6.2.1 Cloning and biotechnology

Question 1

What is cloning?

Answer 1

-process of producing genetically identical cells/organisms from an existing organism

Question 2

Explain natural cloning(aka vegetative propagation)

Answer 2

-production of genetically identical plant clones from non-reproductive tissue i.e roots, stem, leaf
-type of asexual reproduction
-can help plants survive adverse conditions by cloning certain organs involved in food storage/ones that can remain dormant in soil

Question 3

Examples of vegetative propagation

Answer 3

-bulbs: act as underground food storage. new bulbs are able to develop from original bulb and form new shoots + plants
-runners/stolons: i.e strawberries/spider plants, lateral stem grows away from the parent plant + roots develop where the runner touches the ground–} develops a new plant and the runner eventually withers away
-rhizomes: i.e marram grass, specialised horizontal stem running underground away from the parent plant. buds develop and form new vertical shoots which become independent plants
-tubers: large underground plant storage that act as a food storage, buds/’eyes’ on the storage organ sprout to produce new shoots i.e potato

Question 4

Advantages of natural cloning

Answer 4

-conditions that are good for the parent are good for the offspring
-rapid–} population can increase quickly
-reproduction can be carried out with one parent

Question 5

Disadvantages of natural cloning

Answer 5

-offspring may become overcrowded (competition affects yield)
-lack of genetic diversity (unless mutations during DNA replication)
-selection not possible
-whole population susceptible to changes in the environment

Question 6

Uses of natural clones in horticulture

Answer 6

-used by farmers and gardeners to produce new plants
-i.e splitting up bulbs, removing young plants from runners, cutting up rhizomes
-can use grafting(joining shoot of one plant to the growing stem and root of another plant), can use layering(bending a stem of a growing plant downwards so it enters the soil + grows into a new plant)
-food crops are propagated by cloning i.e bananas, sugar cane
-can also take cuttings of plants

Question 7

Method to producing plant clones from cuttings

Answer 7

-use a scalpel to take a cutting between 5 and 10 cm long, from the end of stem of the parent plant
-remove the leaves from the lower end of the cutting, leaving just one at the tip
-dip the lower end of the cutting in rooting powder, which contains hormones that induce root formation
-plant the cutting in a pot containing a suitable growth medium
-provide the cutting with a warm and moist environment by covering the whole pot with a plastic bag
-when your cutting has formed its own roots and is strong enough, you can plant it elsewhere to continue growing

Question 8

What is tissue culture?

Answer 8

-used to clone plants artificially that don’t readily reproduce or are endangered/rare

Question 9

Method of tissue culture technique

Answer 9

-small tissue called explants are taking from the parent plant containing stem cells(totipotent so can differentiate into any type of plant cell)
-cells are sterilised to kill any microorganisms (i.e bacteria and fungi which compete for nutrients with the plant cells, which decrease their growth rate)
-cells are placed on a culture medium containing organic nutrients i.e glucose + aa’s, and a high conc of auxins. carried out under aseptic conditions
-cells proliferate, forming a mass of identical undifferentiated cells called a callus
-mass can be subdivided to produce lots of plant quickly
-when the cells have divided + grown into a small plant, they’re taken out of the medium and planted in soil–} develop into new plants genetically identical to original plant

Question 10

Explain micropropagation using tissue culture

Answer 10

-the process of making large numbers of genetically identical offspring from a single parent plant rapidly using tissue culture techniques
-cells are taken from developing cloned plants and sub cultured(grown on another fresh culture medium)- repeating this process creates large no. of clones
-used to produce plants when a desirable plant: doesn’t readily produce seeds, is rare, has been genetically modified or selectively bred with difficulty, is required to be ‘pathogen-free’
-extensively used in horticulture and agriculture

