Cloning and Biotech Flashcards
vegetative propagation / natural cloning
reproduce asexually using meristem cells
what do you need for vegetative propagation
propagate asexually using tubers, rhizomes, bulbs, suckers, and offsets
Rhizome
specialised horizontal stem running underground + stores food – buds develop
Vegetative organs of plants
enable plants to survive in adverse conditions – contain food + remain dormant
examples of vegetative organs of plants
Root and shoot tips
Axillary buds (where leaves and the stem meet)
Vascular cambium (between xylem and phloem)
how does natural cloning take place
over time miniature plant (a plantlet) / buds forms at these locations + remains attached to its parent plant
clones of their parent
At maturity = detaches
potatoes
Potato tubers are swollen modified roots that form eyes on their surface
Eyes can sprout new growth (called ‘chitting’)
The starch stored in the tuber fuels the early growth of the new plant
ginger
Ginger forms rhizomes, a modified stem that grows horizontally underground
New growth stems from nodes in the rhizome, forming new stems and adventitious roots
The section used in cookery is the rhizome
strawberries / spider plants
have horizontal stems or runners that form over the soil surface, pointing sufficiently far away
new plant = not be overshadowed by its parent, or in competition for water or soil nutrients
Roots form under the nodes of runners, called adventitious roots
The runner dies when the plantlet is self-sustaining
state the type of plant tissue in which clones are produced
meristematic
State methods of natural cloning in plant
runners / suckers / stolons / tubers /
rhizomes / bulbs
how to propagate from cuttings
Short sections of stems taken + planted directly on ground or in pots
cut stems at a slant between nodes
Rooting hormones applied to base of cutting – encourage growth of new roots
remove leaves - reduce transpiration
natural clone
Cuttings vs seeds
Faster
Guarantees quality of plants
Lack of genetic variation
Sugar canes
factors that increase success rate in cuttings
how does using non flowering plant increase success rate
all plant resources available for growing new roots
how does making an oblique cut increase success rate
maximises surface area available for rooting powder/new root development
how does using a hormone rooting powder increase success rate
scientists unsure whether effect is the hormone directly or anti-fungal action but seems to increase success rate
how does reducing leaves increase success rate
minimises loss of water by transpiration whilst maintaining photosynthesis
how does keep cutting well watered increase success rate
reduces water stress
how does covering cutting with a plastic bag increase success rate
keeps air humid and reduces water loss by transpiration
Micropropagation
process of making large numbers of genetically identical offspring from single parent plant using tissue culture
why does micropropagation work
Plant cells – totipotent – entire plant can be reproduced from any of these cells
process of micropropagation
Take small sample of tissue from plant
Sample sterilised by immersing it in sterilising agents
Material removed from plant – explant
Explant placed in sterile culture medium
Balance of plant hormones – e.g auxins + cytokinins – simulate mitosis
Cells proliferate – forms callus = mass of identical cells
where should we take a small sample of tissue from in micropropgation
Meristem tissue from shoot tips + apical buds
Sterile conditions – virus-free
sterilising agents
bleach / ethanol / sodium dichloroioscyanurate
explant
Material removed from plant
Advantages of plant cloning
same genotype + phenotype
The plants produced are free of disease
genetically modified to confer immunity to certain diseases
rapid and can yield large numbers of new plants
Plants that are difficult to grow from their seeds can be produced by plant cloning
Plants can be grown in any country, in any season
Rare and endangered species can be propagated to save them from extinction
Whole plants can be created from genetically modified cells/tissues
Disadvantages of plant cloning
expensive and labour-intensive process
susceptible to microbial contamination
no genetic variation, so all of the offspring are susceptible to the same diseases or other environmental factors
risks large-scale loss of a country’s / continent’s crop of a particular plant
plants have to be carefully screened for abnormalities that could lead to the new plants being infected
natural clones in animals
- invertebrates - regenerate animals from fragments of original
monozygotic twins
why identical twins are referred to as monozygotic
from the same zygote
how are twins formed
egg fertilised by a sperm
forms a zygote
single zygote undergoes a few cell cycles = embryo
embryo splits in two
Two embryos that form are identical
identical offspring, always of the same gender, with identical phenotype
why are identical twins not clones
mutations occur in every cell cycle
why may identical twins look different when born
difference in position + nutrition in uterus
artificial clones in animals
artificial twinning + somatic cell nuclear transfer
artificial twinning principle
artificially split embryo - can be split into more than 2
artificial twinning process
cows – treated with hormones – super ovulates
ova fertilised naturally or via artificial insemination
early embryos flushed out of uterus
OR – eggs fertilised in lab
Before day 6 – cells still totipotent
Cells of embryo split
Grown in the lab for a few days
Implanted into surrogate mother – each different mother for cows– single pregnancies carry less risk
Somatic cell nuclear transfer / reproduction cloning
