CHAPTER 22 - CLONING AND BIOTECHNOLOGY

Question 1

What is vegetative propogation

Answer 1

A form of natural cloning in plants, a structure which develops into a fully differentiated plant, identical to the parent

Question 2

What are some examples of natural plant cloning

Answer 2

Bulbs - leaf bases which swell with stored food from photosynthesis

Runners - from plants like strawberries where a lateral stem grows away from the parent plant and roots develop underneath, eventually creating an independent plant

Rhizomes - a specialised horizontal stem running underground, with stored food

Stem tubers - eg. potato buds on storage organ develop to produce new shoots

Question 3

How are natural clones carried out in horticulture

Answer 3

,Bulbs are split up,

removing young plants from runners

Cutting up rhizomes

Taking a cutting of a plant, with rooting hormone (which is quicker than just using a seed)

Question 4

What are perennating organs and how are they involved in cloning and survival

Answer 4

Organ which contains stored food from photosynthesis e.g., potato

cloning – new bud/plants
may arise from the organ identical to original plant

allows plant to survive adverse conditions

and produce a new shoot

using energy from food store

Question 5

Explain the advantages and disadvantages of propagating crop plants by cutting over using seeds

Answer 5

Cuttings – genetically identical to parent so likely to produce good crops

often shorter time from planting to crop

reliable

don’t have to buy in

can use own plants

Seeds – have genetic variation so more variability in quality of crop

but are more likely to
withstand disease of changes in circumstances

take time and right conditions to germinate and grow to maturity

in some cases can collect seed and reuse for next planting but don’t always get the same quality

Question 6

Suggest why it is important to describe clones as genetically identical to their parent rather than simply identical - and why even this may not always be true

Answer 6

Genetically identical because parts of the same plant

but eventually form will depend on
growing conditions – levels of light, water, temperature etc.

identical suggests appearance is the
same

cloned plants may not be identical because a mutation may take place in stem cells of meristems

changing pattern of growth in the plant

Question 7

When is micropropogation used

Answer 7

When a desirable plant:
Doesnt readily produce seeds
Doesnt respond well to natural cloning
Is very rare
Genetically modified or selectively bred with difficulty
Required to be pathogen-free by growers

Question 8

What are the basic principles of micropropogation and tissue culture

Answer 8

Take a small sample of tissue from the plant you want to lone - meristem from shoot or root tips that is virus free

Sterilising sample, using agents like bleach or ethanol

Explant is then placed in sterile culture medium containing balance of plant hormones, proliferating and forming a mass of identical cells called a callus is divided into individual cells or clumps, transferred into a new culture medium containing a different mix of hormones, stimulating development of tiny genetically identical plantlets

These are potted in compost to grow into small plants

Young plants are planted out to grow and produce a crop

Question 9

What are the advantages for micropropogation

Answer 9

Allows for rapid production of large number of plants with high yield of crops and known genetic makeup

Culturing meristem tissue produces disease-free plants

It makes it possible it produce disease-free plants

Produces viable number of plants are genetic modification of plant cells

Produces very large number of new plants which are seedless and therefore sterile (eg. banana and grape)

It provides a way of reliably increasing the numbers of rare or endangered plants

Question 10

Arguments against micropropagation

Answer 10

Produces monoculture - all susceptible to same disease or change of environment

Relatively expensive process with skilled workers

Vulnerable to infection during production process

If source material is infected, all clones will be infected

Large number of plants can be lost during the process

Question 11

What is the potential of natural cloning for saving important crops such as the banana against disease

Answer 11

Advantages
relatively easy
relatively cheap and readily available
history of use

Disadvantages
any disease in parent plant transferred with cutting etc.
limit to number of new
plants that can be formed so cannot keep up with demand if there is a major threat to crop
still produces clones

Question 12

What is the potential of micropropagation for saving important crops against disease in contrast to natural cloning? give arguments for and against

Answer 12

Advantages can produce disease-free plants

can produce plants engineered to be resistant to
disease

can produce almost limitless numbers of plants fast

Disadvantages
relatively expensive
needs some infrastructure
still produces clones

