Module 6.2.1- Cloning and biotechnology

• Cloning


  A process in which a cell, cell product, or organism is copied from an original source and can be done through asexual reproduction
• Natural cloning- bulbs


  example- daffodils

  leaf bases swell with stored food from photosynthesis

  buds form internally which develop into new shoots and new plants in the next growing season

  
• Natural cloning- runners


  example- strawberry or spider plant

  lateral stem grows away from the parent plant and roots develop where the runner touches the ground. a new plant develops as a result
• Natural cloning- rhizomes


  example- marram grass

  rhizome is a specialised horizontal stem running underground, often swollen with stored food. buds develop and form new vertical shoots becoming new plants
• Natural cloning- stem tubers


  example- potato

  tip of an underground stem becomes swollen with stored food to form a tuber or storage organ. buds on the storage organ develop to produce new shoots
• Using natural clones in horticulture (cuttings advantages and disadvantages)

  + much faster. time taken from planting to cropping is reduced

  + if taken from a good stock, a good plant will yield and grow well since it is genetically identical

  
  

  - lack of genetic variation

  - local disease can wipe out whole population
• Increasing cuttings success rate

  -Use a non-flowering stem

  -make an oblique cut in the stem

  -use hormone rooting powder

  -reduce leaves to 2 or 4

  -keep cutting well watered

  -cover cutting with plastic bag for a few days
• Artificial cloning in animals

  Simplest method:

  - Break up an embryo into more than 1 group of cells at an early stage when it consists entirely of embryonic stem cells. They will each develop into separate genetically identical individuals. At this stage, though, characteristics are mostly unknown.

  
  

  Difficult method:

  - Somatic-cell nuclear transfer
• Artificial cloning- tissue culture


  • done in sterile conditions
  • take a small sample of tissue from desired plant the sample is sterilised
  • place explant (material removed from the plant) on sterile culture medium containing plant hormones(containing auxins and cytokinins) which stimulate mitosis. cells proliferate forming a callus
  • callus is divided and transferred to a new culture medium stimulating development of plantlets
  • plantlets are potted into compost to grow into small plants
• Advantages of micropropagation (artificial cloning)


  • allows for rapid production of large numbers of plants with known genetic makeup which yield good crops
  • culturing meristem tissue produce disease - free plants
  • possible to produce viable numbers of plants after genetic modification
  • can produce large number of seedless(sterile) plants for consumers
  • provides a way of growing naturally relatively infertile /difficult -to - grow - from seed
  • provides a way of reliably increasing numbers of rare/endangered plants
• Disadvantages of micropropagation (artificial cloning)


  • produces a monoculture so all susceptible to a disease
  • relatively expensive process
  • requires skilled workers
  • explants/plantlets vulnerable to infection by moulds/ diseases during production
  • all plants infected if source plant infected
  • large number of new plants can be lost during process
• The main form of animal vertebrae cloning is...

  Monozygotic twins (identical twins) where the early embryo splits to form 2 separate embryos
• Artificial twinning (on a cow)


  the process of producing monozygotic twins artificially

  • a cow with desirable traits is treated with hormones so it super ovulates
  • ova may be fertilised artificially or naturally and then the early embryo is gently flushed out of the uterus
  • whilst cells are still totipotent, they develop further into an embryo
  • each of split embryo is then implanted into the surrogate mother

  
• Somatic cell nuclear transfer (SCNT)


  • nucleus removed from a somatic cell of an adult animal
  • nucleus is removed from a mature ovum harvested from a different female animal (it is enucleated)
  • nucleus from somatic cell is placed in enucleated ovum and given an electric shock (or electrofusion) so it can fuse and divide
  • embryo developed is transferred to uterus of 3rd animal

  
• Arguments for animal cloning


  • High-yielding animals
  • GM embryos can be replicated and develop to give many embryos from the original procedure
  • cloning specific animals enabling rare or extinct animals to be reproduced
• Arguments against animal cloning


  • Very inefficient process
  • many fail to develop
  • miscarriage is common
  • malformed offspring
  • shortened lifespans
• Biotechnology


  A form of technology that uses living organisms, usually genes, to modify products, to make or modify plants and animals, or to develop other microorganisms for specific purposes.
• Bioremediation


  Use of living organisms such as prokaryotes, fungi, or plants to detoxify polluted ecosystems.
• Advantages of microorganisms in biotechnology


  • no welfare issues to consider
  • nutrient requirements of microorganisms are often simple and relatively cheap
  • uses waste materials
  • short life cycle and rapid growth rate
  • genetic engineering allows us to artificially manipulate microorganisms
  • not dependent on climate
  • conditions needed are relatively cheap since microorganisms provide their own catalysts in enzymes
• Disadvantages of microorganisms in biotechnology


  • if conditions are not ideal the microorganisms do not grow properly
  • conditions may be ideal for microorganisms causing food to go off or cause disease, producing toxins
  • ethical issues regarding genetically modified organisms
  • needs sterile conditions
  • protein has to be purified
• Use of microorganisms in biotechnological processes

  • baking - yeast
  • brewing - yeast
  • cheese making - bacteria
  • yoghurt making - bacteria
  • penicillin production -Penicillium
  • insulin production
  • bioremediation

  
• Producing penicillin (?)


