Cloning and Biotechnology Flashcards
E.g. of immobilised enzymes: glucose isomerase
for the conversion of glucose to fructose
E.g. of immobilised enzymes: penicillinacyclase
acyclase for the formation of semi-synthetic penicillins (to which some penicillin-resistant organisms are not resistant)
E.g. of immobilised enzymes: lactase?
for the hydrolysis of lactose to glucose and galactose
E.g. of immobilised enzymes: aminoacyclase?
for production of pure samples of L-amino acids
E.g. of immobilised enzymes: glucoamylase?
- for the conversion of dextrins to glucose
- dextrins are a group of low-moelcular weight carbs produced by the hydrolysis of starch or glycogen
- dextrins are mixtures of polymers of alpha glucose units linked by alpha1,4 or alpha1,6 glycosidic bonds
E.g. of immobilised enzymes: nitrilase?
for the conversion of acrylonitrile to acrylamide (for use in the plastics industry).
What is a clone?
genetically identical copy e.g. monozygotic twins
what is horticulture?
The growing of flowers, fruits, vegs on a relatively small scale - gardening. Contrasts w agriculture which is on a large scale
which organisms reproduce asexually?
- plants
- all prokaryotes
- eukaryotes that reproduce by mitosis
advantages of of cloning?
- quick - no pollination involved
- can happen w/o mate
- all offspring cope in that enivr
disadvantages of cloning?
- no genetic variation
- so any genetic weaknesses passed on
- e.g. selection pressure - all would die
artificial cloning in plants ?
- Gardners take cuttings of plants
- can make use of the fact that plant vascular tissue can join up in the process of grafting
- A scion is cut and grafted onto root stock
- all grape vines are produced like this
natural cloning is a.k.a
vegetative propagation
natural cloning occurs in?
- Bulbs e.g. daffodils
- runners e.g. strawberry
- stem tubes e.g. potato
using natural cloning in horticulture?
- Possible to take cuttings of many plants - short sections of stems are taken and are planted either directly into the ground or in pots
- Rooting powder often applied to the base of the cutting to encourage growth of new roots
Propagation from cuttings advantages over using seeds?
- faster - time from planting to cropping is reduced
* guarantees quality of plants
Propagation from cuttings disad compared to using seeds?
lack of genetic variation
in natural cloning what forms?
a structure that develops into a fully differentiated new plant, which is gen identical to parent
2 basic ways of veg prop?
- cuttings
* runners
⭐ many organisms can reproduce ? and
asexually and sexually
some plamnts can also reproduce sexually which…
leads to variation via seed dispersal. Seed can be carried to a diff area where selection pressures are diff which may lead to speciation.
Artificial cloning?
- storing natural produce (tubers/runners/bulbs)
- cuttings between lead nodes
- grafting
e.g. commercial grape vines
`What is micropropagation?
is the process of making large numbers of genetically identical offspring from a single parent plant using tissue culture cuttings
What kind of tissue is used in micropropagation?
meristematic - totipotent - can differentiate to produce all the different types of cell in a plant
Plants that are cloned using tissue culture?
- that don’t readily produce seeds
- plants that are difficult to take cuttings from
- rare and endangered plants
- plants that have been GE
- plants produced by selective breeding
- plants that must be free of pathogens
Microprop a.k.a ?
tissue culture
Natural cloning in animals?
- Many invertebrates capable - e.g. starfish, sponges
- In vertebrates, monozygotic twins are formed from embryo splitting
- Parthenogenesis
Parthenogenesis?
Natural form of asexual reproduction in which growth and development of embryos occurs w/o fertilisation. In animals means development of an embryo from an unfertilised egg cells
Artificial cloning 2 man techniques:
- Artificial embryo twinning
* Somatic cell nuclear transfer
Embryo splitting?
- early embryo from cow and bull with desirable trait split into totipotent cells
- totipotent cell develops into an embryo
- embryos transferred to surrogate mothers
- identical cloned offspring produced
Somatic cell nuclear transfer?
- Diploid cell taken from mammary gland
- Nucleus removed from somatic cell
- Egg cell taken from egg cell donor
- Nucleus removed
- Nucleus of somatic cell put into egg cell
- Electric pulses used to fuse nucleus and egg cell, causing it to /
- Cell /, forming embryo
- embryo implanted into surrogate mother
offspring produced by somatic cell nuclear transfer?
genetically identical to nuclear donor, but mitochondrial DNA will come from egg cell
+ of SCNT?