Nucleus removed from somatic cell of an adult animal
Nucleus removed from a mature ovum harvested from different female animal of the same species
Enucleated ovum
Nucleus from adult somatic cell placed into enucleated ovum
Milk electric shock – fuses + begins to divide
OR – nucleus from adult cell not removed + placed next to enucleated ovum – divide due to electrofusion – electric current
Embryo – put into uterus of a third animal
DNA of offspring from SCNT
clone of the animal from which the original somatic cell is derived
BUT mitochondrial DNA – come from egg cell
animals required for SCNT
3
The animal to be cloned by donating a cell
The female to donate an egg cell
The surrogate mother
SCNT vs artificial twinning
Artificial twinning – clones embryo
SCNT – clones adult animal
Arguments for animal cloning
Artificial twinning – high yielding farm animals to produce more offspring
Enables success of male animal at passing on desirable genes to be determined
SCNT enables GM embryos to replicate / develop – important in pharming
SCNT – enable rare / endangered / extinct animals to be reproduced
Arguments against animal cloning
SCNT – inefficient – takes many eggs to produce single cloned offspring
Cloned embryos – fail to develop / miscarry / malformed offspring
Animals produced via cloning – shortened lifespans
Cloning destroys embryos which could in theory develop into a healthy adult animal
Biotech
applying biological organisms / enzymes to the synthesis / breakdown / transformation of materials in the service of people
why are microorganisms ideal for biotech
No welfare issues to consider – only optimum conditions for growth
Enormous range of microorganisms capable of carrying of chemical syntheses / degradations
GM – manipulate microorganisms
Short life cycle + rapid growth rate
Nutrient requirements- simple + cheap
Occupy very little space
microorganisms + purpose in baking
yeast
mixed with sugar to respire aerobically
carbon dioxide produced makes bread rise
steps in commercial process - baking
microorganisms + purpose in brewing
yeast
respires anaerobically to form ethanol
GM yeasts ferment at lower + cheaper temp
steps in commercial process - brewing
microorganisms + purpose in cheese making
bacteria
feed off lactose in milk - changing texture + tase
inhibiting growth of bacteria that make milk go off
steps in commercial process - cheese making
microorganisms + purpose in yoghurt making
bacteria
forms ethanal + lactic acid
extracelular polymers that give yoghurt smooth thick texture
steps in commercial process - yoghurt making
advantages of using microoroganisms for food
disadvantages of using microoroganisms for food
making penicillin
P. chryogenum – requires high oxygen levels + rich nutrient medium to grow well
Semi-continuous batch process used
Species of mould from the Penicillium genus can be cultured in industrial fermenters
deep-tank fermentation
Extraction and purification of the product produces large volumes of the drug for therapeutic use
making penicillin conditions
making insulin
Bacteria grown in fermenter + downstream processing results in constant supply of pure human insulin
Recombinant DNA technology can incorporate the gene for human insulin into the genome of the bacterium, Escheriscia coli
Recombinant bacteria are grown in batch fermenters, and each bacterial cell expresses insulin
Insulin is released into the batch medium and purified
Bioremediation
Microorganisms used to break down pollutants + contaminants in soil / water
Naturally occurring microorganisms perform aerobic digestion of the contaminants and release non-polluting products
how does bioremediation work - natural vs GM
Use natural organisms
Many microoganisms naturally break down organic material – CO2 + water
Break down + neutralise contaminants
Oil spill – add nutrients – encourage microbial growth - biostimulation
GM organisms
Break down / accumulate contaminants that they don’t usually encounter
biostimulation
add nutrients – encourage microbial growth
bioventing
process which allows oxygen to reach the contaminants
what does bioremediation rely on
oxidative digestion of pollutants
Naturally occurring microorganisms perform aerobic digestion of the contaminants and release non-polluting products
why do you need to be careful even with harmless microorganisms
Risk of mutation + becoming pathogenic
Contamination with pathogenic microorganisms from environment
nutrient medium
Food provided to bacteria for culturing
liquid form - broth
solid form - agar
Inoculating broth
Make suspension of bacteria to be grown
Mix known volume with sterile nutrient broth in flask
Stopper the flask with cotton wool – prevent contamination
Incubate at temp – shake regularly to aerate the broth – provide oxygen for bacteria
Inoculating agar
Wire inoculating loop – sterilised by holding in Bunsen flame until it grows red hot
Must not be allowed to touch any surfaces as it cools – avoid contamination
Flame the neck of the culture tube
Dip sterilised loop into bacterial suspension in culture tube
Remove lid of petri dish + make a zig-zag streak across surface of agar
Avoid digging loop into agar
Replace the lid of petri dish – held down with tape but not sealed completely
Oxygen can still get in – prevent anaerobic bacteria
Incubate
general aseptic techniques include:
Washing hands thoroughly
No food or drink allowed in the lab
Disinfecting work surfaces with disinfectant or alcohol
Wearing gloves and goggles
Working close to a lit Bunsen burner
Flaming equipment (to kill microorganisms or create updraughts)
Sterilising (in an autoclave) or disposing of all used equipment
why should we work close to a bunsen burner
(this creates an updraught of air so prevents contamination from airborne fungal spores, for example)
