interactions and ionic links
Bound w/ active site exposed and accessible to substrate
Entrapment
Trapped in a matrix (often calcium alginate beads) that doesn’t allow free movement
Encapsulation/membane separation
Separated from reaction mixture by a small permeable membrane - microcapsule
A vs D of adsorption
Simple and cheap
Can be used in a variety of processes
Enzymes v. accessible
May distort active site
Enzymes can detach and leak into reaction mixture
A vs D of covalent bonding
Enzyme less likely to become detached
pH and substrate conc have little effet on enzyme activty
Accessible to substrate
Expensive
Can distort active site, reducing activity
A vs D of encapsulation
Relatively simple
Relatively small effect on enzyme activity
Widely apllicable to diff processes
Expensive
Substrate and product has to be small in order to diffuse through partially permeabe membrane
Diffusion is slow
A vs D of entrapment
Widely applicable to diff processes
May be expensive
Difficult to entrap
Effect of entrapment on enzyme activity dpends on the matrix
Substrate and product needs to be small
Examples of immobilised enzymes
Glucose isomerase Penicillin acylase Lactase Aminoacylase Glucoamylase Nitrile hydratase
Penicillin acylase
Converts naturally produced penicillins to semi-synthetic penicillins
Some resistant microorganims arent resistant to semi-synthetic penicillins
Glucose isomerase
V. commonly use to produce HFCS, sweeter than sucrose and can be used in diet fods
Glucose —> fructose
Lactase
Hyrolyses lactose into glucose and galactose so lactose-intlerant people can drink milk and reduce the risks of developing osteoporosis as a lack of calcium
Aminoacylase
N-acyl-amino acids —> pure sample of L-amino acids
Used in synthesising pharmaceutical compounds
Glucoamylase
Dextrins —> glucose
Immobilised and used to digest sources of starch e.g. corn and cassava
Nitrile hydratase
Nitriles —> amides
Acrylamide can be polymerised to form a plastic and a gel for electrophoresis
Used to treat water, helps to stick many small contaminanrts together so they can be filtered out
Supporting surfaces
Glass
Porous carbon
Clay
Advantages of immobilising enzymes
Lowers temp required
No contamination of end product
Reusable
Protected by immobilisng matrix so high temp or extreme pH has no effect
Disadvanages of immobilising enzymes
Expensive to set up
Bonding may affect active site
Contamination is v. costly as whole system needs to stop
Slower process as enzymes and substrates don’t mix freely
Making yoghurt
Milk is fermented by bacteria
Lactose—> lactic acid
Low pH denatures caesin, causing milk to coagulate and thicknes
Also adds flavour
Makng cheese
Milk is fermented by bacteria
Lactose –> lactic acid, acidifies milk
Rennin coagulates caesin in the presence of Ca2+
Resulted curd separates from liquid whey by curdling, stirring and heating
Microorganisms in baking
Flour is mxed w. water, salt and yeast
Respires anaerobically and produces CO2 bubbles, causing the dough to rise
Making wine
Grapes have yeast on the surface
When crushed uses glucose and fructose to respire and produce CO2 and alcohol
Making beer
Uses malted barley grains that are beginning to germinate
Stored starch to maltose, respiratory substrate for yeast
Produces CO2 and alcohol
Making penicillin
Fermentation of fungus as a batch culture for 6-8 days
Secondary metabolite
Once fermentation is complete, culture is filtered to remove cells
Antibiotics precipitated and purified
Making insulin
Genetic modification of bacteria
Grown in fermenters, continuous culture
Why is the Sheep C treated w/ hormones
Increase thickness and vascularisation of uterine lining
How can SCNT help save endangered species
Doesn’t require fertile females
Female not put at risk during mating
Can subdivide successfully formed embryo
Economic advantages of immobilising enzymes
Reusable so less money required
Higher temp means profit from faster yield