- genetically identical - more sheep w desirable characteristics
- can be used to produce GMA which grow organs that can be used in human transplants
- Allows GM embryos to be replicated and to develop, giving many embryos from 1 GE procedure
- spec animals can be cloned
- rare/ endangered species reproduced
- of SCNT?
- Lack of GV dec robustness of population
- any genetic diseases, mutations passed down
- premature ageing
- inefficient - many eggs to produce a single cloned offspring
- Many clones embryos fail to develop and miscarry
+ of embryo splitting?
- more offspring produced than normal reproduction
* allows success of make animal at passing on desirable genes to be determined
- of embryo splitting?
- Many animals produced by cloning have red. life spans
* many cloned embryos fail to develop and miscarry or produce malformed offspring
Non - reproductive cloning?
• Cloning specific cells to replace others, not the whole animal`
Why clone cells?
- testing to see effect of external factors on the cells
- research effects of radiation
- cosmetics (reduced need for animal testing)
- now used experimentally for gene therapy
What is biotechnology?
using biological organisms/ enzymes for the synthesis, breakdown or transformation of materials for the benefit of humans
e.g. yeast - fermentation
Cheese?
- ester bonds broken by enzymes in bacteria
* FA = acidic, causes milk to curdle
What is most commonly used in biotechnology?
Fungi e.g. yeast or bacteria e.g. E coli
Why are microorg used in biotechnology?
- reproduce quickly - rapid growth and v short life cycle
- cheap molecular req. - can often use the wate products of other industry
- no ethical issues
- convenient - no welfare issues
- easier to store
- wide range available
- can be genetically modified - 1 chromosome in bacteria
- grow in simple conditions - high T and P not needed
E.g. of bacteria used in biotech?
E. coli - produce GM insulin and lactase
E.g. of archae used in biotech?
Thermoccocus - heat resistant enz e.g. Taq polymerase
E.g. of fungi used in biotech?
Penicillium - penicillin
E.g. of protoctists used in biotech?
algae - food thickening agents
E.g of biotech processes: baking?
- yeast
- anaer to produce ethanol and CO2
- CO2 makes bread rise
- yeast added to flour, left in warm envir to rise
- excess air removed, left to rise again
- cooked in hot oven, CO2 bubbles expand so bread rises more
E.g. of biotech processes: brewing?
- yeast, produced ethanol
- enzymes hydrolyse starch to sugars that can be used by the yeast
- enzymes break down starch to wort
- wort sterilised and cooled
- wort inoculated w yeast
- beer is conditioned for 4-29 days at 2-6 in tanks
E.g. of biotech processes: cheese making?
- bacteria feed on lactose in milk, changing texture and taste and inhibiting the growth of bacteria which make milk go off
- Milk pasteurised and homogenised
- mixed with bacterial cultures and kept until the milk separated into solid curds and liquid whey
E.g. of biotech processes: Yoghurt making?
- Bacteria to produce ethanal and lactic acid
- skimmed milk powder added to milk, mixture pasteurised, homogenised, cooled
- milk mixed with a 1:1 ratio w lactobasillus bulgarius and streptococcus thermophilus and incubated at around 45 degrees for 4-5 hrs
E.g. of biotech processes: Penicillin ?
- produced by the mould penicillium notatum
- semi-conservative batch process used
- first stage of production: fungus grows
- 2nd stage: penicillin produced
- then drug extracted and purified
E.g. of biotech processes: insulin?
- less side effects
- Used by more religious groups
- Bacteria grown in a fermenter and downstream processing results in a constant supply of pure human insulin
E.g. of biotech processes: bioremediation?
- MO used to break down pollutants and contaminants in the soil in water
- natural organisms can be used - many MO naturally break down organic material producing CO2 +H2O
- Or GM MO - scientists trying to develop GM bacteria which can break down or accumulate contaminants which they would not naturally encounter
Advantages of using MO to produce human food: fast
reproduce fast and produce proteins faster than plants and animals
Advantages of using MO to produce human food: WASTE
can be fed on waste, reducing costs
Advantages of using MO to produce human food: PROTEINS
MO can be genetically modified to produce req proteins
Advantages of using MO to produce human food: welfare issues
no welfare issues w growing MO
Advantages of using MO to produce human food: not dependent on
- production of MO is not dependent on weather, breeding cycles etc
- takes place constantly
- and can be inc or dec to meet demand
Advantages of using MO to produce human food: protein and fat content?
