Cloning & Biotechnology Flashcards
Define clone
- Genetically identical organisms
- Derived from a single original parent cell
Define cloning
Production of an organism genetically identical to another organism
Outline natural methods of cloning in some eukaryotes
- Asexual reproduction in plants (e.g. tubers)
- Budding in fungi
- Identical twins are clones - originate from the same cell
Define asexual reproduction
- One parent needed
- Offspring are genetically identical to each other and the parent
- Product of mitosis
Give examples of asexual reproduction in plants
Strawberry runners
- New plant develops at end of lateral stem
Potato tubers
- Buds on storage organ develop to produce new shoots
Bulbs (e.g. daffodils)
- Buds form internally
- Develop into new shoots and plants in next growing season
Rhizomes (e.g. marram grass)
- Horizontal stem that grows underground
- Buds develop to from new vertical shoots
Describe the process of taking a cutting
- Use a scalpel to take a small piece of plant
- Dip in rooting powder
- Replant in soil
Why are plant cuttings often used in horticulture?
- Faster than planting seeds
- Guarantees quality of plants
What is the disadvantage of using plant cuttings rather than seeds?
Reduced genetic variation in population
Describe an experiment to investigate the factors which affect rooting in stem cuttings
Possible independent variables:
- How many leaves are left on a cutting
- Temperature used
- Whether rooting powder is used
- Whether the cuttings are covered in a plastic bag
Possible dependent variables:
- Number of roots formed
- Length of the shoot formed after 10 days
Possible control variables:
- Temperature
- Carbon dioxide levels
- Humidity
- Plant species
Define artificial cloning
- Production of large number of clones
- Performed on industrial scale
Define micropropagation
- Making large numbers of genetically identical offspring
- From a single parent plant
- Using tissue culture techniques
Define explant
Small sample of tissue taken from chosen plant
Describe the process of cloning plants by tissue culture
- Cut plant material into explants e.g. meristem tissue, axial buds
- Sterilise explant with bleach or alcohol
- Place on growth medium containing glucose, amino acids, nitrates, phosphates
- Callus made from undifferentiated cells forms
- Auxins and cytokinin hormones added to stimulate callus formation
- Divide callus
- Treat with more hormones and nutrients to induce roots and shoots
- Transfer to soil when developed into small plants
Why is it necessary to use an aseptic technique when carrying out micropropagation?
Reduces contamination by microorganism
Define totipotent
- Cells that can differentiate into any other type of cell
- Early embryo (zygote) cells are totipotent
Give the advantages and disadvantages of cloning plants by tissue culture
Advantages
- Quick
- Can reproduce infertile plants
- Can save rare species from extinction
Disadvantages
- Expensive and labour intensive process
- All offspring susceptible to same pest / disease (little genetic variation)
- If source plant infected with virus, all clones will also be infected
Compare the equipment and techniques of taking cuttings with those used for micropropagation
Equipment
- Cutting needs less, micropropagation needs more
Skills and staff
- Cutting needs less, micropropagation needs more
Cloned offspring
- Cutting produces less, micropropagation produces more
Aseptic technique
- Micropropagation requires more aseptic discipline
Define natural twinning
- Splitting of early embryo naturally
- Forms monozygotic (identical) twins
Define totipotent
- Cells that can differentiate into any other type of cell
- Early embryo (zygote) cells are totipotent
Describe the process of embryonic cloning by artificial twinning
- Female animal with desirable traits selected
- Treated with hormones to produce more egg cells
- Egg cells fertilised naturally or by artificial insemination
- Embryo removed from mother’s uterus at early stage of development
- Split embryo up into individual totipotent cells
- Split embryos developed in lab for a few days
- Ensures embryos developing correctly
- Implant embryos into the uterus of different animals of the same species
Compare the process of natural twinning and artificial twinning
Natural twinning
- Early embryo splits
- Two foetuses develop
- From the two halves of divided embryo
Artificial twinning
- Split in early embryo is produced manually
- Number of identical embryos replaced in surrogate mothers
- Produces a number of identical high quality animals
Describe the process of the somatic cell nuclear transfer (SCNT) cloning technique
- Remove body (somatic) cell from animal to be cloned and remove nucleus
- Remove egg cell from animal of same species and remove nucleus
- Insert body cell nucleus into empty egg cell
- Fuse with electric shock
- Fused cell initially grown in vitro
- Early embryos split
- Embryos inserted into the uterus of surrogate mothers
