cloning & biotechnology 6.2.1
genetically identical organisms
e.g.
bacteria - binary fision
eukaryotic organisms - mitosis
advantages of asexual reproduction
Quick
No need to find mate
Can act as back up if sexual reproduction doesn’t work
Cause organisms are adopted to environment all offspring should be adapted as well
Not wasting energy on gamete production
disadvantages of gamete production
No genetic variation
Risk of disease
Any changes in environment will affect all individuals - if kills 1 could kill all
Offspring may become overcrowded as if parent successful all offspring will be successful
natural clones in plants
Vegetative propagation
= when a new plant grows from the parent plant (circumnavigated seed process)
types:
leaves - clones grow on leaf margins, immature plants drop off & take root. e.g. Kalanchoe plant
tubers - swollen underground stems, new shoots grow from buds or eyes. e.g. potatoes
sucker - new stems will grow from the roots of a plant. e.g. English Elm - why Dutch elm disease was such a problem root suckers weren't resistant as contained same genes as parent plant
bulbs - underground stem of fleshy leaf bases, new plants grow from buds. e.g. onions
rhizome - underground stems that grow sideways in soil, 1 or more stems will grow in spring. e.g. ginger
corms - often mistaken for bulbs, solid not fleshy, underground stem with scaly leaves & buds, in spring buds grow to produce 1 or more plants, e.g. croci
runner - horizontal stem that grows over soil & forms buds, each bud grows into a new plant. e.g. strawberries
artificial clones in plants
1) cuttings
A section of stem is cut between leaf joints
Cut end is treated with hormones to encourage roots to grow
E.g. geraniums are produced this way
2) grafting
A shoot is cut & joined to the root section of another plant
Genes of roots different to genes of clones
3) tissue cultures (micropropagation by callus culture)
method
3) cells divide by mitosis to form a mass of cells called callus (mass of totipotent cells)
4) single cells removed from callus & placed into growing medium with hormones to encourage root growth. different ratios are used
2) explant placed in nutrient growth medium (glucose, amino acids, phosphates, auxins/cytokines)
5) growing plants transferred to greenhouse to acclimatise before planting outside
1) piece of tissue (explant) taken from plant. meristem tissue usually chosen cause its actively dividing & usually free of virus infection. explant is sterilised in alcohol/bleach cause microbes would compete for nutrients
auxin:cytokin at 4:2 stimulates shoot growth
auxin:cytokin at 100:1 stimulates root grwoth
advantages
- extremely cost effective
- easy to transport or store large numbers of plants under sterile conditions
- enables large nums of plants to be produced from single parent
- has conservation use in recovery programmes of endangered plants
- ensures good quality of stock plants are retained, e.g. resistant to disease & insects or high yield
- helps eliminate plant diseases since only healthy stock selected & sterile conditions used
- its time effective, since it circumvents need for pollination & seed production
- eliminates seasonal restriction on germination
cloning in agriculture
advantages
High yield
Resistance
Drought, disease, pest
Taste, texture, colour, nutrition
Rapid growth compared to seeds
Used where sexual reproduction isn’t possible
E.g. bananas
disadvantages
Tissue culture expensive to set up, needs large labs & equipment
Labour intensive
Often fails cause of contamination
Clones at risk of disease cause of no variation
cloning animals
somatic cell nuclear transfer
1) egg obtained & enucleated
2) body cell ( somatic) from adult to be cloned is isolated
3) complete adult cell is fused with empty egg cell by applying electric shock
4) shock also triggers cell to start dividing
5) cell undergos mitosis to produce a ball of cells
6) young embryo placed into uterus of surrogate mother
7) clone baby (like dolly the sheep clone) is born, its a clone of its 'genetic' mother
embryo splitting
1) zygote created using IVF
2) zygote allowed to divide by mitosis to produce ball of cells
3) cells separated & allowed to continue dividing
4) each small mass of cell is placed into uterus of surrogate mother
this process used to clone elite farm animals or for scientific research
exact phenotype will be unknown until birth cause it depends on which egg & sperm were used
advantages
A whole herd of animals with desired chariteristics
E.g. cows producing spider silk in their milk
Drug tested high value or rare animals can be produced
Drug tested on cloning animals
Genotypes are the same
Increases validity
Control variable
Cloned human or animal tissue avoids the need for tests on human/animal
Can produce cells & tissues identical to the donor for organ repair without rejection
disadvantages
Poor genetic diversity
Ethic: meat producing chicken cannot walk
Adult cloning isn’t very successful & the clones have shorter life & not as healthy
reproductive cloning VS non-reproductive cloning (therapeutic cloning)
reproductive cloning
non-reproductive cloning (therapeutic cloning)
= cloning to produce a whole organism
e.g. embryo transplantation (dolly)
using clones to produce clones
e.g. stem cell research, production of cells. tissues or organs
biotechnology
uses of microbes in biotechnology
why use microbes?
