CHAPTER 22 - CLONING AND BIOTECHNOLOGY Flashcards
What is vegetative propogation
A form of natural cloning in plants, a structure which develops into a fully differentiated plant, identical to the parent
What are some examples of natural plant cloning
Bulbs - leaf bases which swell with stored food from photosynthesis
Runners - from plants like strawberries where a lateral stem grows away from the parent plant and roots develop underneath, eventually creating an independent plant
Rhizomes - a specialised horizontal stem running underground, with stored food
Stem tubers - eg. potato buds on storage organ develop to produce new shoots
How are natural clones carried out in horticulture
,Bulbs are split up,
removing young plants from runners
Cutting up rhizomes
Taking a cutting of a plant, with rooting hormone (which is quicker than just using a seed)
What are perennating organs and how are they involved in cloning and survival
Organ which contains stored food from photosynthesis e.g., potato
cloning – new bud/plants
may arise from the organ identical to original plant
allows plant to survive adverse conditions
and produce a new shoot
using energy from food store
Explain the advantages and disadvantages of propagating crop plants by cutting over using seeds
Cuttings – genetically identical to parent so likely to produce good crops
often shorter time from planting to crop
reliable
don’t have to buy in
can use own plants
Seeds – have genetic variation so more variability in quality of crop
but are more likely to
withstand disease of changes in circumstances
take time and right conditions to germinate and grow to maturity
in some cases can collect seed and reuse for next planting but don’t always get the same quality
Suggest why it is important to describe clones as genetically identical to their parent rather than simply identical - and why even this may not always be true
Genetically identical because parts of the same plant
but eventually form will depend on
growing conditions – levels of light, water, temperature etc.
identical suggests appearance is the
same
cloned plants may not be identical because a mutation may take place in stem cells of meristems
changing pattern of growth in the plant
When is micropropogation used
When a desirable plant:
Doesnt readily produce seeds
Doesnt respond well to natural cloning
Is very rare
Genetically modified or selectively bred with difficulty
Required to be pathogen-free by growers
What are the basic principles of micropropogation and tissue culture
Take a small sample of tissue from the plant you want to lone - meristem from shoot or root tips that is virus free
Sterilising sample, using agents like bleach or ethanol
Explant is then placed in sterile culture medium containing balance of plant hormones, proliferating and forming a mass of identical cells called a callus is divided into individual cells or clumps, transferred into a new culture medium containing a different mix of hormones, stimulating development of tiny genetically identical plantlets
These are potted in compost to grow into small plants
Young plants are planted out to grow and produce a crop
What are the advantages for micropropogation
Allows for rapid production of large number of plants with high yield of crops and known genetic makeup
Culturing meristem tissue produces disease-free plants
It makes it possible it produce disease-free plants
Produces viable number of plants are genetic modification of plant cells
Produces very large number of new plants which are seedless and therefore sterile (eg. banana and grape)
It provides a way of reliably increasing the numbers of rare or endangered plants
Arguments against micropropagation
Produces monoculture - all susceptible to same disease or change of environment
Relatively expensive process with skilled workers
Vulnerable to infection during production process
If source material is infected, all clones will be infected
Large number of plants can be lost during the process
What is the potential of natural cloning for saving important crops such as the banana against disease
Advantages
relatively easy
relatively cheap and readily available
history of use
Disadvantages
any disease in parent plant transferred with cutting etc.