- Embryos divide by mitosis
- Example: Dolly the sheep
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
Artificial twinning
- Gametes meet outside the body (or early
embryos flushed from mother)
- Egg cell contributes all maternal DNA
- Embryos produced from gametes
- Embryos genetically related to two parents
SCNT
- Nucleus removed from somatic cell and added to enucleated egg cell in laboratory
- Egg cell only contributes mitochondrial DNA
- Embryos produced from somatic nucleus and enucleated egg cell
- Embryos genetically related to one parent
Give the potential uses of adult cell cloning
- Produce animals with best characteristics
- To save endangered animals
- To grow stem cells or tissues / organs for human treatment
- To investigate the treatment and development of disease
State the advantages and disadvantages of using clones to test a treatment for a disease
Advantages
- Genetically identical so all subjects react the same
- Genetic variable controlled
Disadvantages
- Expensive
- Don’t see varied response to drug like in real populations
Outline the arguments for and against animal cloning
For
- Artificial twinning enables high-yielding animals to produce more offspring than normal
- SCNT can potentially enable endangered or
extinct animals to be reproduced
Against
- SCNT is very inefficient - many eggs needed to produce single cloned offspring
- Cloned animals tend to have shortened
lifespans
- Many cloned animal embryos fail to develop
properly
Define biotechnology
- Large-scale use of biological organisms or enzymes
- To synthesis, breakdown or transform materials
- To make suitable for human use
- e.g. food, drugs
What are the most commonly used organisms in biotechnology?
- Bacteria
- Yeast
Why are microorganisms used in biotechnology?
- No welfare issues
- Large range of microorganisms - Can carry out many different processes
- Can be genetically engineered to carry out other reactions
- Short life cycle and rapid growth rate
- Large quantities can be produced in short period of time
- Nutrients required are simple and relative cheap
- Conditions required are cheap and safe to maintain
- Relatively low temperature, supply of oxygen and food, removal of waste products
Define indirect food production
- Action of microorganisms on food to create a different type of food
- e.g. acting on milk to create cheese
What are the disadvantages of using microorganisms to produce human food?
- Require ideal conditions for process to work
- Pathogenic microorganisms can also grow
- Process has to be sterile
- Ethical concerns if GM organisms used
How is bread made?
- Yeast respires aerobically, producing CO2
- CO2 bubbles expand when cooked, making
bread rise - Yeast cells killed during cooking
How is beer made?
- Yeast mixed with malted barley and hot water
- Respiration (fermentation) continues for days in anaerobic conditions
- Ethanol produced as waste product
- Yeast eventually inhibited by rising pH
How is cheese made?
- Milk pasteurised and homogenised
- Mixed with bacterial cultures to separate milk into solid curds and liquid whey
- Curds cooked and pressed to produce cheese
How is yoghurt made?
- Skimmed milk powder added to milk
- Pasteurised, homogenised and cooled to 47°C
- Milk mixed with bacteria and incubated for 4-5 hours
- Yoghurt stored in cool conditions
Why is milk pasteurised before being used to make cheese and yoghurt?
- Microorganisms killed
- Enzymes denatured
- No pathogenic bacteria or competitors present
Why is milk homogenised before being used to make cheese and yoghurt?
- To spread fat droplets evenly through milk
- So cream doesn’t separate out
- Creates uniform product
Define direct food production
- Production of microorganisms for direct consumption
- e.g. Quorn (made from fungus)
Discuss the advantages and disadvantages of using microorganisms to produce human food
Advantages
- Reproduce and produce protein faster than animals and plant
- No welfare issues
- High protein content with little fat
Disadvantages
- Some microorganisms produce toxins if conditions aren’t optimal
- Protein has to be purified to remove any toxins or contaminants
- Need sterile conditions - increases cost
Define bioremediation
- Use of microorganisms or plants
- To break down pollutants and contaminants in soil or water
Why is bioremediation often carried out at the site of contamination?
- Area of contamination may be very large so not practical to remove contaminated material
- Organisms involved in bioremediation grown and break down contaminants in situ
- Living organisms used so they grow and spread
- May be harvested and contaminants retrieved
Give an example of bioremediation using natural organisms
- Microbes will break down crude oil
- Oil spills treated with nutrients to encourage microbial growth
Give an example of bioremediation using GM organisms
Modifying bacteria to remove mercury from contaminated sites
Why must aseptic techniques be used when culturing microorganisms in the laboratory?
