GENETIC ENGINEERING

• Recombinant DNA technology


  The process of genetic engineering involving combining DNA from different sources or different organisms, in a single organism
• Steps in genetic engineering

  1. Obtain the required gene

  2. Place a copy of the gene in a vector

  3. The vector carries the gene to the recipient cell

  4. The recipient expresses the gene via protein synthesis
• Vector


  A means of delivering a gene into a cell, and a carrier into which the required gene is inserted, resulting in recombinant DNA
• Common vectors


  • Bacterial plasmids
  • Viral DNA
  • Liposomes
• Methods to get the gene into target cells

  1. Electroporation

  2. Microinjection

  3. Liposomes
• Restriction enzymes


  Used to cut through DNA at specific points, acting as a natural defence mechanism in bacteria against viral pathogens
• Sticky ends


  Unpaired and exposed bases along the DNA molecule after restriction enzyme cutting
• DNA ligase


  Enzyme used to catalyse the condensation reaction joining the sugar-phosphate backbones of the double helix together
• Only fragments cut with the same restriction enzyme can be joined by DNA ligase due to complementary sticky ends
• Plasmid


  A DNA molecule which is entirely separate from the chromosomal DNA found in bacteria (and a very small amount of eukaryotes)
• Bacterial genetic engineering process

  1. Restriction enzymes cut gene from DNA

  2. Gene inserted into plasmid

  3. Plasmids with gene mixed with bacterial cells

  4. Transformed bacteria contain new DNA
• Transgenic


  An organism which has added DNA due to genetic engineering
• Conjugation


  A process where genetic material may be exchanged between bacteria, often involving plasmids carrying antibiotic resistance genes
• Griffith experiment conditions


  • Infect mice with S-strain (smooth, deadly)
  • Infect mice with R-strain (rough, not deadly)
  • Infect mice with heat-killed S-strain (not deadly)
  • Infect mice with mixture of heat-killed S-strain and R-strain (deadly)
• Griffith's experiment showed bacteria can take up DNA from their environment, transforming the R-strain to be toxic
• Reverse transcriptase


  Enzyme used to synthesise a complementary DNA strand from the mRNA coding for the insulin gene
• Bacterial genetic engineering is used to produce human insulin more cheaply and quickly than extracting it from pigs
• Rough strain


  Mice do not die as their immune systems reject and destroy the bacteria
• Smooth strain


  Protective capsule means bacteria are not destroyed by immune system, so mice die
• Heat-killed smooth strain


  Bacteria are killed and so do not harm the mice, mice survive
• Heat-killed strain and rough strain

  Although alone are harmless, the mixture of the two kills the mice, mice die
• Producing human insulin


  1. Find mRNA strand coding for insulin gene

  2. Use reverse transcriptase to synthesise complementary DNA strand

  3. Add free nucleotides and DNA polymerase to make single-stranded molecule double-stranded

  4. Mix cDNA with open plasmids and DNA ligase enzyme to create recombinant plasmids

  5. Mix recombinant plasmids with bacteria so they take them up
• Bacteria can then be grown on an agar plate, and transformed bacteria will be capable of producing human insulin
• Genetic markers


  Plasmid vectors carry genes that make them resistant to two different antibiotics (ampicillin and tetracycline)
• Identifying transformed bacteria


  1. Grow all bacteria on standard agar plate

  2. Transfer colonies to ampicillin agar plate, only those with plasmid will grow

  3. Transfer some cells from ampicillin plate to tetracycline agar plate, only those without insulin gene will grow

  4. Colonies that grew on ampicillin but not tetracycline have taken up insulin gene
• Vitamin A deficiency can lead to blindness and 2 million deaths annually worldwide
• Vitamin A


  Comes from animal sources or beta-carotene which is converted to vitamin A in the human gut, requires lipids in diet for absorption
• Golden Rice


  Genetically engineered rice plants to accumulate beta-carotene in the grain part that we eat

  
• Producing Golden Rice


  Insert two genes (for phytoene synthetase and Crt-1 enzyme) near a specific promoter region to switch on beta-carotene production in the endosperm
• Golden Rice is said to be biofortified, containing higher than regular concentrations of beta-carotene
• The usefulness of Golden Rice in treating and preventing vitamin A deficiencies is questioned, as many believe you would have to eat much more rice than normal for any effect
• Organisations such as Greenpeace have criticised Golden Rice, arguing that genetic modification of crops will reduce biodiversity and the safety to humans is unknown

  
• After food safety investigations, full field trials of growing Golden Rice in a natural environment are expected to begin by 2012