limit to number of new
plants that can be formed so cannot keep up with demand if there is a major threat to crop
still produces clones
What is the potential of micropropagation for saving important crops against disease in contrast to natural cloning? give arguments for and against
Advantages can produce disease-free plants
can produce plants engineered to be resistant to
disease
can produce almost limitless numbers of plants fast
Disadvantages
relatively expensive
needs some infrastructure
still produces clones
How does natural animal cloning occur in invertebrates
Some animals eg. starfish, can regenerate entire animals from fragments of the original if they are damaged
Flatworms and sponges fragment and form new identical animals as part of their normal reproductive process
Hydra produce small buds on the side of their body which will develop into genetically identical clones
Some insects can produce offspring without mating - although high level of mutation so these arent true clones
How does natural animal cloning occur in vertebrates
Formation of monozygotic twins, when the early embryo splits to form two seperate embryos - differences occur in nutrition and position in uterus
Some female amphibians and reptiles will produce offspring when no male is available - but they are often male so they arent clones, but all of the genetic material is from the mother
How are invertebrates cloned
Liquidising a sponge or chopping up a starfish and a new animal will regenerate from most of the fragments
What are the two methods of vertebrate cloning
Artificial twining
Somatic cell nuclear transfer (SCNT)
Describe the process of artificial twinning, using cattle as an example
Cow with desirable traits is treated with hormones so she super-ovulates, releasing more mature ova than normal
Ova may be fertilised naturally or by artificial insemination, by a bull with good traits, then embryo is flushed out of the nucleus
Or mature eggs are removed and fertilised by top-quality bull semen in the lab
Usually before or around day 6 - cells are totipotent, and early embryo is split to produce several smaller embryos - each capable of producing a healthy full-term calf
Each of the split embryos is grown in the lab for a few days to ensure healthy growth before they are implanted into a surrogate mother
Embryos develop into foetuses and are born normally, so a number of identical cloned animals are produced by different mothers
Describe the stages of Somatic cell nuclear transfer
Nucleus is removed from a somatic cell of an adult animal
Nucleus is removed from a mature ovum harvested from a different female of the same species (it is enucleated)
The nucleus from adult somatic cell is placed into enucleated ovum and given a mild electric shock so it fuses and begins to divide
Embryo that develops is transferred into the uterus of a third animal, where it develops to term
New animal is a clone of the animal from which the original somatic cell is derived, but mitochondrial DNA will come from egg cell
What are the arguments for animal cloning
Produces more offspring than normal reproduction
Allows success of passing on desirable genes
Allows scientist to replicate a specific animal eg, top class race horse
Enables rare or endangered or extinct animals to reproduce
Arguments against animal cloning
Inefficient process - most animals take any eggs to produce a single cloned offspring
Cloned embryos fail to develop and miscarry or produce malformed offspring
Many animals produced by cloning have shortened lifespans
Unsuccessful in increasing populations of rare organisms or to bring back extinct animals
How is artificial twinning different from natural twinning
Natural twinning early embryo splits
and two fetuses go on to develop
from the two halves
of divided embryo
Artificial twinning split in early embryo is produced manually
number of identical embryos may be replaced in surrogate mothers
to produce a number of identical high
quality animals
The evidence suggests that monozygotic twins do not occur naturally in cattle. Suggest ways in which this might be investigated
Observing births and recoding twin births when animals appear the same
genetic testing of any
twin cattle of the same gender
Explain the similarities between artificial twinning and SCNT
Both processes involve removing eggs from an animal
both involve surrogate parents
both potentially produce a number of genetically identical organisms
Explain the differences between artificial twinning and SCNT
In twinning either gametes meet outside the body
and early embryo develops before being split
or early embryos flushed from the mother
egg cell contributes all maternal DNA
embryos produced from gametes
embryos genetically related to two parents
What is biotechnology
Applying biological organisms or enzymes to the synthesis, breakdown or transformation of materials in the service of people
What are some reasons why using microorganisms are ideal
No welfare issues to consider
Enormous range of microorganisms capable of carrying out many different reactions
