Cloning and biotechnology

• Clones - genetically identical organisms or cells

  (made by asexual reproduction)
• Natural cloning of plants

  > involves vegetative propagation where vegetative parts of the plant can reproduce.

  • This includes: runners, rhizomes , suckers, bulbs, corms and leaves

  > Used in:

  • horticulture and agriculture to increase number of plants.
  • medicine - producing chemicals/hormones for treatments (e.g. insulin)
• Vegetative propagation

  > plants grow horizontal stems that can form roots

  • runners if they grow on the surface
  • rhizomes if they are underground

  > suckers are new stems that grow from the roots of plants

  > in all, the horizontal branch may die leaving the new stem as a separate individual

  > Bulbs (onions) have an underground stem which grow leaf bases and apical buds which will grow into a new plants

  > Corms (croci) remain in the ground and produce buds which produce new plants

  > some plants grow clones on leaf margins which grow immature plants that drop off and take root
• Evaluation of natural cloning

  (+)

  • advantageous traits can be carried to next generation
  • relatively rapid so population can increase rapidly and take advantage of environmental conditions
  • reproduction can be carried out with one parent

  (-)

  • offspring may become overcrowded
  • no genetic diversity
  • population has little variation
  • selection not possible
  • whole population is susceptible to less advantageous environmental changes
• Natural cloning in animals

  > clone when identical twins are formed

  zygote divides and daughter cells split to become two separate cells which develop into two individuals
• Simple cloning technique - cuttings in vegetative propagation

  > stem is cut at the nodes

  > cut end of the stem is placed in moist soil

  • dipping cut stem in rooting hormone stimulates root growth
  • also use cuttings from roots and leaves

  > helps produce large numbers of plants rapidly
• > Tissue culture - growing new tissues, organs or plants from certain tissues cut from a sample plant

  > Micropropagation - growing large numbers of new plants from meristem tissue taken from a sample plant
• Tissue culture technique: micropropagation

  1> explants taken from suitable plant material and sterilised with alcohol or bleach

  2> Explants are placed on a sterile growth medium (agar gel) containing

  • nutrients: glucose, amino acids and phosphates
  • growth hormones: cytokinins and auxins

  3> cells of explant divide by mitosis forming a callus

  4> callus is divided producing a larger number of small clumps of undifferentiated cells

  5> clumps stimulated to grow and differentiate into new plant tissues by moving to different growth media

  6> plantlets form and moved to greenhouse
• Why is meristem usually used as an explant in micropropagation?

  they are always free from virus infection
• Why are the explants sterilised with bleach or alcohol?


  kills bacteria and fungi which would impact growth of plant
• What is a Callus?

  a mass of undifferentiated, totipotent cells
• Advantages of artificial cloning in plants

  (+)

  • rapid method of producing new plants compared to growing from seed
  • can be carried out using asexual reproduction
  • offspring will inherent same desirable characteristics (high yield, resistance to disease, phenotype)
  • using meristem as an explant for tissue culture ensures new plants are free from viruses
• Disadvantages of artificial cloning in plants

  (-)

  • tissue culture is labour intensive and requires certain skills
  • expensive equipment and facilities to perform
  • culture can fail due to microbial contamination
  • cloned offspring/ monoculture are genetically identical and susceptible to same diseases or pests
  • no genetic variation (unless introduced by a mutation in DNA)
• Artificial cloning in animals uses totipotent cells which can divide and differentiate into any types of cells.

