6.4 - Cloning and biotechnology

Question 1

Advantages and disadvantages of natural asexual reproduction

Answer 1

• All offspring have genes to survive in the environment
• Quick – many offspring made in short period of time, more likely that species survives
• Possible when sexual reproduction fails or isn’t possible – more likely that species survives
• No genetic variation – if one individual is susceptible to a disease then they all will be –
  could lead to local extinction of whole population

Question 2

Natural cloning in plants – English Elm Tree

Answer 2

• Clones by vegetative propagation (from meristem tissue via mitosis)
• Parent plant of English Elm trees produce root suckers (basal sprouts)
• If the trunk of the parent plant gets damaged e.g. clear felling it triggers the growth of the
  root suckers
• This gives the root suckers a competitive advantage compared to other species when
  competing for light as root suckers are already growing and are taller

Question 3

Artificial cloning in plants -

Cuttings

Answer 3

• Growing tip is cut from plant you want to clone
• Cut between nodes
• Dipped in rooting powder which contains auxins to stimulate root and root hair growth
• Planted in sterile soil and grows into clone of parent plant
• Large numbers of clones can be produced very simply

Question 4

Artificial cloning in plants - Grafting

Answer 4

• A shoot section of a plant is cut (graft)
• This is joined to an already growing root and stem (rootstock)
• Vascular tissue is lined up and then graft and rootstock are bound together
• Graft is genetically identical to parent plant but rootstock is genetically different
• Useful for plants which will not sexually reproduce to produce desired product, will produce product e.g. fruit quicker than growing from plantlet

Question 5

Artificial cloning in plants - Tissue culture (micropropagation)

Answer 5

• Tissue from apical buds (an explant) taken because it is meristematic and therefore undifferentiated and can still undergo mitosis
• Surface is cleaned using alcohol to ensure sterile conditions so that no bacteria can grow as it could compete with the plant tissue
• Explant is placed onto nutrient medium to encourage mitosis this produces a callus (mass of undifferentiated cells)
• The callus is subdivided and placed in a new nutrient medium which will encourage differentiation of tissue. It contains:
• auxins - stimulate formation of root hairs o cytokinins - stimulate shoot growth
• magnesium - helps the plant make chlorophyll o nitrates needed for protein synthesis
• sucrose - converted to glucose for respiration
• The callus cells will grow into plantlets and can be then placed sterile soil to grow

Question 6

Advantages and disadvantages of cloning (via tissue culture) (rather than seeds)

Answer 6

Advantages

• genetically identical plants made so maintains favourable characteristics
• quicker to produce
• disease free plants created
• can be used for cloning infertile plants
• easy to genetically manipulate and make GM plants
• easy to transport/store

Disadvantages

• All susceptible to same diseases as they are genetically identical
• Loss in genetic diversity
• Labour intensive and so very expensive

Question 7

Cloning animals by nuclear transfer

Answer 7

• Egg cell enucleated
• Adult somatic cell diploid nucleus from a different animal removed and injected into
  enucleated egg cell (or adult cell fused with enucleated egg cell)
• Cell give a small electric shock to stimulate mitosis
• Cell grows into an embryo in vitro
• (embryo can be split into several embryos – to produce artificial identical twins)
• Embryo(s) implanted into surrogate mother(s)

Question 8

Advantages and disadvantages of cloning animals

Answer 8

Advantages

• All animals inherit the desirable characteristics
• Fast way of getting many individuals in a short period of time
• Avoids mating risks

Disadvantages

• no genetic variation in population
• All individuals susceptible to same diseases
• Cloned animals may have shorter life spans than other animals
• Success rate of cloning is very low
• Very expensive process and is very labour intensive

Question 9

Collecting eggs for cloning (or IVF!)

Answer 9

• Female treated with hormone (FSH)
• Superovulation
• Collect eggs from ovaries

Question 10

Why a clone is not entirely genetically identical to the nucleus donor…

Answer 10

• DNA is also found in mitochondria
• Only get DNA from nucleus in clones (not their cytoplasm)
• Mitochondrial DNA is found in cytoplasm

Question 11

How surrogates could be prepared for implantation of embryo…

Answer 11

• Hormone treatments
• To prepare uterus for implantation by causing the lining to thicken so there is an increased
  blood supply for the placenta

Question 12

Cloning to preserve an endangered species

Answer 12

• increases rate of reproduction
• does not require species’ eggs
• so does not require fertile female
• female not put at risk by mating / pregnancy (if different sp. Used for surrogacy)
• successfully formed embryo can be subdivided / cloned to produce more offspring

Question 13

T Non reproductive cloning

Answer 13

• Not cloning to produce new organisms, cloning to produce new cells or tissues to replace
  damaged ones in people who have got diseases or had accidents
• E.g. could be used to produce new heart muscle after heart attack, new neurones to
  repair damage to spinal cord causing paralysis, treat diseases such as Parkinson’s disease

Advantages:
- Will not be rejected if they are cloned from person’s own cells
- Removes problem of shortage of donor organs for transplants
- If embryonic cells are used they could differentiate into any type of cell and could
treat numerous diseases that adult cells could not

Disadvantages:
- Some people e.g. Catholics have an ethical objection to using cloning embryonic material as they see the embryos as living but they will not becoming a baby and so they see this as the same as murder.