Biotechnology is any technological process that makes use of living organisms or parts of living organisms to make useful products or provide useful services.
Includes:
Domestication of animals
Selective breeding (plants and animals)
Beer produced for 7000 years
Yoghurt, cheese, bread
Definition is shifting towards manufacture of drugs.
Gene technology/modification/therapy
Cloning
Use of enzymes in industry
Immunology
Rapid growth - bacteria double every 30 mins
Produce useful proteins which can be harvested
Can be genetically modifies to produce specific proteins
Can work at low temperatures
Grown anywhere in the world (inside a fermenter)
Products are very pure
Can be fed on waste
Fewer ethical considerations
yogurt production
- acidity denatures milk protein causing to to coagulate
- proteins now partially digested therefore easier for us to fully digest - fermentation also produces flavour
- lactose converted into lactic acid
- other bacteria are probiotic & may be added - aid in gut fermentation & boost immune system
- milk fermented by Lactobacillus bulgaricus & streptococcus thermophilus
cheese making
3) resulting curd is separated from liquid by cutting, string & heating
4) curd is pressed into moulds
2) Ca ions precipitate the casein out of solution & bind molecule together
5) inoculation with penicillium will give additional flavour & produce blue colour
1) kappa-casein (soluble) broken down to insoluble casein
often lactobacillus added to milk at start to aid in solidifying
milk is mixed with rennet (found in stomach of young mammals). rennet contains enzyme chymosin that coagulates milk protein casein in presence of calcium ions
bread
Bread is a mixture of flour, water, salt and yeast (Saccharomyces cerevisiae)
1) Mix ingredients by kneading to produce a dough
2) Proving / fermenting: - dough left in warm place for 3 hours. Yeast respires anaerobically producing carbon dioxide bubbles. Dough rises because of the bubbles.
3) Cooking: - Alcohol evaporates during this process.
wine
Produced by anaerobic respiration of S.cerevisiae. wine is made using grapes (contains fructose and glucose). Grapes are crushed and the yeast uses sugars to produce carbon dioxide and alcohol
beer
Germinating barley grains are used. The grains break down starch to maltose.
This is called 'malting'. The maltose is respired by the yeast anaerobically to produce carbon dioxide and alcohol.
Hops are added to give a bitter taste.
Quorn
Quorn is made using a fungus (Fusarium venenatum). The fungal protein produced is known as a single-cell protein. An example is Quorn and was first made in the 1980s.
Single-cell protein has no animal fat or cholesterol. It is suitable for vegetarians.