MO have a high protein content with little fat
Advantages of using MO to produce human food: taste?
can be made to taste like anything
Disadvantages of using MO to produce human food: toxins?
can produce toxins if conditions not maintained at optimum
Disadvantages of using MO to produce human food: separated?
MO have to be sep. from nutrient broth and processed to make food
Disadvantages of using MO to produce human food: sterile conditions?
need sterile conditions that are carefully controlled adding to costs
Disadvantages of using MO to produce human food: GM organisms?
often involve GM organisms and many people have concerns about eating GM foods
Disadvantages of using MO to produce human food: purification?
the protein has to be purified to ensure it contains no toxins or contaminants
Disadvantages of using MO to produce human food: waste?
many ppl dislike the thought of MO grow on waste
Disadvantages of using MO to produce human food: natural flavour?
little natural flavour - need additives
What is culturing MO?
growing large pops of MO can be done using agar plates, broth or bioreactors
Stages of a growth curve?
- the lag phase
- exponential phase
- stationary phase
- death phase
The lag phase?
- no. slowly inc (this is b4 growth is detected)
* bc adjusting to envir - synthesising enzymes needed e.g. lac operon
Exponential phase?
• growth taking place at the max rate (doubling)
stationary phase?
- death rate = growth rate
* decreased availability of space, nutrients running out
death phase?
- death rate . growth rate
- nutrients ran out
- toxic build up
when does a sigmoidal growth curve occur?
always happens when MO are grown in a culture where the nutrients are limited
but if nutrients could be kept from running or what would happen?
stationary phase wouldn’t be reached, would stay in exponential phase
equation to work out pop sizes?
• Nt = No x 2^n
Nt = no. of cells at end
No = no. of cells at start
• n = no. of generations
why is 2 used?
pop doubles every generation
if want to work out no. of generations?
use log little 2 (…)
how many mm3 per c3?
1000
how many cm3 per dm3?
1000
What is a metabolite ?
• a molecule made by a cell in the course of its metabolism
primary metabolite?
- molecule produced by all of the cells in a culture all of the time
- involved in normal metabolic processes like resp and growth
secondary metabolite?
only produced by some cells or at a particular growth phase. Not directly involved in normal metabolic processes
e.g. of primary metaboloute?
- pyruvate produced in glycolysis
* urea by ornithine cycle
secondary metabolite e.g.
penicillin produced by Penicillium fungus and Morphine produced by the opium poppy
Batch culture?
- MO inoculated into a fixed V of medium
- As the culture is abut to reach the death phase the products are harvested
- the whole system is then cleaned and sterilised and new batch set up
Product of batch culture?
The product of a batch culture is often a secondary metabolite, produced in the stationary phase, when the population is under environmental stress.
continuous culture?
• MO are inoculated into sterile 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, MO, products
- keeping the culture volume constant
Product of continuous culture?
The product of a continuous culture is often a primary metabolite, or the MO itself produced in the log phase, when the population is not under environmental stress
in both batch and continuous culture?
- as growth takes place, nutrients are used uo and waste products build up
- as the culture reaches the SP, the overall growth ceases but end products still produced
Bioreactor is a.k.a ?
fermenter
B: why is there steam?
to clean valve
B: nutrient or inoculant?
e.g. AA
B: nutrient medium is ?
sterile, to get rid of comp from other species
B: impeller?
mixes, preventing MO from settling at the bottom, motor causes it to turn
B: O2 conc probe?
- depends on MO and how they respire
* if are, high and if aner, low
B: cooling jacket?
high level of metabolic activity and impeller moving which generates heat so needs to be cooled down
B: cold water inlet and cold water outlet?
coldness maintained
B: steam near harvest pipe?
steriliser of liquid being harvested
B: antifoam?
preventing from foaming which could generate pressure
B: pressure guage?
so doesn’t explode
B: filtered waste gases?
to avoid removing MO too
B: pH probe
- pH change = enzymes denatured
* acid/ base added to alter pH
B: T probe
T change can cause denaturation
B: sparger?
air vent -allows bubbles ofgas to be put in. Can be used as a stirrer.