  P.chrysogenum needs relatively high oxygen levels and rich nutrient medium to grow well

  Sensitive to pH and temperature affecting way it is produced commercially

  Semi-continuous batch process used

  First stage = fungus grows

  Second stage = fungus produces penicillin

  Drug extracted from medium and purified
• Making insulin (summary)


  (Genetically modified summary) A gene responsible for making insulin is isolated from cells of the human pancreas and is inserted via vectors into bacteria that can rapidly reproduce
• Approaches to bioremediation


  using natural organisms - many naturally break down organic material producing carbon dioxide and water

  
  

  using GM organisms - break down contaminants they would not naturally encounter

  e.g. genetically modifying bacteria to break down mercury in water
• Inoculating broth- nutrient medium (Aseptic technique)

  1. Make a suspension of the bacteria to be grown

  2. Mix a known volume with the sterile nutrient broth in the flask

  3. Stopper the flask with cotton wool to prevent contamination from the air

  4. Incubate at a suitable temperature, shaking regularly to aerate the broth providing oxygen for the growing bacteria

  
• Inoculating agar- nutrient medium (Aseptic technique)


  1. Wire inoculating loop must be sterilised by holding above bunsen burner until it glows red, not touch surfaces when cooling

  2. Dipsterilised loop in bacterial suspension, remove lid of the Petri dish and make zig-zag streak across agar surface - avoid digging into agar/agar must stay in tact

  3. Replace lid of Petri dish, should be held downwith tape but not sealed completely so oxygen can still get in-prevents the growth of anaerobic bacteria

  4. Incubate at suitable temp
• The growth of bacterial colonies (stages)

  Lag phase:

  • bacteria are adapting to the new environment, growing and synthesising new enzymes etc.

  Exponential phase:

  • the rate of bacterial reproduction is close to or at its theoretical maximum

  Stationary phase:

  • when total growth rate is 0 new cells formed by binary fission= cells dying

  Death phase:

  • reproduction has ceases and death rate of cells increase

  
• Limiting factors that prevent exponential growth in a culture of bacteria


  - nutrients available

  - oxygen levels

  - temperature (affects enzymes)

  - build up of waste

  - change in pH (affects enzymes)
• Primary metabolites


  production of products essential for microbial growth like amino acids, organic acids, alcohol, certain enzymes, etc.
• Secondary metabolites


  organic compounds that are not directly involved in the normal growth, development, or reproduction of an organism e.g. many pigments, toxic plant chemicals etc.
• Batch fermentation


  • the microorganisms are inoculated into a fixed volume of medium
  • as growth takes place, nutrients are used up and both new biomass and waste products
  • as the culture reaches the stationary phase, overall growth ceases but during this phase the microorganisms often carry out biochemical changes to form the desired end products
  • the process is stopped before the death phase and the products are harvested. the whole system is then cleaned and sterilised and a new batch culture started up
• Continuous culture


  • microorganisms inoculated into sterile nutrient medium and start to grow sterile
  • nutrient medium added continuallyto the culture once it reaches the exponential point of growth
  • culture broth is continually removed: medium, waste products, microorganisms, and product (keeping culture volume in bioreactor constant)

  enables continuous balanced growth
• Continuous vs Batch fermentation

  
  

  
  

  • Continuous kept in exponential phase vs stationary phase
  • Nutrients continuously added vs used up
  • Products continuously harvested and is difficult and expensive vs batch is just left there
  • Significant loss from contamination vs 1 batch lost only from contamination
  • More efficient vs less efficient
  • Primary metabolites produced vs primary and secondary metabolites
• Revise serial dilutions


  • Use a Gilson's pipette or graduated pipette
  • Transfer out 1 cm^3 of broth solution into a boiling tube
  • Using a pipette transfer 9 cm^3 of water to the same boiling tube
  • Add a bung and invert the tube to form a 10% dilution
  • Repeat steps in orange until you are able to count bacterial colonies
  • Carry out necessary calculations (e.g. multiply colonies by 10 ^n where n is the number of dilutions you took)

  
• Controlling bioreactors- temperature

  If the temperature is too low, the microorganisms will not grow quickly. If it is too high, enzymes can begin to denature

  
  

  Heating/cooling system linked to temperature sensors and a negative feedback system maintains optimum temperature
• Controlling bioreactors- nutrients and oxygen

  Probes or sample tests taken to detect change in nutrients and oxygen
• Controlling bioreactors- mixing and asepsis

  most bioreactors have a mixing mechanism and is stirred continuously to ensure all microorganisms receive enough food and oxygen

  
  

  bioreactors are also sealed, aseptic units to prevent contamination
• Controlling bioreactors diagram


  diagram

  
• Formula for growth of microorganisms in a closed culture

  N= N0 x 2^n

  
  

  population= initial population x 2^n
• Advantages of isolated enzymes


  Less wasteful- can be reused

  More efficient- work at higher concentrations

  More specific

  Maximise efficiency

  Less downstream processing- pure product formed