The fungi Kluyveromyces Scytalidium and Candida are now being tried. They produce proteins with similar amino acid profiles to animal and plant protein. They can be grown on almost any organic material including wastes e.g. paper, and the watery part (whey) from cheese-making.
advantages:
1) Production much faster than rearing animals or growing plants
2) High protein content of food (85% dry mass)
3) Easy to increase or decrease production dependent on demand
4) No animal welfare issues
5) Protein contains no animal fat or cholesterol
6) Easy to genetically modify to adjust amino acid content
7) Can produce in all seasons
8) Not much land needed
9) The microbes can be fed on waste
disadvantages:
1) People may not eat food which has been fed on waste
2) Difficulty isolating the protein from the substrate
3) Difficulty purifying the product
4) Product can have too many nucleic acids which can cause gout if not removed
5) Amino acid profile can be different from plant or animal proteins. Ouorn is often deficient in Methionine
6) The culture medium / conditions are perfect for pathogens. Can be difficult to ensure aseptic conditions
7) Quorn does not have the taste or texture of traditional sources
bacterial growth curve
1) Lag phase
Taking in water
Adjusting to environment
Activating genes
Making enzymes
Length of lag phase depends on how good growing conditions are
2) log phase
Exponential growth
Population doubles each generation
Limiting factors:
Space
How quickly nurience can be taken up
Speed of division
How much food is available
3) stationary phase
Nutrients are decreasing
Waste is building up
E.g. CO2 or ethanol
Rate of formation of new cells is equal to death rate
4) decline or death phase
Nutrients exhaustion
Toxic waste build up
All microbes will eventually die in an enclosed environment
E.g. making wine yeast will die at 10% ethanol concentration
metabolites
primary metabolites
Substances made by organisms as part of its growth
E.g.
Amino acids
Proteins
Enzymes
Nucleic acids
Ethanol
Lactate
If growth increases so do these products
secondary metabolites
Made by organisms but not part of its growth
Starts after main growth period
E.g.
Antibiotics made by fungi
Only a few organisms produce secondary metabolites
industrial fermenting
In order to achieve max yield we must ensure good growth of the microbes…
Must maintain:
Temperature
too cold = slow
Too hot = death
Temp probes/paddle to distribute heat
Nutrients
Sources of carbon, nitrogen, vitamins/minerals.
Amount & timing of nutrients is important
Oxygen concentration
For sufficient respiration & growth
Anaerobic respiration may lead to unwanted products
pH
Must be optimum & adjusted if needed
Various probes to measure O2, pH & temp
Concentration of product
If its builds up may affect process & reduce yield
Need to remove regularly
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- thousands of litres
- idea to produce huge yield of product
- huge tanks
aseptic conditions
Means any unwanted microbes are kept out. Only chosen microbe is grown
Achieved in fermenter by…
Steam clean apparatus
Filter air entering
Sterilise any nutrients
High polished steel to stop microbes sticking
Why asepsis is important
Competition from other microbes reducing yield
Spoilage
Toxic materials made
Many will destroy your chosen microbes &/or products
batch vs continuous culture
batch
continuous
Fermenter is set up & run for specified time. Then stopped. Products removed. Repeat
Penicillin made this way from PENICILLIUM FUNGUS
Pros:
Easy to set up & maintain
If you contaminate only 1 batch is lost
Very useful for secondary metabolite production
Cons:
Slow & less effective
Nutrients added regularly
Products removed regularly. Or contumely
Insulin made this way
Pros:
High yield
Nutrients added all the time
Used for primary metabolites production
Cons:
More difficult to control
Contamination destroys huge volume
penicillin production
Florey and Chain devised the method to mass produce penicillin using the fungus Penicillium chrysogenum.
Penicillin is a secondary metabolite.
It is only produced when the population has reached a certain size. It is produced using Batch Culture
1) Fermenter run for about 7 days. Culture is filtered to remove cells.
2) Potassium compounds added to precipitate out penicillin crystals
3) The antibiotic is mixed with inert substances and prepared for different forms such as tablets, syrups or for injection.
insulin production
steps:
1) The DNA for insulin is first isolated.
2) A plasmid made of DNA is removed from a bacterial cell.
3) A restriction enzyme cuts the plasmid DNA open, leaving sticky ends.
4) The insulin gene, with complementary sticky ends is added.
5) DNA ligase enzyme splices (joins) together the plasmid DNA and the Insulin DNA.