Question 14

Why microbes are used in biotechnology

Answer 14

• Reproduce quickly so produce products very quickly
• Useful products easy to separate
• Can be easily genetically manipulated to produce products such as insulin
• Processes occur at low temperatures – so cheap to maintain
• Can be grown on very cheaply on unwanted food waste
• Growth is not dependent on climate so can produce products irrespective of
  weather conditions

Question 15

Standard growth curve (complete curve only occurs in closed culture)

Answer 15

• Lag phase – no reproduction as microbes acclimatise to new conditions, time taken for
  first reproductive cycle
• Log/exponential phase
• no competition for nutrients
• very high reproductive rate (very low death rate)
• primary metabolites are made – needed for normal growth e.g. enzymes
• Stationary phase
  *Nutrients depleted and limited – competition
  *Reproductive rate = death rate
  *Secondary metabolites made – not essential for normal growth so produced after
  exponential growth phase e.g. antibiotics e.g. penicillin
• Death phase – death rate greater than reproductive rate
• Toxic products killing microbes
• Lack of nutrients e.g. glucose as respiratory substrate

Question 16

Continuous (open) fermentation vs batch (closed) culture

Answer 16

In continuous culture, nutrients and products added and removed from culture
continuously and at the same rate. In batch culture starter population mixed with fixed quantity of nutrients at start and no more are added. At end products removed and tank emptied. Process started again.
• Continuous culture produces primary metabolites during log phase because nutrients do not deplete and culture stays in log phase e.g. Insulin from E.coli. Produces secondary metabolites after log phase (during stationary phase) because nutrients deplete e.g. Penecillium making antibiotic Penicillin
• Faster growth in continuous culture
• Continuous cultures more difficult to set up and maintain as conditions must be
monitored and controlled
• Contamination of continuous culture leads to loss of much more product but batch
culture contamination will only led to loss of one batch

Question 17

Monitoring and controlling conditions in continuous fermenter

Answer 17

• Thermometer measures temperature.
• Water jacket pumps cool water around fermenter, excess heat released from respiration
  removed by cool water
• Temperature must be controlled to ensure optimum temperature for enzymes of microbes to ensure they do not denature and fermentation does not stop
• pH probe monitors pH
• buffers can be used to maintain constant pH
• pH must be controlled to ensure optimum pH for enzymes of microbes to ensure they do
  not denature and fermentation does not stop
• Stirrers to ensure even distribution of microbes and nutrients and temperature and avoid
  clumping of cells
• Air inlet lets in sterile air. sterile to avoid contamination. Air to supply oxygen for aerobic
  respiration of microbes
• Nutrient input to supply glucose as a respiratory substrate and nitrates as a source of
  nitrogen for protein production
• Air out – removes waste gases and maintains constant pressure
• Harvest pipe – to remove product

Question 18

Fed Batch fermentation

Answer 18

• Limited amounts of glucose are added at intervals
• Enough glucose added for respiration to keep the culture alive.
• Limited amount keeps culture in stationary phase for longer and prevents death phase
• Will allow more secondary metabolites to be produced than a normal batch culture

Question 19

Changing equipment to make a batch culture continuous

Answer 19

• ref to method for addition of nutrients and removal of waste / products
• nutrient addition and waste / product removal at a constant rate
• continually throughout fermentation period
• keep volume constant
• thermometer and pH probe for monitoring temperature and pH
• use a buffer to maintain constant pH
• use a water jacket to maintain constant temperature
• use a stirrer to avoid clumping of cells
• ref. to need to maintain sterility

Question 20

Importance of maintaining aseptic conditions in fermenter

Answer 20

• to avoid unwanted presence of microbes
• so no competition for nutrients
• so no decrease in yield
• so no contamination of product

Question 21

Advantages of isolated enzymes over cells as a source of enzymes and visa versa

Answer 21

Isolated enzymes

• no other unwanted by products made
• no contamination from cell
• don’t respire any reactants

Cells as a source of enzymes

• Not necessary to start with pure enzyme
• Cell produce the enzyme
• Enzymes may be expensive or difficult to isolate

Question 22

Methods of immobilising enzymes

Answer 22

• Encapsulation - trapped in alginate beads
• Adsorption - stuck onto collagen/clay/resin/ porous glass
• Covalent bonding to cellulose/collagen fibres
• Trapped inside gel e.g. silica lattice
• Partially permeable membrane microspheres

Question 23

Advantages of immobilising enzymes

Answer 23

• product is uncontaminated by enzyme therefore no downstream processing needed -
  LESS COST
• Not lost during process and therefore reusable - REDUCES COST
• Matrix protects the enzyme so enzyme works at higher temperatures so reaction can be
  faster as done at higher temp
• Matrix protects the enzyme so enzyme works in changed pHs

Question 24

Disadvantages of immobilising enzymes

Answer 24

• Immobilisation difficult/expensive
• Can be less active as substrate has to diffuse through matrix which can take time so don’t form ESCs as readily – lower yield in same time