Question 11

Advantages of micropropagation

Answer 11

-allows for the rapid production of large numbers of plants with known genetic makeup which will yield good crops
-culturing meristem tissue produces disease-free plants
-makes it possible to produce viable numbers of plants after genetic modification of plant cells
-provides a way of producing very large numbers of new plants which are seedless and therefore sterile
-provides a way of growing plants which are naturally infertile/difficult to grow from seed
-provides a way of reliably increasing the numbers of rare/endangered plants

Question 12

Arguments against micropropagations

Answer 12

-produces a monoculture= many plants which are genetically identical–} all susceptible to the same diseases or changes in growing conditions
-expensive process
-if the source plant is infected with a virus, all of the clones will also be infected
-large numbers of new plants are lost during the process

Question 13

What is natural animal cloning?

Answer 13

-can be produced naturally as a result of sexual reproduction—} fertilised egg can split in the early stages of development into multiple embryos with the same genetic info
-these can develop as normal to produce genetically identical offspring i.e identical twins

Question 14

Natural cloning in invertebrates

Answer 14

-starfish can regenerate entire animals from fragments of damaged original
-flatworms and sponges fragment
and form new identical animals as part of their normal reproductive
process
-hydra produce small ‘buds’ on the
side of their body which develop into genetically identical clones

Question 15

Natural cloning in vertebrates

Answer 15

-formation of monozygotic twins: early embryo splits to form two separate embryos
-some female amphibians and reptiles reproduce asexually(however the offspring are often male so not a clone of the mother but share her genetic material)

Question 16

What is artificial embryo twinning?

Answer 16

-similar to what happens in natural cloning(when an early embryo splits and two foetuses go on
to develop from the two halves) except the split in the embryo is manual
-used by the farming community to produce the maximum offspring as the early embryo may be split into
more than two pieces and create a no. of identical offspring

Question 17

What are the stages of artificial twinning?

Answer 17

-an egg cell is extracted from a female cow and fertilised in a petri dish
-fertilised egg is left to divide at least once, forming an embryo in vitro(outside a living organism)
-the individual cells from the embryo are separated and each is put into a separate petri dish—} each cell divides and develops when the cells are still totipotent, forming an embryo in each dish
-embryos are implanted into female cows, which act as surrogate mothers
-embryos continue to develop inside the surrogate cows, and eventually genetically identical offspring are born

Question 18

Somatic cell nuclear transfer(SCNT)

Answer 18

-process of cloning an adult animal by taking the nucleus from an adult somatic
(body) cell and transferring it to an enucleated egg cell (an oocyte
which has had the nucleus removed)
-steps:
1) a somatic cell(one that isn’t reproductive) is taken from an animal and the nucleus is removed
2) an oocyte(immature egg cell) is taken from another sheep and its nucleus is removed to form an enucleated oocyte
3) the nucleus from the somatic cell is placed in the enucleated oocyte and given a mild electric shock(electrofusion) so it fuses and begins to divide—} produces an embryo
4) embryo is then implanted into a surrogate mother and a new animal is formed which is the clone of the somatic cell animal(but the mitochondrial DNA will come from the egg cell)

Question 19

Uses of animal cloning

Answer 19

-scientists use cloned animals for research purposes i.e genetically identical so less variables for drug trials
-cloning can be used in agriculture so farmers can increase the no. of animals with desirable characteristics to breed from
-animals that have been genetically modified to produce a useful substance that they wouldn’t normally produce could be cloned i.e goat milk with beneficial protein
-cloning can be used to save endangered animals from extinction by cloning new individuals

Question 20

Arguments for animal cloning

Answer 20

-artificial twinning enables high-yielding farms to produce more offspring than normal reproduction
-desirable genetic characteristics are always passed on to clones(no independent assortment or crossing over, which generate genetic variation during meiosis)
-infertile animals can be reproduced
-SCNT enables GM embryos to be replicated and develop many embryos
-cloning helps develop new treatments for disease, which could mean less suffering for some people

Question 21

Arguments against animal cloning

Answer 21

-very difficult, time consuming and expensive
-undesirable characteristics are always passed on to clones, susceptible to the same diseases
-inefficient process= often takes many eggs to produce a single cloned offspring
-many animals produced by cloning have shortened lifespans, unethical
-use of cloned human embryos as a source of stem cells is controversial(embryos often get destroyed after stem cells are harvested)

Question 22

What is biotechnology and what are the most commonly used organisms used in it?