Question 13

How does natural animal cloning occur in invertebrates

Answer 13

Some animals eg. starfish, can regenerate entire animals from fragments of the original if they are damaged

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 will develop into genetically identical clones

Some insects can produce offspring without mating - although high level of mutation so these arent true clones

Question 14

How does natural animal cloning occur in vertebrates

Answer 14

Formation of monozygotic twins, when the early embryo splits to form two seperate embryos - differences occur in nutrition and position in uterus

Some female amphibians and reptiles will produce offspring when no male is available - but they are often male so they arent clones, but all of the genetic material is from the mother

Question 15

How are invertebrates cloned

Answer 15

Liquidising a sponge or chopping up a starfish and a new animal will regenerate from most of the fragments

Question 16

What are the two methods of vertebrate cloning

Answer 16

Artificial twining
Somatic cell nuclear transfer (SCNT)

Question 17

Describe the process of artificial twinning, using cattle as an example

Answer 17

Cow with desirable traits is treated with hormones so she super-ovulates, releasing more mature ova than normal

Ova may be fertilised naturally or by artificial insemination, by a bull with good traits, then embryo is flushed out of the nucleus

Or mature eggs are removed and fertilised by top-quality bull semen in the lab

Usually before or around day 6 - cells are totipotent, and early embryo is split to produce several smaller embryos - each capable of producing a healthy full-term calf

Each of the split embryos is grown in the lab for a few days to ensure healthy growth before they are implanted into a surrogate mother

Embryos develop into foetuses and are born normally, so a number of identical cloned animals are produced by different mothers

Question 18

Describe the stages of Somatic cell nuclear transfer

Answer 18

Nucleus is removed from a somatic cell of an adult animal

Nucleus is removed from a mature ovum harvested from a different female of the same species (it is enucleated)

The nucleus from adult somatic cell is placed into enucleated ovum and given a mild electric shock so it fuses and begins to divide

Embryo that develops is transferred into the uterus of a third animal, where it develops to term

New animal is a clone of the animal from which the original somatic cell is derived, but mitochondrial DNA will come from egg cell

Question 19

What are the arguments for animal cloning

Answer 19

Produces more offspring than normal reproduction

Allows success of passing on desirable genes

Allows scientist to replicate a specific animal eg, top class race horse

Enables rare or endangered or extinct animals to reproduce

Question 20

Arguments against animal cloning

Answer 20

Inefficient process - most animals take any eggs to produce a single cloned offspring

Cloned embryos fail to develop and miscarry or produce malformed offspring

Many animals produced by cloning have shortened lifespans

Unsuccessful in increasing populations of rare organisms or to bring back extinct animals

Question 21

How is artificial twinning different from natural twinning

Answer 21

Natural twinning early embryo splits

and two fetuses go on to develop

from the two halves
of divided embryo

Artificial twinning split in early embryo is produced manually

number of identical embryos may be replaced in surrogate mothers

to produce a number of identical high
quality animals

Question 22

The evidence suggests that monozygotic twins do not occur naturally in cattle. Suggest ways in which this might be investigated

Answer 22

Observing births and recoding twin births when animals appear the same

genetic testing of any
twin cattle of the same gender

Question 23

Explain the similarities between artificial twinning and SCNT

Answer 23

Both processes involve removing eggs from an animal

both involve surrogate parents

both potentially produce a number of genetically identical organisms

Question 24

Explain the differences between artificial twinning and SCNT

Answer 24

In twinning either gametes meet outside the body

and early embryo develops before being split

or early embryos flushed from the mother

egg cell contributes all maternal DNA

embryos produced from gametes

embryos genetically related to two parents

Question 25

What is biotechnology

Answer 25

Applying biological organisms or enzymes to the synthesis, breakdown or transformation of materials in the service of people

Question 26

What are some reasons why using microorganisms are ideal

Answer 26

No welfare issues to consider Enormous range of microorganisms capable of carrying out many different reactions Genetic engineering allows manipulation of microorganisms to carry out desirable reactions eg. production of human insulin Short life cycle and rapid growth rate Simple nutrient requirements and is often cheap Low temperature conditions

Question 27

What are some examples of indirect microorganism action on food production

Answer 27

Baking - yeast Brewing - yeast Cheese making - bacteria Yoghurt making - bacteria