B: compressed air?
the gas supply
B: harvest pipe?
how products are harvested
Advantages of batch culture?
- Easy to set up - min attention needed during culture
- fermenter can be used for diff cultures
- only 1 batch needs to be discarded if cotamination happens
disadvantages of batch culture?
- down time between cultures
* large fermenters needed for profitable yields
Advantages of continuous culture?
- no down time
* small vessels can be used bc of continuous input and output
disadvantages of batch culture?
- sig problems if culture becomes contaminated
* culture can sometimes block input and output pipes
What happens inside a bioreactor?
culture of MO grows
what is a bioreactor?
vessel where conditions can be monitored and controlled very precisely
hameocytometer?
- used to estimate pop size of MOs
- cells in a small precise V of suspension are counted
- then answer multiplies up to the total volume of the pop
- cover slip put over haemocytometer which sits exactly 0.1mm above the counting grid, so the liquid containing the cells to be counted is 0.1mm deep
problems w culturing MO?
- MOs potentially harmful - espec. if grown in anaer conditions
- GM MOs must be contained inside bioreactors by law - biohazard centre
- cultures easily contaminated with unwanted MOs
What is done to avoid these issues?
aseptic techniques used
e.g. of aseptic techniques - sterlilised?
- all equip and agar sterilised beforehand using steam/ disinfectant/ ethanol
- autoclave can be used for small items
e.g. of aseptic techniques - protective clothing?
worn to prevent contamination FROM humans
e.g. of aseptic techniques - methods which stop MO from settling down?
- airflow upwards and out to prevent MOs settling on sterile surfaces
- Lit Bunsen on bench
- Higher pressure maintained inside bioreactor room with air pumps
- Cultures prepared in fume cupboard
e.g. of aseptic techniques - flaming?
• bottle openings and inoculating loops flamed
e.g. of aseptic techniques - opening lids of agar plates and bottles?
kept to a min.
e.g. of aseptic techniques - disposal of agar plates?
done carefully
culturing MO- which conditions are prevented?
anaerobic, to prevent growth of dangerous toxin producing anaerobes
other e.g. of aseptic techniques ?
- steam cleaning
* washing w ethanol
e.g. of enzymes used in industry?
baby food, biological washing powder
immobilised enzymes?
can be done in a no. of diff ways so that the products can easily be separated again after the reaction, leaving the enzyme molecule to be reused
IE - beads are?
totally porous - solution can go thru it
Methods of immobilising enzymes?
- adsorption to carrier
- covalent boding to carrier
- entrapment
- encapsulation
adsorption to carrier?
• enzyme bonded ionically or with hydrophobic interactions w carrier - gold, glass beads
adv of adsorption to carrier?
+ simp and cheap
+ can be used w many diff processes
+ high reaction rates
disadv of absorption to carrier?
- bonds not that strong so some leakage can occur
covalent bonding to carrier?
• enzyme molecules covalently bond to carrier and to other molecules
adv of covalent bonding to carrier?
+ very little leakage because bonding is strong
+ enzymes very accessible to substrate
disadv covalent bonding to carrier?
- active site may be modified in the process, making it less efficient
- cost varies
entrapment?
• enzymes trapped in gel or network of cellulose fibres
advantages of entrapment?
widely applicable to diff processes
disadvantages of entrapment?
active site less easily available so reaction rates slower
- can b difficult to entrap
- effect of entrapment on enzyme activity very variable depending on matrix
encapsulation?
• enzyme molecules separated from substrate by a partially permeable membrane, often in the form of a capsule
disadvan of encapsulation?
- may be expensive
- diffusion of the substrate to and product from the active site can be slow and hold up the reaction
advan of encapsulation?
- simple to do
- widely applicable to diff processes
general advantages of IE?
+ Enzyme molecules stabilised by immobilisation - more resistant to high T and extreme pH
general disadvantages of IE?
• Can slow reaction rates bc active site not as freely available as free enzyme
Advantages of micropropagation?
- sterile = infection would damage plant - disease free plant
- large no. of gen identical plants produced
- new plants which are seedless produced to customer’s tastes
- new way of growing infertile plants
disadvantages of micropropagation?
- monoculture produced - plants are genetically identical - all susceptible to the same diseases
- expensive, skilled workers req
- explants and plantlets vulnerable to infection during production
- if source infected by virus all clones will be infected