Answer 22

-the industrial use of living organisms to produce food, drugs and other products
-most commonly used organisms are fungi and bacteria because:
-easy to grow successfully any time of year(as long as they have the right temp, pH, nutrients, moisture levels + availability of gases)
-inexpensive to grow
-grow rapidly under ideal conditions due to short life cycle(products can be made quickly)
-can be genetically engineered to carry out reactions they wouldn’t normally do

Question 23

Uses of microorganisms in biotech: Indirect food production

Answer 23

-microorganisms have an indirect effect when being used to make food–} it is their actions on food that is important
- Baking: the microorganism yeast makes the bread rise by respiring anaerobically, CO2 produced by fermentation of sugars in the dough makes sure it doesn’t stay flat
- Brewing: yeast respires anaerobically using glucose from the grain it is added to i.e barley to produce ethanol and CO2(fermentation)
- Cheesemaking: lactic acid bacteria converts the lactose in milk into lactic acid(makes it sour and helps solidify), chymosin enzyme can also be extracted from yeast which clots the milk
- Yoghurt: lactic acid/ ethanal bacteria causes milk to clot and thicken(both types of bacteria produce extracellular polymers that make yoghurt smooth + thick)

Question 24

Uses of microorganisms in biotech: direct food production

Answer 24

-microorganisms can be used to directly produce protein you can eat
- Quorn(type of single-cell protein) is produced by a single celled fungus that is grown in large fermenters
:) suitable for vegetarians, high in protein and low in fat

Question 25

Advantages of using microorganisms to produce food

Answer 25

-making single-celled proteins can be a mechanisms to get rid of waste products--} can be used as organic substrates -less land required compared to crop growing or livestock -reproduce fast and produce protein faster than animals/plants -can be considered healthier as microorganisms have high protein content and little fat -production not reliant on climate/seasons -no welfare issues when growing microorganisms -nutrient requirements i.e oxygen + glucose are cheap

Question 26

Disadvantages of using microorganisms to produce food

Answer 26

-some microorganisms can also produce toxins in conditions are not optimum -maintaining sterile conditions is expensive and requires lots of effort -concerns about eating GM foods i.e texture, taste, grown on waste products -protein has little natural flavour -if consumed in high quantities, health problems could be caused due to high levels of uric acid when large amounts of AA are broken down

Question 27

Producing penicillin

Answer 27

-fungi(usually P.chrysogenum) from the Penicillium genus produce an antibiotic that stops bacteria from growing + competing for resources -produced under stress using industrial fermenters: • relatively small fermenters because it is very difficult to maintain high levels of oxygenation in very large bioreactors. • The mixture is continuously stirred to keep it oxygenated • There is a rich nutrient medium. • The growth medium contains a buffer to maintain pH at around 6.5 • The bioreactors are maintained at about 25-27°C -penicillin is then collected and purified to be used in medicine

Question 28

Producing insulin

Answer 28

-crucial for treating people with type 1 diabetes -made by genetically modified bacteria, which have had the gene for human insulin production inserted into their DNA -bacteria are grown in an industrial fermenter on a large scale + the insulin produced is collected and purified

Question 29

Bioremediation

Answer 29

-microorganisms are used to break down pollutants from contaminated sites i.e soil or water • can be done using natural organisms: pollutant removing bacteria that occur naturally at the site are provided with extra nutrients + enhanced growing conditions to break down and neutralise many contaminants(pollutants broken down into less harmful products) • GM organisms: developing GM bacteria which can break down or accumulate contaminants which they would not naturally encounter i.e mercury in water

Question 30

What are cultures of microorganisms?