- Risk of mutation producing pathogenic strains
- May be contamination with pathogenic microorganisms from environment
How are nutrients supplied to microorganisms grown in the laboratory?
- Broth - liquid
- Agar - solid
How is broth inoculated?
- Make a suspension of the bacteria to be grown
- Mix a known volume with sterile nutrient broth in flask
- Stopper flask
- To prevent contamination
- Incubate at a suitable temperature
- Shake regularly to aerate
- Prevents growth of anaerobic bacteria
How is agar inoculated?
- Wire inoculating loop sterilised in bunsen flame
- Do not touch any surfaces as it cools to avoid contamination
- Dip cooled, sterilised loop in bacterial suspension
- Remove lid of petri dish and streak suspension across surface of agar
- Make sure to keep agar surface intact
- Replace lid of petri dish
- Hold down with tape but do not seal completely
- Allows oxygen to still get in
- Prevents growth of anaerobic bacteria
- Incubate at suitable temperature
Compare the processes of culturing bacteria in broth and on agar
- Both provide nutrients, suitable pH and moisture for bacterial growth
- 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
Outline the four stages of microbe growth in a closed system
- Lag phase
- Microorganism adapting to new environment
- Growing and synthesising enzymes
- Not reproducing at maximum rate
- Log/exponential phase
- Microorganism reproduction at maximum rate
- Stationary phase
- Number of new cells formed equal to number of cells dying
- Microorganisms produce secondary metabolites e.g. penicillin
- Death phase
- Reproduction almost ceased
- Death rate increasing
Describe the pattern of growth of microbes in a closed system
- Lag phase - slow increase in population at start
- Log phase - exponential increase
- Rate of increase slows
- Stationary phase - population levels off
- Death phase - population falls
Why is a logarithmic scale used to represent size of bacterial populations?
- Difference in numbers from initial organism to maximum population size too great to
represent using standard numbers
Explain the factors that can prevent exponential growth in a culture of bacteria
- Nutrient availability
- Initially plenty of food
- As nutrients used up, insufficient amounts left to support exponential growth
- Oxygen levels
- As population rises, so does oxygen consumption
- Not enough oxygen for aerobic respiration
- Temperature
- Low temperature slows rate of growth and reproduction
- High temperature denatures enzymes and kills microorganisms
- Build-up of waste
- Build up of toxic material can poison and kill cells in culture
- Change in pH
- CO2 produced by respiration lowers pH
- If pH too low, enzyme activity reduces and population growth slows
Why is the theoretical bacterial growth rate not sustainable in a closed system?
- In large closed culture nothing gets in or out
- Initially, growth can be at theoretical maximum rate as no factors are limiting
- As culture continues, numbers increase
- Food and oxygen are used up
- Microorganisms run out of food or oxygen
- Waste products build up, affecting pH
- Denatures enzymes or poisons bacteria
Why does putting food in a fridge prolong how long it can be eaten for?
Low temperatures slow rate of bacterial growth
Why does food eventually go bad in a fridge?
- Bacterial growth slows but does not stop at low temperatures
- Bacteria grow slowly and eventually destroy food in fridge
Why is vinegar a good preservative of foods?
- Vinegar is ethanoic acid - lowers pH
- Inhibits bacterial growth
Why are serial dilutions used when counting bacteria?
- If too many colonies present, impossible to count how many there are
- Dilutions reduce the number of colonies on the agar plate
How is the total viable cell count calculated when using serial dilutions?
Multiply number of colonies by dilution factor
What is the equation for the theoretical maximum number of bacteria at a given time?
Nt = N0 x 2n
- Nt = number of cells at time t
- N0 = number of cells at time t = 0
- 2n = Number of divisions
Define primary metabolite
- Substance produced by microorganism
- Formed as part of essential functions
- Produced in all growth phases
- e.g. amino acids, enzymes, ethanol (produced by anaerobic respiration)
Define secondary metabolite
- Substance produced by microorganism
- Not essential for normal growth
- Produced in stationary phase
- e.g. penicillin, pigments
What is penicillin?
Antibiotic
What type of organism produces penicillin?
Fungus
What stage of the microorganism’s growth curve is penicillin made?