6) The plasmid (now genetically modified) is inserted back into the bacterium
7) The bacterium host cell divides and produces copies of the plasmid?
8) The Bacterium makes human insulin using the gene in the plasmid.
9) The insulin is extracted from the bacierial culture.
Insulin used to be extracted from the pancreas of animals to treat type 1 diabetes. It was less effective. It was also expensive to extract.
In 1978, synthetic human insulin was developed by genetically modifying a bacterium.
Vast quantities can be produced at low cost. Insulin is manufactured by continuous culture as it is a primary metabolite
bacterium used was E. coli.
aseptic technique
mobilised & immobilised enzymes
bioremediation
This is the use of microbes to clean soil and underground water on polluted sites.
The toxic pollutants are converted to less harmful substances
In 1971 a modified Pseudomonas bacterium was produced which could digest crude oil.
Others can digest pesticides and solvents.
To function, the microbes need water, suitable temp and pH.
Sometimes additional food is given e.g. sugar to ensure good growth.
Nutrients and extra oxygen is sometimes added.
advantages:
- treatment is 'in situ'
- few waste products formed
- less labour or equipment needed
- less harmful than getting people to clean up waste
- natural meathod
disadvantages:
- cant be used to clean up heavy metals, e.g. cadmium or lead
why are enzymes useful
- work at low temps
- specific so fewer biological products formed - less purification
Immobilised enzymes
Mixing the enzyme and substrate together is costly as they need to be separated afterwards. Better to immobilise the enzyme.
Advantages
- Processing costs low - don't need to be separated
- Enzymes can be reused immediately. Continuous process possible.
- Enzymes are more stable as immobilising matrix protects them
Disadvantages
- Immobilisation more expensive and time consuming to set up
- Less activity as the enzyme and substrate do not mix freely - fewer successful collisions so fewer ESCs per second, so less product formed per second
methods of immobilisation
Adsorption
Collagen matrix used. Enzyme binds to it because of hydrophobic interactions and ionic links. Adsorbing agents include carbon, glass beads, clays and resins.
Advantage: very high reaction rates
Disadvantage: enzymes may become detached (leakage)
Covalent bonding
Bonded to a support material, e.g. Cellulose or clay fibres. Usually an insoluble material.
Advantage: binding is very strong. Little to no leakage
Disadvantage: only a low quantity of enzyme can be bonded. Yield is fairly low.
Entrapment
Enzyme trapped within gel beads. They are still in natural state so enzyme active site not affected.
Advantage: more resistant to changes in pH and temperature.
Disadvantage: lower reaction rate as substrate and products need to pass through trapping barrier.
Membrane separation
Partially permeable membrane. Substrate molecules small enough to pass through. Product molecules small enough to pass out.
Disadvantage: Product and substrate must be small enough to go through. Lower rates of reaction.
industrial uses of immobilised enzymes
penicillin acylase
glucose isomerase
Converts glucose to fructose
Much sweeter taste
Used in diet food because you don't need to use so much to get same taste
Used to make High Fructose Corn Syrup. Many uses in the food industry
Cheaper than sucrose
Used in soft drinks, jams, cereals
Used to make semi synthetic penicillin e.g.
Amoxicillin and ampicillin.
Some microbes are resistant to penicillin but not resistant to these modified antibiotics.
lactase
Converts lactase into glucose and galactose by hydrolysis
Makes lactose--free milk
People who are lactose intolerant can therefore still drink milk.
This will help prevent weak bones or osteoporosis
aminoacylate
A hydrolase is used to produce L-amino acids
from N-acyl-amino acids.
Needed as the building block for agrochmicals
Pharmaceuticals, food additives
glucoamylase
Speeds up the breakdown of starch to glucose.
During starch breakdown by amylase, short polymers of glucose are produced called dextrins.
Glucoamylase converts these to glucose.
Used to convert starch pulp to gasohol for alterkative motor fuel.
Used in the food industry to make High Fructose Corn Syrup
nitrile hydratase
Converts nitriles to amides e.g. acrylamide, a type of plastic.