kill micro-organisms
purpose of these steps
Primary metabolites
substances formed as an essential part of the normal functioning of microorganism
E.g. ethanol
Secondary metabolites
substances produced that are not essential for normal growth but still used by cell
E.g. pigments
significance of primary + secondary metabolites
depending on which you want - determine the time in which you will harvest the culture
Batch fermentation
Microorganism inoculated onto fixed volume of medium
Growth takes place
Nutrients used up
New biomass + waste products build up
Culture reaches stationary phase – overall growth stops
Often carry our changes to make desired products
Process stopped before death phase + products harvested
Continuous fermentation
Microorganisms inoculated into sterile nutrient medium
Medium added continually to culture once it reaches exponential point of growth
Culture broth continually removed – medium / waste products / microorganisms / product
Keep culture volume in bioreactor constant
Downstream processing
Bioreactors – produce mixture of unused nutrient broth / microorganisms / primary + secondary metabolites / waste
Useful part has to be separated – downstream processing
factors that will max yield of products in bioreactors
temp
nutrients
oxygen
mixing
asepsis
controlling temp
Maintain optimum temp – rate + denature
Heating / cooling systems linked to temp sensors
Negative feedback system
E.g. – use water jacket
Max enzyme activity = max yield
controlling nutrients
Added + circulated to ensure access
Probes / sample tests indicate levels dropping
controlling oxygen
Sterile air pumped in
Provided max oxygen – max respiration – max yield
mixing
Large volumes of liquid – viscous
Simple diffusion not enough to ensure food + correct temp
Mixing mechanisms – stirred continuously with paddles
Even distribution
asepsis
Sealed / aseptic units
Must be cleaned between cultures – prevent contamination
Contamination – interspecific competition – reduce yield
state the growth phases of bacterial colonies in a closed system
lag
log
stationary
death
describe the growth of bacterial colonies in a closed system
measuring bacterial populations
Direct counting – includes all cells – living or dead
Viable counting – culturing samples + counting colonies that grow – only takes living samples into account
Turbidity – measure of living + dead in solution
turbidity
Grow in broth
Turbidity – measure of cloudiness of a suspension
As population grows – becomes more turbid
changing turbidity - monitored by measuring how much light can pass through the suspension at fixed time intervals after the initial inoculation – colorimeter – plot a curve
why log phase
high availability of nutrients + plenty of space
why death phase
due to lack of nutrients + toxic wase builds up
how to calculate the number of bacteria after divisions
factors that limit the log phase
nutrients - as bacteria multiply - nutrients used up + becomes insufficient to support growth
oxygen - as population increases - demands for respiratory oxygen increases
temp - too low / too high
build up of waste - toxic material inhibit growth + kill culture
change in pH - carbon dioxide produced by respiration = pH falls - effects enzyme activity
advantages of isolated enzymes over whole organism
less wasteful – whole microorganism use up substrate growing + reproducing – makes biomass
more efficient – isolated work at much higher conc
more specific – no unwanted enzymes + no wasteful wide reactions
less downstream processing – pure product produced // whole organisms produce variety of products difficult + expensive to purify
immobilised enzyme
enzyme that is attached to an insoluble material to prevent mixing with the product
advantages of immobilised enzymes
held stationary during reactions – can be recovered from mixture + reused
enzymes do no contaminate end product - no downstream processing
greater temp tolerance – less easily denatured by heat – optimum over a much wider temp range – bioreactor less expensive to run
disadvantages of immobilised enzymes
Specialist expensive equipment required – high cost of bioreactor
more costly to buy - unlikely to be financially worthwhile for smaller industries // higher initial cost of materials
rate of reaction is sometimes lower - as the enzymes cannot freely mix with the substrate
4 ways of immobilising enzymes
adsorption
surface immobilisation
adsorbed to inorganic carriers - cellulose / silica / carbon nanotubes
advantages + disadvantages of adsorption
covalent / ionic bonding
surface immobilisation
covalent bonding - carriers with amino / hydroxyl /carboxyl groups
ionic bonding - polysaccharides - cellulose
advantages + disadvantages of covalent / ionic bonds
entrapment - in matrix
polysaccharides / gelatin
advantages + disadvantages of entrapment in matrix
entrapment - capsules
membrane entrapment in microcapsules
encapsulation
advantages + disadvantages of entrapment - encapsulation
example of immobilised enzymes forming lactose free dairy products
Enzyme: Lactase
Converts lactose to glucose and galactose
example of immobilised enzymes forming Semi-synthetic penicillin
Enzyme: Penicillin acylase
Converts the original form of penicillin into one which is effective against penicillin-resistant organisms
example of immobilised enzymes forming sweetened / thickened food
Enzyme: Glucoamylase
Converts starch and other dextrins into glucose
example of immobilised enzymes forming sweetened foods with low sugar
Enzyme: Glucose isomerase
Converts glucose into the sweeter sugar, fructose
example of immobilised enzymes forming purified samples of L amino acids
Enzyme: Aminoacylase
Separates out L-amino acids from D-amino acids