  Main techniques:

  • embryo twinning
  • somatic cell nuclear transfer (SCNT)
• Artificial cloning in animals: embryo splitting

  precise genotype and phenotype depends on which sperm and egg used so wont be known until offsprings are produced

  1> gametes are collected from two parents with desirable characteristics

  2> a zygote is created via in vitro fertilisation and allowed to divide by mitosis forming groups of cells

  3> groups are separated and continue division

  4> each mass of cells are placed in uterus of surrogate mothers producing genetically identical offspring
• Artificial cloning in animals: Somatic cell nuclear transfer

  can clone an adult and phenotype is known before cloning

  1> egg cell is obtained from one animal and nucleus is removed via enucleation

  2> somatic cell from the adult to be cloned is isolated

  3> complete somatic cell or its nucleus is fused with empty egg cell via an electric shock which triggers it to start developing as it would after fertilisation

  4> cell undergoes mitosis to produce ball of cells

  5> young embryo is placed into the uterus of a surrogate mother and produces offspring genetically identical to nucleus donor
• Arguments for artificial cloning in animals

  (+)

  • can produce herd of animals with a high yield or unusual characteristics that could be lost through normal sexual reproduction
  • individuals from endangered species can be cloned to increase numbers
  • produces tissues and organs genetically identical to the donor which wont be rejected
  • cloned tissues can be tested with medicinal drugs avoiding use of animals or people
  • using genetically identical tissues for scientific research allows effects of genes/ hormones to be assessed with no interference from different genotypes
• Arguments against artificial cloning in animals

  (-)

  • lack of genetic variation may expose herd to diseases/pests
  • animals may be produced with little regard for their welfare with undesirable characteristics: shorter lifespan and be less healthy
  • success rate of adult cell cloning is poor and is expensive
  • ethical/ religious objections: creating an embryo with potential of life to then destroy it
  • cloning endangered species does not increase their genetic diversity
• Why microorganisms are used in biotech

  • relatively cheap and easy to grow
  • production process takes place at lower temperatures and normal atmospheric pressures saving fuel costs and is safer
  • production not dependent on climate so takes place anywhere
  • fed by-products from food industries (waste water, starch) BUT these need to be pre-treated, adding to cost
  • have a short life cycle and reproduce quickly so a large population can grow rapidly, increasing efficiency
  • can be genetically modified easily
  • fewer ethical considerations
  • product is purer/ easier to isolate, reducing downstream costs
• Yoghurt production

  > milk undergoes fermentation:

  > bacteria convert lactose to lactic acid

  • the acidity denatures lactose causing it to coagulate

  > the bacteria partially digest the milk making it easier to digest once consumed

  > other bacteria may be added as probiotics improving human health by improving digestion of lactose and stimulating the immune system
• Cheese making

  > milk is pre-treated with bacteria converting lactose into lactic acid

  > once acidified, milk is mixed with Rennet containing rennin

  > rennin coagulates the milk protein, casein, in presence of Ca2+

  • kappa-casin is broken down making casein insoluble
  • this is precipitated by action of Ca2+ , binding the molecules together

  >Resulting compound is curd and is separated from liquid by cutting, stirring and heating

  >bacteria continue to grow producing more lactic acid and curd is pressed into moulds

  >flavour is determined by maturing process and inoculation with fungi
• Coagulate -clot or change to solid state
• Bread making

  1> mixing: ingredients are mixed by kneading producing dough

  2> fermenting: dough left in warm place while yeast respires aerobically producing co2 bubbles causing dough to rise

  3> cooking: risen dough is baked and alcohol evaporates
• Brewing

  Wine:

  • grapes have yeast on their skin and contain fructose and glucose
  • when crushed, yeast uses the sugars in aerobic respiration producing co2 and alcohol

  Beer: malting

  • as barely grains germinate, stored starch is converted into maltose
  • maltose is respired by the yeast
  • anaerobic respiration produces co2 and alcohol
• Mycoproteins/Single cell protein (SCP)

  > microorganisms can be used to manufacture protein directly as food

  • e.g: QUORN

  > Fungal/ bacterial sources can produce proteins with similar amino acid profile to animal and plant protein
• Advantages of using SCPs for human consumption

  • more efficient production than animal and plant protein
  • biomass produced has high protein content
  • no animal welfare issues
  • microorganisms = good source of protein
  • contains no animal fat or cholesterol
  • can easily be GM to adjust amino acid content
  • SCP can be combined with removal of waste products
  • not much land is required
  • production is independent of climate requirements
• Disadvantages of using SCPs for human consumption