Genetic engineering allows manipulation of microorganisms to carry out desirable reactions eg. production of human insulin
Short life cycle and rapid growth rate
Simple nutrient requirements and is often cheap
Low temperature conditions
What are some examples of indirect microorganism action on food production
Baking - yeast
Brewing - yeast
Cheese making - bacteria
Yoghurt making - bacteria
What are some examples of direct microorganism action on food production
Eating fungi eg. mushrooms
Producing Single-cell protein eg. Quorn from a fungus and fermenter and added with egg whites
Advantages of using microorganisms to produce human food
Reproduce fast and produce protein faster than animals or plants
High protein with little fat content
Can use variety of waste materials, including animal and human waste
Genetically modified to produce protein required
Production of microorganisms isnt present on weather or breeding cycles
No welfare issues when growing
Made to taste like anything
Disadvantages of using microorganisms to produce human food
Some microorganisms can also produce toxins if not maintained in optimum conditions
Microorganisms have to be separated to make food
Involve GM organisms and many people have concerns eating GM food
Protein has to be purified
Dislike the thought of eating microorganisms grown on waste
Has little natural flavour - needs additives
Compare the way yeast is used in the process of baking and brewing
Baking:
Mixed with sugar and water
Respires aerobically
Carbon dioxide produced used to
make bread rise
Yeast killed by heating as bread cooks
Takes a couple of hours
Brewing:
Mixed with malted barley and hot water
Respiration (fermentation ) Continues for days in anaerobic
conditions (1);
Ethanol produced as waste product Yeast eventually inhibited (not killed) by rising pH
Build-up of ethanol and lack of oxygen
Why is milk pasteurised before being used commercially to make cheese and yoghurt
To destroy bacteria that would make it go bad rapidly
or cause diseases such as TB
Why is milk homogenised before being used commercially to make cheese and yoghurt
The fat droplets are spread evenly through milk so cream doesn’t separate out
and creates a uniform product
Give two important differences between the production processes of cheese and yoghurt
Cheese whole milk used
bacteria used to separate the curds from the whey
changing texture, and bacteria ripen or mature the cheese in controlled slow reactions at low temperatures to change taste
out-compete bacteria that would make the cheese go bad
takes weeks, months
or years
can last for years
Yoghurt skimmed milk powder added to milk to enrich it
specific bacteria added and incubated at 45°C for 4–5 hours to produce extracellular polymers that
give the texture to yoghurt
lasts 2–3 weeks in a fridge
What are the different approaches to bioremediation
Using natural organisms -
Many microorganisms naturally break down organic material producing carbon dioxide and water
GM organisms -
Breakdown or accumulation of contaminants which they wouldnt usually encounter
What conditions are needed to produce penicillin
Small Fermenters
Mixture continuously stirred to keep oxygenated
Rich nutrient medium
Growth medium contains a buffer to maintain pH at around 6.5
medium temperature
What was the main difference between the use of fungi to produce penicillin and the use of bacteria to produce human insulin
Fungi produce penicillin naturally
bacteria genetically engineered/modified to produce human insulin
What is bioremediation? why it is often carried out on the site of contamination
Use of microorganisms or plants to break down pollutants and contaminants in soil or water
often carried out on site
because area of contamination may be very large so not practical to
remove contaminated material/too expensive to remove contaminated material
organisms involved in bioremediation grown and break down contaminants in situ
living organisms so they
grow and spread
may be harvested and contaminants retrieved
What are the risks of using microorganisms as a culture
There is always a risk of mutation taking place making the strain pathogenic
There may be contamination with pathogenic microorganisms
What are the processes by which bacteria must be added
Inoculating broth
Inoculating agar
How do you make inoculating broth
Make suspension of the bacteria to be grown
Mix a known volume with the sterile nutrient broth in the flask
Stopper flask with cotton wool to prevent contamination from the air
Incubate at a suitable temperature shaking regularly to aerate the broth providing oxygen for the growing bacteria
How do inoculating agar prepared
Wire inoculating loop must be sterilised by holding it in a bunsen flame until it glows red hot
Dip the sterilised loop in the bacterial suspension, remove lid of petri dish and make a zig-zag streak across the surface of the agar
Replace lid of petri dish, held down and sealed with tape, but not completely so oxygen can still get in
