Cloning and biotechnology chp 22 ONLY DONE 22.6 + .8 Flashcards
why should you take care when culturing microorganisms
- risk of mutation making bacteria pathogenic
- may be contamination with pathogenic microorganisms from environment
define inoculation
- adding of bacteria to nutrient medium (agar or broth)
explain steps to inoculate broth
- make suspension of bacteria to be grown
- mix known vol of bacteria with sterile nutrient broth
- stopper flask with cotton wool to prevent contamination
- incubate at suitable temperature
^shake regularly to aerate broth and provide bacteria with oxygen
explain steps to inoculating agar
- use sterile inoculating loop (Bunsen burner, until glow red hot)
^don’t let it touch surface to avoid contamination - dip loop in bacteria suspension
- remove lid of Petri dish
- apply in zig zag pattern (gently)
- replace lid of dish
- secure lid with tape and store upside down to avoid condensation dripping
- incubate
what’s the 1st phase of growth of bacterial colonies
- lag phase
^growing,
^synthesising enzymes
^not producing at max rate
what’s the 2nd phase of growth of bacterial colonies
- log/exponential phase
^rate of reproduction at max
whats the 3rd phase of growth of bacterial colonies
features of
- stationary phase
^total growth rate = 0
^number of cells made from binary fission = number of cells dying
what’s the last phase of growth of bacterial colonies
- death stage
^reproduction almost ceased
^death rates of cells increasing
what are some limiting factors to growth of bacteria
- nutrients available
- oxygen levels
- temperature (can denature)
- build up of waste (raises toxicity of environment)
- change in pH (can denature)
what are some examples of aseptic techniques
- closing doors + windows + turning off fans
- roll up sleeves, take jewelry off, tie up hair
- sanitise surface with 70% alcohol
- wash hands and forearms
- steralise all equipment before and after
- once equipment picked up don’t put it down until finished with
- open bottles with pinky
- flaming (bottle necks, inoculating loops)
- keep lid on petri dish for as long as possible (if opening keep close to dish)
- incubate agar unside down
define isolated enzymes
Enzymes separated from their natural environment
what are some advantages of using isolated enzymes instead of whole organisms
- less wasteful
- more efficient
- more specific (no unwanted enzymes present)
- maximises efficiency (can give optimum conditions for that enzyme)
- less downstream processing (pure product formed so no need to purify)
what type of enzymes are isolated enzymes usually
- extra cellular
why are extracellular enzymes easier to form into isolated enzymes
- are secreted making them easier to isolate
- organisms produce relatively few extracellular enzymes so easy to identify
- tend to be much more robust
when would intracellular enzymes be used as isolated enzymes
- bigger range of intracellular enzymes
^so can be more specific
^in this case pros > cons
what is an example of an intracellular isolated enzyme
- penicillin amylase for converting natural penicillin into drugs which are more effective
define immobilised enzymes
- isolated enzymes attached to inert support system over which substrates passes
what are the advantages of using immobilised enzymes
- immobilised enzymes can be reused (cheaper)
- easily separated from reactants + products so less need for downstream processing
- more reliable (high degree of control of process)
- greater temperature tolerance
- ease of manipulation (properties can be altered)
what are some disadvantages of using immobilised enzymes
- reduced efficiency (process of immobilising may reduce rate)
- higher initial cost of materials of both enzymes + bioreactor
- more technical issues (bioreactors with immobilised are complex)
what are the 4 types of immobilisation
- adsorption
- covalent bonding
- entrapped in matrix
- encapsulated
define adsorption immobilisation
- process of attachting enzyme to inert inorganic support system without use of covalent bonding
define covalent/ioninc bonding immobilisation
- covalent/ionic bonding of enzyme to inorganic carriers
what is an example of a matrix within which enzymes can be immobilised
- gelatin
- activated carbon
define encapsulation immobilisation
- membrane entrapment in microcapsules or semi-permeable membrane
what are the advantages of adsorption immobilisation
- simple and cheap to do
- can be used with many different processes
- enzyme accessible to substrate
- activity unchangedby process
what are the disadvantages of adsorption immobilisation
- enzymes can be lost from matrix relatively easily
what are the advantages of using covalent/ionic adsorption immobilisation
- cost varies (can be cheap)
- enzymes strongly bound (unlikely to be lost)
- enzymes accessible to substrate
- pH and substrate conc have little effect on rate
what are the disadvantages of using covalent/ionic adsorption immobilisation
- cost varies
- active site of enzyme may be modified in process
^can make it less effective
what are some advantages of entrappment in matrix immobilisation
- widely applicable to different processes
what are some disadvantages of entrappment in matrix immobilisation
- can be expensive
- can be difficult
- diffusion of substrate to and product from active site can slow down reaction
- effect of entrapment on enzyme activity variable (depending on matrix)
what are some advantages of encapsulation immobilisation
- relatively simple to do
- relatively small effect of enzyme activity
- widely applicable to different processes
what are some disadvantages of encapsulation immobilisation
- relatively expensive
- diffusion of substrate to and product from active site can slow reaction
what is an example of immobilised enzymes in industry
- immobilised lactase used to produce lactose free milk
^hydrolyses lactose to glucose and galactose