  • fungal protein grown on waste is not appealing to people
  • protein needs to be purified to ensure it is uncontaminated
  • need to be isolated from material in fermenters which they grow
  • amino acid profile may be different from traditional animal protein
  • risk of infection - conditions needed to grow microorganisms ideal for pathogenic organisms
  • protein does not have same taste/texture as traditional protein sources
• Controlled conditions of fermenters in drug production

  • temperature: enzymes can denature when too hot and growth will be limited when too cold
  • pH: enzyme activity and growth are affected by pH extremities
  • o2 availability: microorganisms respire aerobically
  • nutrient availability (carbon, nitrogen, minerals, vitamins) : required for microorganisms to grow and synthesise products
  • conc. of product: if product accumulates, synthesis decreases
  • asepsis
• Batch fermentation - large quantities of a product is produced

  • secondary metabolites produced when cells are under stress due to high population density or limited nutrient availability
  • culture left with limited nutrients and fermented for limited time
  • product can then be extracted
• Continuous culture- growth of microorganisms in a nutrient broth at a specific rate

  • primary metabolites are synthesised from microorganisms whilst growing
  • these are continuously extracted from fermenting broth and population density is maintained
  • broth is topped up with nutrients to keep cells fermenting
• Evaluation of batch culture

  (+)

  • easier to manage and operate
  • short duration
  • less chance of contamination as no nutrients are added

  (-)

  • more expensive and less productive than continuous culture
  • bioreactors need to be consistently sterilised which is time consuming
  • culture has a long lag phase so little yield is produced
• Evaluation of continuous culture

  (+)

  • cheaper and more productive than batch culture
  • produces high yields through cell recycle and immobilisation

  (-)

  • highly likely to be contaminated due to long cultivation period and long-term changes in culture
  • more difficult to manage
• Asepsis - maintenance of sterile conditions in bioreactors


  Nutrient medium may support growth of unwanted microorganisms reducing population as they can:

  • compete with culture for nutrients and space
  • reduce yield of metabolites and spoil them
  • produce toxic chemicals
  • destroy cultured microorganisms
• Penicillin production

  • fungi have been selectively bred to be more productive than early strains
  • penicillin is a secondary metabolite and is produced once population reaches a certain size so is made by batch culture

  1> fermenter run for a week and culture is filtered to remove cells

  2> antibiotic is precipitated as crystals by addition of K+

  3> this is mixed with inert substances and prepared for administration in desired form
• production of insulin

  • synthetic human insulin is produced from the genetically modified bacteria E.coli
  • gene for human insulin was combined with a plasmid to act as a vector so it could be inserted into the bacterium
  • result enabled production of large quantities of human insulin at low costs so is made by continuous culture
• Bioremediation

  > use of genetically modified/natural microorganisms to clean the soils and underground water on polluted sites

  • convert toxic pollutants (crude oil, pesticides)-> less harmful substances
  • e.g: natural - algae can process sewage water

  > it stimulates growth of microbes that use contaminants as a food source

  • requires suitable : o2,(if aerobic bacteria) water, pH and temperature
• Advantages of bioremediation

  • uses natural systems
  • less labour/equipment is needed
  • treatment in situ
  • few waste products
  • less risk of exposure to clean-up personnel

  BUT cannot treat heavy metals (lead)
• In the laboratory, microorganisms are grown in either medium - agar or broth

  growing microorganisms on agar plates involves 3 steps:

  • sterilisation
  • inoculation
  • incubation
• Aseptic procedure to reduce contamination

  > wash hands and disinfect working area

  > have a bunsen burner operating causing air to rise preventing air-borne microorganisms settling and create area of sterile air

  > open vessel and pass bottle over flame to prevent bacteria in air entering bottle and flame as its closed

  > lift lid slightly off petri dish to introduce desired organism

  > glassware and metal equipment should be passed through flame before/after contact with microorganism
• Aseptic techniques - Sterilisation

  • nutrient agar medium sterilised in autoclave with water under pressure at 121.c
  • when cooled its poured into sterile petri dishes and left to set with lid down