Stationary phase
Describe the process batch fermentation
- Fermenter set up with fixed quantity of nutrients
- Microorganisms inoculated into fixed volume of nutrients
- Left alone for culture to grow
- Culture reaches stationary phase
- Nutrients used up, waste products build up
- Secondary metabolites (e.g. penicillin) made
- Process stopped before death phase
- Product harvested
- System cleaned, sterilised and new batch culture started
Explain the importance of maintaining aseptic conditions in manufacturing penicillin by
fermentation
- Avoid unwanted microbe entry
- No competition for nutrients
- No decrease in yield
- No contamination of product
Describe the process of continuous fermentation
- Microorganisms inoculated into sterile nutrient medium
- Nutrients continually added and product regularly harvested
- Culture kept in log phase of growth
- Maximises production of primary metabolites or of the microorganisms
By what process is insulin produced?
- Continuous fermentation
- GM E. Coli grown in fermentor
- Insulin continuously harvested
What must be kept constant in the bioreactor during continuous fermentation?
- Nutrient levels
- pH
- Metabolic products
- Temperature
Explain why temperature needs to be controlled in a bioreactor
- Temperature too low - microorganisms will not grow quickly enough
- Temperature too high - enzymes start to denature, microorganisms may die
Explain why nutrients needs to be controlled in a bioreactor
If food supply used up - microorganisms will start to die
Explain why oxygen needs to be controlled in a bioreactor
If oxygen used up - microorganisms will start to die as cannot respire aerobically
Explain why pH needs to be controlled in a bioreactor
- If waste products (e.g. carbon dioxide) build up then pH will decrease
- Change in pH can affect enzyme action and stop growth
Why is it necessary for bioreactors to have stirring mechanisms?
- Simple diffusion not fast enough
- Ensures all microorganisms receive enough food and oxygen
Describe the differences between continuous and batch fermentation
Continuous
- 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
- Everything added at beginning in fixed volume
- Nutrients used up - microorganisms, products, and waste products build up
of nutrient medium
- May be stationary phase when secondary
metabolites formed - process stopped, products extracted, reactor cleaned, and new process begun
Define isolated enzyme
Enzyme removed from host microorganism
Explain the advantages of using isolated enzymes rather than whole microorganisms
- Less wasteful
- No microorganisms to use up substrate for metabolic processes
- More efficient
- Work at higher concentrations than when part of microorganism
- More specific
- No unwanted enzymes present
Why are most enzymes used in industrial processes extracellular?
- Easier to isolate as they are secreted
- Relatively few produced - easier to identify
- More robust than intracellular enzymes
Define immobilised enzyme
Enzyme attached to another substance e.g. glass, gel, alginate beads
Discuss the benefits of using immobilised enzymes for large-scale production
- Enzyme can be re-used - reduces cost
- Product uncontaminated - reduces downstream processing costs
- Immobilised enzyme works at higher temperature - reaction can be faster
- Immobilised enzyme works in changed pH
Discuss the disadvantages of using immobilised enzymes for large-scale production
- Reduced efficiency - immobilisation may reduce activity rate
- Higher initial costs - immobilised enzymes more expensive
- Higher costs of bioreactor - needs to be suitable for immobilised enzymes
- More technical issues - reactors more complex for immobilised enzymes
Describe the different methods of immobilising enzymes
- Entrapment - e.g. in alginate beads or cellulose matrix
- Adsorption - stuck to insoluble solid (e.g. carbon / clay / resin / glass)
- Covalent bonding - cross-link enzymes to each other and inorganic carriers (e.g. cellulose)
- Membrane separation - enzyme and substrate either side of partially permeable membrane
How can immobilisation increase the effectiveness of an enzyme?
- Enzymes arranged to be accessible to substrate
- Allow continuous production by continuous flow of medium over enzyme
- Conditions can be tightly controlled over enzyme beds
- Changes in pH and temperature have less effect
How can immobilisation decrease the effectiveness of an enzyme?
- Immobilising enzyme may affect ability to catalyse reaction
- Matrix or capsule can inhibit diffusion of substrate to and from active site
- Reactions may be slowed
- In surface immobilisation enzymes may be lost from matrix relatively easily
What is lactose?
sugar found in milk
Write an equation for the hydrolysis of milk
Lactose → glucose + galactose
Explain the production of lactose-free milk
- Milk poured over immobilised lactase
- Lactose broken down into glucose and galactose
- Suitable for people who are lactose intolerant
What is the advantage of using lactase in an immobilised state in the food manufacturing industry?
- Less likely to become denatured - higher temperatures can be used to increase rate
- Enable enzymes to be reused - reduces costs
- Enables reaction to flow continuously
What is glucose isomerase immobilised enzyme used for?
Glucose isomerase
- Used to produce fructose from glucose
- Fructose used as sweetener in food industry