Answer 30

-population of one type of microorganism that’s been grown under controlled conditions -done to have large enough no.s of the microorganisms for them to be seen clearly with the naked eye

Question 31

Why must safety procedures be followed when culturing microorganisms?

Answer 31

-always the risk of a mutation taking place —} making the strain pathogenic -may be contamination with pathogenic microorganisms from the environment

Question 32

what is needed to culture microorganisms?

Answer 32

-right conditions of temp, oxygen and pH -nutrient broth/agar to provide a better medium for microbial growth--} allows samples containing a very small no. of organisms to multiply rapidly

Question 33

What are aseptic techniques and give examples?

Answer 33

-used to prevent contamination of cultures by unwanted microorganisms that may affect growth of cultures - disinfecting work surfaces to minimise contamination - work near Bunsen burner(microorganisms in the air are drawn away from culture) - sterilise equipment used to transfer cultures by passing it through flame before + after(kills any microorganisms) - pass the neck of the broth container through the flame after opening + before closing(air moves out of container, preventing unwanted organisms moving in) -minimise time agar plate is open(reduces chance of airborne microorganisms contaminating the culture)

Question 34

Inoculating broth

Answer 34

-make a suspension of the bacteria to be grown -mix a known vol with the sterile nutrient broth in the flask -stopper the flask with cotton wool to prevent contamination from the air -incubate at a suitable temp, shaking regularly to aerate the broth, providing oxygen for the growing bacteria

Question 35

Inoculating agar: streaking

Answer 35

-inoculating loop must be sterilised by holding it in a Bunsen flame until it glows red -dip inoculating loop in the bacterial suspension -remove lid of petri dish + make a zigzag streak across the surface of the agar(avoid loop digging into agar by holding it horizontal) -replace lid of the petri dish + seal it with tape -inoculate at a suitable temp

Question 36

Inoculating agar: spreading

Answer 36

-open the petri dish + pipette the bacteria onto the agar -use a glass spreader to evenly spread this on the agar -replace the lid of the petri dish + seal it with tape -incubate at a suitable temp

Question 37

Investigating the effect of temp on the growth of bacteria (method can be reused for other factors)

Answer 37

-use a sterile pipette to add the same vol of bacteria broth to each of 6 agar plates(discard pipette/leave in beaker of disinfectant) -spread the broth across the surface of the agar using a sterile plastic spreader(discard after use) -put the lids on the plates and tape shut -place 3 plates in a fridge at 4° and put 3 in an incubator at 23°(plates placed upside down to stop any condensation forming on the lid dropping onto the agar) -put another lidded agar plate that is uncultured in each temp as a negative control -leave all plates for the same amount of time--} count the no. of bacterial colonies that have formed on each plate and record results on a table -work out the mean no. of colonies formed at each temp

Question 38

What is a closed culture?

Answer 38

-when growth takes place in a vessel that's isolated from the external environment (extra nutrients aren't added + waste products aren't removed from the vessel during growth)

Question 39

Why are logs used?

Answer 39

-to represent the bacterial population because the difference in numbers from the initial organism to the vast offspring is sometimes too great to represent using standard numbers

Question 40

Growth curve: lag phase

Answer 40

-when bacteria are adapting to their new environment -they are growing + synthesising the enzymes they need -population size increases slowly as reproduction rate is slow in this phase

Question 41

Growth curve: exponential (log) phase

Answer 41

-when the rate of bacterial reproduction is close to or at its theoretical maximum -population size increases quickly because the culture conditions are at their most favourable for reproduction--} no. of microorganisms doubles at regular intervals

Question 42

Growth curve: stationary phase

Answer 42

-occurs when the total growth rate is zero--} no. of new cells formed by binary fission is cancelled out by the no. of cells dying(due to lack of food + poisonous waste products building up) -population size stays level

Question 43

Growth curve: death phase

Answer 43

-occurs when reproduction has almost ceased and the death rate of cells is increasing greater than the reproductive rate -happens because food is very scarce + waste products are at toxic levels

Question 44

How can we estimate the number of cells in a culture?