What are the phases of growth in bacterial colonies
Lag phase - when bacteria are adapting to their new environment
Exponential phase - Rate of bacterial reproduction is close to or at its theoretical maximum
Stationary phase - total growth is 0 (in dynamic equilibrium)
decline phase - reproduction has ceased and rate of death of cells is increasing
What are the several limiting factors which prevent exponential growth
Nutrients available
Oxygen levels
Temperature
Build-up of waste
Change in pH
Compare the processes of culturing bacteria in broth and on agar
Both provide nutrients, suitable pH, moisture etc
both need to be maintained at optimum
temperature for growth
both must be kept sterile until inoculated with microorganisms
both can be shaken at intervals to aerate it
agar plates remain closed once made up
broth is mixed
with known volumes of culture medium
agar plates inoculated using sterile wire loop and culture
medium
numbers in broth counted using turbidity, serial dilutions, and microscope graticules
numbers on agar calculated using colony counting
Why are there such clear differences between the theoretical growth curve of a bacterial colony and the actual growth curve in a closed culture
In large closed culture nothing gets in or out
initially, growth can be at theoretical rate as no
factors are limiting
as culture continues, numbers increase, food and oxygen are used up and
waste products build up often affecting pH
microorganisms run out of food or oxygen, are
inactivated by pH changes affecting enzymes or poisoned by waste products
so whilst theoretical
growth curve is exponential, real growth curve reaches a peak, plateaus, and declines
Explain why vinegar is a very good preservative
Vinegar is ethanoic acid therefore has a low pH
and inhibits bacterial growth
Explain why food eventually goes bad in the fridge
As temperatures fall bacteria growth slows but does not stop
so in fridge bacteria grow slowly
and eventually destroy food
Explain why in the northern hemisphere, material placed in a compost heap rots down much faster in August than it does in december
Reactions in bacteria and fungi that act as decomposers affected by temperature
in August
temperatures relatively high so decomposition reactions relatively fast. In December, the
temperatures are much cooler so slower reactions in decomposers and rotting slower
What is primary and secondary metabolites from microorganism culture
Primary are the substances wanted which form the essential functioning of the microorganism
Secondary metabolites are non essential for growth, but still used by the cells, eg. pigments and toxins against herbivores. Extracted in bioprocess eg. penicillin and other antibiotics
What is batch fermentation
Microorganisms are inoculated into a fixed volume of medium
As growth takes place, nutrients are used up and both new biomass and waste products build up
As the culture reaches the stationary phase, overall growth ceases, carries out biochemical changes to form desired end products
Process is stopped before death phase and the products are harvested
How does continuous culture work
Microorganisms are inoculated into sterile nutrieinnt medium and start to grow
Sterile nutrient medium is added continually to the culture once it reaches the exponential point of growth
Culture broth is continually removed, the medium, waste products, microorganisms, and product - keeping the culture volume in the bioreactor constant
What factors need to be controlled in bioreactors
Temperature
Nutrients and oxygen
Thickness
Asepsis
What is the structure of a bioreactor
page 600
Bioreactors may run batch or continuous process. What is the difference
Continuous processes run continuously once fermentation is started
sterile nutrient medium
added continuously once culture is growing exponentially
culture broth continually removed so product can be processed and culture volume remains the same
Batch process everything added
at beginning in fixed volume of medium
nutrients used up and microorganisms, products, and
waste products build up
may be stationary phase when secondary metabolites formed, process
stopped, products extracted, reactor cleaned, and new process begun
Choose three factors which need to be controlled in a bioreactor to give the maximum yield of product and for each explain why it is important and how it might be controlled
Temperature
if temperature too low microorganisms will not grow
quickly enough, too high and enzymes will start to denature and microorganisms are inhibited or
destroyed. Bioreactors often have a heating and/or a cooling system linked to temperature sensors
and a negative feedback system to maintain optimum conditions.
Nutrients
if microorganisms use up
food supply they will start to die off so need a mechanism to keep food supplied, nutrient medium can be added in controlled amounts to broth when probes or sample tests indicate that levels are decreasing to be mixed in using stirrers/paddles as will not spread through fast enough by diffusion
alone.