Question 28

What are some examples of direct microorganism action on food production

Answer 28

Eating fungi eg. mushrooms Producing Single-cell protein eg. Quorn from a fungus and fermenter and added with egg whites

Question 29

Advantages of using microorganisms to produce human food

Answer 29

Reproduce fast and produce protein faster than animals or plants High protein with little fat content Can use variety of waste materials, including animal and human waste Genetically modified to produce protein required Production of microorganisms isnt present on weather or breeding cycles No welfare issues when growing Made to taste like anything

Question 30

Disadvantages of using microorganisms to produce human food

Answer 30

Some microorganisms can also produce toxins if not maintained in optimum conditions Microorganisms have to be separated to make food Involve GM organisms and many people have concerns eating GM food Protein has to be purified Dislike the thought of eating microorganisms grown on waste Has little natural flavour - needs additives

Question 31

Compare the way yeast is used in the process of baking and brewing

Answer 31

Baking: Mixed with sugar and water Respires aerobically Carbon dioxide produced used to make bread rise Yeast killed by heating as bread cooks Takes a couple of hours Brewing: Mixed with malted barley and hot water Respiration (fermentation ) Continues for days in anaerobic conditions (1); Ethanol produced as waste product Yeast eventually inhibited (not killed) by rising pH Build-up of ethanol and lack of oxygen

Question 32

Why is milk pasteurised before being used commercially to make cheese and yoghurt

Answer 32

To destroy bacteria that would make it go bad rapidly or cause diseases such as TB

Question 33

Why is milk homogenised before being used commercially to make cheese and yoghurt

Answer 33

The fat droplets are spread evenly through milk so cream doesn’t separate out and creates a uniform product

Question 34

Give two important differences between the production processes of cheese and yoghurt

Answer 34

Cheese whole milk used bacteria used to separate the curds from the whey changing texture, and bacteria ripen or mature the cheese in controlled slow reactions at low temperatures to change taste out-compete bacteria that would make the cheese go bad takes weeks, months or years can last for years Yoghurt skimmed milk powder added to milk to enrich it specific bacteria added and incubated at 45°C for 4–5 hours to produce extracellular polymers that give the texture to yoghurt lasts 2–3 weeks in a fridge

Question 35

What are the different approaches to bioremediation

Answer 35

Using natural organisms - Many microorganisms naturally break down organic material producing carbon dioxide and water GM organisms - Breakdown or accumulation of contaminants which they wouldnt usually encounter

Question 36

What conditions are needed to produce penicillin

Answer 36

Small Fermenters Mixture continuously stirred to keep oxygenated Rich nutrient medium Growth medium contains a buffer to maintain pH at around 6.5 medium temperature

Question 37

What was the main difference between the use of fungi to produce penicillin and the use of bacteria to produce human insulin

Answer 37

Fungi produce penicillin naturally bacteria genetically engineered/modified to produce human insulin

Question 38

What is bioremediation? why it is often carried out on the site of contamination

Answer 38

Use of microorganisms or plants to break down pollutants and contaminants in soil or water often carried out on site because area of contamination may be very large so not practical to remove contaminated material/too expensive to remove contaminated material organisms involved in bioremediation grown and break down contaminants in situ living organisms so they grow and spread may be harvested and contaminants retrieved

Question 39

What are the risks of using microorganisms as a culture

Answer 39

There is always a risk of mutation taking place making the strain pathogenic There may be contamination with pathogenic microorganisms

Question 40

What are the processes by which bacteria must be added

Answer 40

Inoculating broth Inoculating agar

Question 41

How do you make inoculating broth

Answer 41

Make suspension of the bacteria to be grown Mix a known volume with the sterile nutrient broth in the flask Stopper flask with cotton wool to prevent contamination from the air Incubate at a suitable temperature shaking regularly to aerate the broth providing oxygen for the growing bacteria

Question 42

How do inoculating agar prepared

Answer 42

Wire inoculating loop must be sterilised by holding it in a bunsen flame until it glows red hot Dip the sterilised loop in the bacterial suspension, remove lid of petri dish and make a zig-zag streak across the surface of the agar Replace lid of petri dish, held down and sealed with tape, but not completely so oxygen can still get in