Answer 44

-the no. of cells in a culture of microorganisms double at regular intervals during the exponential growth phase -use **N=Nᵒ x 2n** to work out how many cells in a population after a certain no. of divisions - Nᵒ= initial no. of cells - n= no. of divisions

Question 45

How do you use logarithms in growth curves?

Answer 45

-to avoid having really large values to plot, 'logs' are used -log tells you how many times 10 has been multiplied by 10 to give you that number -press log, then the number you're using, then equals

Question 46

Interpreting data values from a a logarithmic scale

Answer 46

-the units on the y-axis tell you the total no. of cells rather than the log values(makes it easier to work out how many cells are present at a given time) -its possible to work out how many cells are present at a given time if the y-axis only has log values using the **antilog** --} 10x button on calc then enter the log value at your chosen time, pressing equals gives you the no. of cells

Question 47

Limiting factors which prevent exponential growth in a bacteria culture

Answer 47

- nutrients available: as the no. of microorganisms multiply, the nutrients are used up + the level becomes insufficient to support further growth + reproduction - O2 levels: as the population rises, so does the demand for respiratory oxygen, which eventually becomes limiting - temperature: enzyme-controlled reactions within the microorganisms are affected by the temp of the culture medium(too high= denatures enzymes + kills the microorganisms) - waste products: build-up of toxic material may inhibit further growth + may poison/kill the culture - change in pH: as CO2 produced by respiration increases, pH falls to a point that it affects enzyme activity + inhibits population growth

Question 48

How is the no. of bacteria worked out during serial dilution technique?

Answer 48

-number of colonies x dilution of sample

Question 49

What are primary metabolites?

Answer 49

-substances that are wanted in order to form an essential part of the normal functioning of a microorganism i.e ethanol

Question 50

What are secondary metabolites?

Answer 50

-substances produced that are not essential for normal growth, but still used by the cells of organisms i.e pigments -often the required product in a bioprocess i.e antibiotics

Question 51

What is a fermentation vessel?

Answer 51

-a large container where cultures are grown in to either obtain lots of the microorganism or to collect lots of useful product that the organism makes

Question 52

Steps of batch fermentation

Answer 52

-microorganisms are inoculated into a fixed volume of medium -nutrients are used up and + waste products build up -as the culture reaches the stationary phase, overall growth stops but the microorganisms carry out biochemical changes to form desired end products -process is stopped before death phase and products are harvested -system is cleaned + sterilised before new batch culture

Question 52

Methods for culturing microorganisms: Batch fermentation

Answer 52

-where microorganisms are grown in individual batches(closed cultures) in a fermentation vessel, when one culture ends it's removed and a different batch of microorganisms is grown in the vessel

Question 53

Methods for culturing microorganisms: Continuous fermentation

Answer 53

-where microorganisms are continually grown in a fermentation vessel without stopping -nutrients are put in and waste products are taken out at a constant rate -enables continuous balanced growth, with levels of nutrients, pH and metabolic products kept roughly constant -largely used for the production of a single-celled protein + in some waste water treatment processes

Question 54

steps of continuous fermentation

Answer 54

-microorganisms are inoculated into sterile nutrient medium and start to grow -sterile nutrient medium is added continually to the culture once it reaches the exponential growth point -culture broth is continuously removed, keeping the culture volume in the vessel constant

Question 55

Why is it important to control the conditions of the fermentation vessels?

