Cloning and Biotech Flashcards
What does penicillin need (FUNGUS)
High oxygen levels and rich nutrient medium
What is the mould penicillin sensitive to?
pH and temperature
First stage of penicillin production
Fungus grows
Second stage of penicillin production
Produces penicillin and is then extracted and purified
Process penicillin and reasons?
Small fermenters to maintain high levels of oxygenation
Continuously stirred to keep it oxygenated
Buffer solution
Bioreactors are maintained at about 25-27
Problems with animal insulin
Allergies
Supply erratic - demand for meat
Peak activity made was way later after injection - hard to predict
Faith groups no pig products
GM insulin now
Constant supply of pure insulin from downstream processing and bacteria grown in a fermenter
Natural bioremediation
Oil spill/sewage - microbial growth naturally breaks down organic material producing carbon dioxide and water
GM organism bioremediation
GM bacteria that can break down or accumulate contaminants which they would not encounter - remove mercury contamination from water
Risks whenever culturing
Mutation making a pathogenic strain
Contamination from pathogens in environment
Liquid form
Broth
Solid form
Agar
Inoculation
Process of adding bacteria/microorganism to agar/broth
Inoculating broth
Suspension of bacteria mixed with sterile nutrient
Stopper the flask with cotton wool to prevent contamination from air
Incubate and shake regularly to aerate and provide oxygen for bacterial growth
Inoculating agar
Wire (nichrome) - sterilised by holding in flame
Dip loop in bacterial suspension and make zig zag streaks
Replace lid of Petri dish - not sealed completely so oxygen can come in and prevent anaerobic growth of bacteria
Bacterial growth formula
N = No * 2^n
n is number of generations (divisions)
Lag phase
Synthesising enzymes - birth = death - acclimatising
Log phase
Rate is not limited - exponential no limiting factors
Stationary phase
Death = birth
Decline
Toxic build up of metabolic waste products
Nutrients limited/run out
Limiting factors with bacteria
Nutrients available
Oxygen levels
Temperature - denature enzymes
Build up of waste - build up of toxic material inhibit further growth and poisons/kills culture
Change in ph - carbon dioxide produces increases ph falls - enzyme activity
When do you want as much microorganisms as possible
For example with Quorn
Primary metabolites
Substances are wanted which are formed as an essential part of normal functioning - ETHANOL as a product of anaerobic respiration
Secondary metabolites
Substances which are not essential for normal growth but are still used by the cells - penicillin and other antibiotics
When are primary metabolites formed
During active growth - log phase
When are secondary metabolites formed
Stationary phase
Batch fermentation
Microorganisms inoculated into fixed volume of medium and nutrients are used up - biomass and waste products build up
SECONDARY METABOLITES
Culture reaches stationary phase - forms end products now (antibiotics/enzymes)
End products are harvested and whole system is cleaned/sterilised with new batch culture set up
Continuous culture
Inoculated and sterile nutrient medium added continuously once reaches exponential point of growth
BROTH IS CONTINUALLY REMOVED - MEDIUM, WASTE PRODUCTS, MICROORGANISMS AND PRODUCT - PRIMARY METABOLITES
CULTURE VOLUME REMAINS CONSTANT IN BIOREACTOR
Quorn uses
Continuous culture
Continuous reactors
Produce a mixture of unused broth/microorganisms/primary metabolites and possible secondary ones and waste products - downstream processing is very difficult and expensive here
Temperature in bioreactor
If temp too low then microorganisms will not grow quickly enough
If temp too high then denature
Negative feedback heating/cooling system to maintain optimum conditions
Nutrients and oxygen
Oxygen and nutrient medium can be added in controlled amounts to the broth when probes indicate they are dropping
Mixing?
Simple diffusion is not enough to ensure all microorganism receive food/oxygen - stirred continuously
Asepsis
Most bioreactors are sealed to prevent contamination from air
Steam inlet
Sterilising
Gas exhaust outlet
Prevent pressure building up
Cooling water inlet
Cooling water through outer jacket maintains temperature at an optimum
Filtered air inlet
Needs oxygen to respire aerobically
Which one has a fixed volume of medium
Batch
Advantage of batch
Chance of contamination is minimum because it is closed and can be easily separated
Which culture lasts longer
Continuous culture
Which one is easier to control and growth stops when nutrients are used up
Batch
Which one is very costly when contaminated and uses smaller vessels
Continous
Metabolites produced during log phase
Primary metabolites - involved in supporting growth of microorganisms and help them reproduce
Enzymes/nucleotides
Metabolites produced during stationary phase
CURVE UPWARDS AT THE START OF STATIONARY PHASE
Secondary metabolites - not involved in growth and reproduction - advantages of microorganisms
Antibiotics/toxins (defence mechanism)
Primary metabolites
produced in large quantities
Secondary metabolites
Produced in small quantities
Why is bag placed on during vegetative propagation
Reduces transpiration
Why cut between nodes during vegetative propagation
This is the area that will produce the roots
Dolly the sheep
THE EMBRYOOOOOO WAS IMPLANTED BACK INTO SURROGATE MOTHER
Correlation
Does not imply causation
Control variables for SCNT
Age of surrogate/health of surrogate
Age of embryo
Age of nucleus donor/egg donor
Advantage of microorganisms extra
Low cost - require low temperatures/pressure
Better for the environment since no pollution/land being used to grow food
Holding lid of Petri dish
Prevents contamination from air
Bacterial growth at 35 degrees
Could encourage growth of human pathogens
Immobilised enzymes
Less easily denatured
Immobilised enzymes downsides
Fewer active sites exposed
Higher initial start up