Question 43

What are the phases of growth in bacterial colonies

Answer 43

Lag phase - when bacteria are adapting to their new environment Exponential phase - Rate of bacterial reproduction is close to or at its theoretical maximum Stationary phase - total growth is 0 (in dynamic equilibrium) decline phase - reproduction has ceased and rate of death of cells is increasing

Question 44

What are the several limiting factors which prevent exponential growth

Answer 44

Nutrients available Oxygen levels Temperature Build-up of waste Change in pH

Question 45

Compare the processes of culturing bacteria in broth and on agar

Answer 45

Both provide nutrients, suitable pH, moisture etc 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

Question 46

Why are there such clear differences between the theoretical growth curve of a bacterial colony and the actual growth curve in a closed culture

Answer 46

In large closed culture nothing gets in or out initially, growth can be at theoretical rate as no factors are limiting as culture continues, numbers increase, food and oxygen are used up and waste products build up often affecting pH microorganisms run out of food or oxygen, are inactivated by pH changes affecting enzymes or poisoned by waste products so whilst theoretical growth curve is exponential, real growth curve reaches a peak, plateaus, and declines

Question 47

Explain why vinegar is a very good preservative

Answer 47

Vinegar is ethanoic acid therefore has a low pH and inhibits bacterial growth

Question 48

Explain why food eventually goes bad in the fridge

Answer 48

As temperatures fall bacteria growth slows but does not stop so in fridge bacteria grow slowly and eventually destroy food

Question 49

Explain why in the northern hemisphere, material placed in a compost heap rots down much faster in August than it does in december

Answer 49

Reactions in bacteria and fungi that act as decomposers affected by temperature in August temperatures relatively high so decomposition reactions relatively fast. In December, the temperatures are much cooler so slower reactions in decomposers and rotting slower

Question 50

What is primary and secondary metabolites from microorganism culture

Answer 50

Primary are the substances wanted which form the essential functioning of the microorganism Secondary metabolites are non essential for growth, but still used by the cells, eg. pigments and toxins against herbivores. Extracted in bioprocess eg. penicillin and other antibiotics

Question 51

What is batch fermentation

Answer 51

Microorganisms are inoculated into a fixed volume of medium As growth takes place, nutrients are used up and both new biomass and waste products build up As the culture reaches the stationary phase, overall growth ceases, carries out biochemical changes to form desired end products Process is stopped before death phase and the products are harvested

Question 52

How does continuous culture work

Answer 52

Microorganisms are inoculated into sterile nutrieinnt 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, microorganisms, and product - keeping the culture volume in the bioreactor constant

Question 53

What factors need to be controlled in bioreactors

Answer 53

Temperature Nutrients and oxygen Thickness Asepsis

Question 54

What is the structure of a bioreactor

Answer 54

page 600

Question 55

Bioreactors may run batch or continuous process. What is the difference

Answer 55

Continuous processes 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 process everything added at beginning in fixed volume of medium nutrients used up and microorganisms, products, and waste products build up may be stationary phase when secondary metabolites formed, process stopped, products extracted, reactor cleaned, and new process begun

Question 56

Choose three factors which need to be controlled in a bioreactor to give the maximum yield of product and for each explain why it is important and how it might be controlled

Answer 56

Temperature if temperature too low microorganisms will not grow quickly enough, too high and enzymes will start to denature and microorganisms are inhibited or destroyed. Bioreactors often have a heating and/or a cooling system linked to temperature sensors and a negative feedback system to maintain optimum conditions. Nutrients if microorganisms use up food supply they will start to die off so need a mechanism to keep food supplied, nutrient medium can be added in controlled amounts to broth when probes or sample tests indicate that levels are decreasing to be mixed in using stirrers/paddles as will not spread through fast enough by diffusion alone. Oxygen if microorganisms use up oxygen they will start to die off so need a mechanism to keep nutrient medium oxygenated, oxygen is bubbled through broth when probes or sample tests indicate that levels are dropping to be mixed in using stirrers/paddles as will not spread through fast enough by diffusion alone. pH if waste products of microorganisms e.g., carbon dioxide build up then pH of mixture will decrease. Change in pH can affect enzyme action and stop growth, buffers are added to mixture and stirred in or alkaline solution added to maintain optimum pH.