Answer 55

-maximises the yield of microorganisms and desirable products

Question 56

Controlling fermentation vessels

Answer 56

-pH: monitored on pH probe, optimum so enzymes can work efficiently, maintaining high rate of reaction and product yield -temp: kept optimum by water jacket to increase product yield(enzyme, RoR)--} too low= microorganisms won't grow quickly enough, too high= denature, microorganisms are inhibited/destroyed -nutrients + oxygen= fresh medium/sterile air can be added in controlled amounts to the broth(increases product yield as microorganisms can respire for energy/access nutrients for growth) -contamination= vessels are sealed and are sterilised between uses with hot steam(kills unwanted microorganisms + prevents competition)

Question 57

Advantages of isolated enzymes

Answer 57

-less wasteful: whole microorganisms use up substrate whilst growing + reproducing to produce biomass AND product -more efficient: isolated enzymes work at much higher concs than possible when they are part of the whole microorganism -more specific: no unwanted enzymes present, so no wasteful side reactions -maximise efficiency: isolated enzymes can be given ideal conditions for maximum product formation, which may differ from those needed by the whole microorganism. -less downstream processing: isolated enzymes produce pure product making this process less expensive

Question 58

How are extracellular isolated enzymes easier to use than intracellular?

Answer 58

-secreted which makes them easy to isolate and use -each microorganism produces relatively few extracellular enzymes making them easier to identify and isolate than intracellular - tend to be adapted to cope with greater variations in temp and pH than intracellular enzymes

Question 59

what are immobilised enzymes and why are they used?

Answer 59

-Enzymes that are attached to an insoluble material so they can’t become mixed with the products -substrate solution is run through a column of immobilised enzymes, active sites are still available but the solution flowing out the column were only contained the desired product -use of isolate enzymes are complicated and expensive -better suited for large-scale production

Question 60

What are the three main ways that enzymes are immobilised?

Answer 60

• encapsulated in matrix/semi-permeable membrane i.e. jelly like alginate beads (doesn’t allow free movement, remains fully active, substrate diffusers into matrix and products diffuse out) • surface immobilisation(adsorption to inorganic carriers) i.e trapped in a silica gel matrix (remains fully active, substrate diffusers into alginate matrix + products diffuse out) • covalent bonding: enzymes bonded to a supporting surface i.e cellulose/collagen (not likely to become detached, expensive, can distort active site which reduces activity)

Question 61

Advantages of immobilised enzymes

Answer 61

-can be washed + reused, cheaper -easily separated from the reactants and products of the reaction they are catalysing so reduced downstream processing - cheaper -more reliable —} insoluble support provides a stable environment for the immobilised enzymes -greater temperature tolerance= less easily denatured + work at optimum levels over a wider range of temperatures —} less expensive to run

Question 62

Disadvantages of immobilised enzymes

Answer 62

-reduced efficiency= process of immobilising an enzyme may reduce its activity rate -higher initial costs of materials - more expensive than free enzymes or microorganisms BUT don’t need to be replaced frequently -extra equipment required, which can be expensive -more technical issues - vessels which use immobilised enzymes are more complex than simple fermenters

Question 63

Uses of immobilised enzymes: immobilised lactase

Answer 63

-used to produce lactose-free milk—} lactase hydrolyses lactose into glucose and galactose, which can be passed over immobilised lactase

Question 64

Uses of immobilised enzymes: Immobilised penicillin acylase

Answer 64

-used to make semi-synthetic penicillins from naturally produced penicillins -effective against penicillin resistant bacteria

Question 65

Uses of immobilised enzymes: conversion of dextrins to glucose

Answer 65

-immobilised glucoamylase can be used to complete the breakdown of starch to glucose syrup -Amylase first breaks down starch into dextrins

Question 66

Uses of immobilised enzymes: immobilised glucose isomerase

Answer 66

-used to produce fructose from glucose -fructose means that less sugar is needed to obtain the same level of sweetness

Question 67

Uses of immobilised enzymes: Immobilised aminoacylase

Answer 67

-used to produce pure samples of L-amino acids used in the production of pharmaceuticals, organic chemicals, cosmetics, and food

Question 68

Uses of immobilised enzymes: Immobilised nitrile hydratase

Answer 68

-used to convert acrylonitrile to acrylamide—} important compound which is used in the production of many plastics