Oxygen
if microorganisms use up oxygen they will start to die off so need a mechanism to
keep nutrient medium oxygenated, oxygen is bubbled through broth when probes or sample tests
indicate that levels are dropping to be mixed in using stirrers/paddles as will not spread through fast
enough by diffusion alone.
pH
if waste products of microorganisms e.g., carbon dioxide build up then
pH of mixture will decrease. Change in pH can affect enzyme action and stop growth, buffers are added to mixture and stirred in or alkaline solution added to maintain optimum pH.
Why are immobilised enzymes so efficient
Less wasteful - do not produce biomass
More efficient - isolated enzymes work at much higher concentrations
More specific - no unwanted enzymes present, no wasteful side reactions
Maximise efficiency - can be given ideal conditions for maximum product formation as opposed to ideal condition for organism
Less downstream processing - so it is therefore cheaper
Why are extracellular enzymes cheaper and easier to use for isolating enzymes than intracellular
Secreted, making them easy to isolate and use
Microorganims produce few extracellular enzymes, so it is easy to identify, but produce hundreds of intracellular enzymes
More robust than intracellular, as conditions outside a cell are less tightly controlled
What are some advantages of using immobilised enzymes
Immobilised enzymes can be reused - so cheaper
Easily separated from the reactants and products of the reaction
More reliable - higher control over the process
Greater temperature tolerance
Easy manipulation
What are some disadvantages of using immobilised enzymes
Reduced efficiency by immobilising an enzyme by changing its active site
Higher initial cost of materials
Higher initial cost of bioreactor
More technical issues
What is adsorption surface immobilisation, and its advantages and disadvantages
Adsorption to inorganic carrier eg. silica
+ves:
Simple, cheap
can be used for many processes
Accessible to substrate and activity is almost unchanged
-ves:
enzymes can be lost from the matrix fairly easily
(pg 603)
What is covalent surface immobilisation, and its advantages and disadvantages
Covalent or ionic bonding to an inorganic carrier, polysaccharide or polymer
+ves:
Cost varies,
Strongly bound so unlikely to be lost
Enzymes accessible to substrate
pH and substrate conc often have little effect on activity
-ves:
Cost varies
Active site of the enzyme may be modified
(pg 603)
What is entrapment in a matrix, and its advantages and disadvantages
Entrapment in a matrix eg. gelatin
+ves:
Widely applicable to different processes
-ves:
May be expensive
Can be difficult to entrap
Diffusion to the substrate to and product from active site
Variable enzyme activity depending on matrix
(pg 603)
What is entrapment in a microcapsule, and its advantages and disadvantages
Entrapment - membrane entrapped in a semipermeable membrane eg. a polymer
+ves:
Simple to do
Small effect on activity
Widely applicable to different processes
-ves:
Expensive
Diffusion of the substrate to and product from active site can be slow and hold up the reaction
(pg 603)
What are some examples of immobilised enzyme products
Penicillin
Fructose
lactose free milk
L-amino acids
Dextrins
Plastics
What is meant by an immobilised enzyme
Enzymes attached to an inert support system
over which the substrate passes and is converted to product
What are the main advantages of immobilised enzymes over whole microorganisms
More efficient
more specific; can optimise conditions for specific enzyme
less downstream processing
What are the main advantages of immobilised enzymes over free enzymes
Can be reused
easily separated from reactants and products
more reliable as control over process
greater temperature tolerance
Summarise the ways in which enzymes are immobilised
Surface immobilisation – absorption onto inorganic carriers
covalent or ionic bonding onto inorganic carriers
entrapment in a matrix
entrapment in membrane bound microcapsule
How can immobilisation:
Increase the effectiveness of an enzyme
Enzymes are accessible to substrates
allow continuous production by a continuous flow of medium over the enzyme
conditions can be very tightly controlled over the enzyme beds
changes in pH and temperature have less effect
How can immobilisation:
decrease the effectiveness of an enzyme
Immobilising an enzyme may affect its ability to catalyse a reaction
diffusion of substrate to and from active site of enzyme can be inhibited
by immobilising matrix or capsule and so slow reaction
in surface immobilisation enzymes may be lost from matrix relatively easil