Question 57

Why are immobilised enzymes so efficient

Answer 57

Less wasteful - do not produce biomass More efficient - isolated enzymes work at much higher concentrations More specific - no unwanted enzymes present, no wasteful side reactions Maximise efficiency - can be given ideal conditions for maximum product formation as opposed to ideal condition for organism Less downstream processing - so it is therefore cheaper

Question 58

Why are extracellular enzymes cheaper and easier to use for isolating enzymes than intracellular

Answer 58

Secreted, making them easy to isolate and use Microorganims produce few extracellular enzymes, so it is easy to identify, but produce hundreds of intracellular enzymes More robust than intracellular, as conditions outside a cell are less tightly controlled

Question 59

What are some advantages of using immobilised enzymes

Answer 59

Immobilised enzymes can be reused - so cheaper Easily separated from the reactants and products of the reaction More reliable - higher control over the process Greater temperature tolerance Easy manipulation

Question 60

What are some disadvantages of using immobilised enzymes

Answer 60

Reduced efficiency by immobilising an enzyme by changing its active site Higher initial cost of materials Higher initial cost of bioreactor More technical issues

Question 61

What is adsorption surface immobilisation, and its advantages and disadvantages

Answer 61

Adsorption to inorganic carrier eg. silica +ves: Simple, cheap can be used for many processes Accessible to substrate and activity is almost unchanged -ves: enzymes can be lost from the matrix fairly easily (pg 603)

Question 62

What is covalent surface immobilisation, and its advantages and disadvantages

Answer 62

Covalent or ionic bonding to an inorganic carrier, polysaccharide or polymer +ves: Cost varies, Strongly bound so unlikely to be lost Enzymes accessible to substrate pH and substrate conc often have little effect on activity -ves: Cost varies Active site of the enzyme may be modified (pg 603)

Question 63

What is entrapment in a matrix, and its advantages and disadvantages

Answer 63

Entrapment in a matrix eg. gelatin +ves: Widely applicable to different processes -ves: May be expensive Can be difficult to entrap Diffusion to the substrate to and product from active site Variable enzyme activity depending on matrix (pg 603)

Question 64

What is entrapment in a microcapsule, and its advantages and disadvantages

Answer 64

Entrapment - membrane entrapped in a semipermeable membrane eg. a polymer +ves: Simple to do Small effect on activity Widely applicable to different processes -ves: Expensive Diffusion of the substrate to and product from active site can be slow and hold up the reaction (pg 603)

Question 65

What are some examples of immobilised enzyme products

Answer 65

Penicillin Fructose lactose free milk L-amino acids Dextrins Plastics

Question 66

What is meant by an immobilised enzyme

Answer 66

Enzymes attached to an inert support system over which the substrate passes and is converted to product

Question 67

What are the main advantages of immobilised enzymes over whole microorganisms

Answer 67

More efficient more specific; can optimise conditions for specific enzyme less downstream processing

Question 68

What are the main advantages of immobilised enzymes over free enzymes

Answer 68

Can be reused easily separated from reactants and products more reliable as control over process greater temperature tolerance

Question 69

Summarise the ways in which enzymes are immobilised

Answer 69

Surface immobilisation – absorption onto inorganic carriers covalent or ionic bonding onto inorganic carriers entrapment in a matrix entrapment in membrane bound microcapsule

Question 70

How can immobilisation: Increase the effectiveness of an enzyme

Answer 70

Enzymes are accessible to substrates allow continuous production by a continuous flow of medium over the enzyme conditions can be very tightly controlled over the enzyme beds changes in pH and temperature have less effect

Question 71

How can immobilisation: decrease the effectiveness of an enzyme

Answer 71

Immobilising an enzyme may affect its ability to catalyse a reaction diffusion of substrate to and from active site of enzyme can be inhibited by immobilising matrix or capsule and so slow reaction in surface immobilisation enzymes may be lost from matrix relatively easil