Acrylamide is used to treat water. It causes contaminants to stick together so they are easy to filter out.
Polyacrylamide is used in paper making and to make gel for electrophoresis
1) Wash your hands
2) Disinfect your working area
3) Have a Bunsen burner on. This causes air to rise & prevents airborne microbes from setting. It creates a sterile environment where you can work
4) As you open a bottle, flame the opening to prevent bacteria entering. Also flame before closing
5) Don’t lift lid off Petri-dish completely. Just enough as required
6) Any glassware or equipment should be passed through the flame before and after contact with desired microbe
techniques used in microbiology
1) Sterilisation
Nutrients agar medium & any equipment must be sterilised in an autoclave (121’C for 15 mins)
This kills all living organisms & their more resistant spores
When agar is cool, pour into sterile dishes & leave to set, keep lids on to prevent contamination
2) Inoculation
Streaking - a wire look is used to transfer a drop of liquid medium onto the surface of the agar. The drop is then drawn out into a streak
Seeding - a sterile pipettes transfers a small drop of liquid medium to the surface of the agar or to a Petri dish BEFORE the agar is poured on
Spreading - a sterile glass spreader is used to spread a drop over the surface of the agar
3) Incubation
Label your Petri dish
Use 2 strips of tape to keep lid on
Don’t seal completely or unwanted anaerobic bacteria could grow and they can be dangerous
Place in incubator (upside down - this stop condensation falling onto agar and stops drying out)
After 24hrs the plates can be examined
Each colony results from a single bacterium
Filamentous fungi appear fluffy an grow in to a mass of hyphae
Wash hand after & sterilise Petri dishes
estimating the number of cells in a culture
Num of cells in population = initial Num of cells x 2^n
Cells divide every 30 mins
300 cells present at start
How many are present after 7hrs
300x2^14=4,915,200
n = num of cell divisions
using logs:
Press log button on calculator then type original number and press equal - gives you log number (that is the log number, which is what put as a power of 10 to get original number)
measuring growth rate of bacteria
Use aseptic technique throughout
1) Inoculate a bottle of sterile broth with chosen bacterium
2) Incubate broth at 25’C
3) At time 0 take a standardised drop of broth (0.1 cm^3) & spread over agar plate using a spreader
4) Incubate plate at 25’C
5) At regular intervals (e.g. 1hr) take another 0.1cm^3 sample from the broth bottle & inoculate a new agar plate as before
6) Repeat a this several times
7) After 24hrs (from the start of each dish), count the number of colonies on each plate
8) Uses the volume (0.1cm^3) to calculate the density of colonies per cm^3
9) Post population density (cfu/cm^3 [colony forming units]) against time
10) Y axis should be in log10 scale
the need of serial dilutions
Often the populations are too high to count. The colonies are so vase in number that they merge together making counting impossible
To overcome this problem we inoculate several agar plate at each time interval using a serial dilution.
At least 1 of these plates will produce colonies that can be counted.
We then use the serial dilution value to calculate the true number of colonies
18 colonies in final dilution. Volume of dilution used on agar plate is 0.1
To find volume in final dilution times 18 x 100
Then x10 3 time to get back to volume at 1st dilution
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18 x 100 = 1,800
1,800 x 10 = 18,000
18,000 x 10 = 180,000
180,000 x 10 = 1,800,000
other ways of counting cells
Spectrophotometer:
A spectrophotometer shies a light through a sample
The more bacteria there are the less light will pass through
The machine measures turbidity (cloudiness) of the broth
The more turbid, the more cells present therefore more replication has taken place
Petroff-Hausser cell counter:
Microscopic quadrat that allows you to count cells in a tiny volume of liquid
Can measure rate of growth in different liquids
factors to investigate
What could be the independent variables when investigating bacterial growth:
PH - add different pH buggers to the broth
Temp - use a range of incubators set a different temps
Nutrient availability - use different preparations with a different nutrient missing or at different concentrations