genetic engineering

• what is genetic engineering?

  • modifying an organism's genome for a desired outcome
  • involves the isolation of genes from one organism and transferring them into another using vectors
• state the steps involved in gene isolation of a desired gene

  • isolation of desirable gene: use restriction endonuclease to cut the desired gene from organism
  • some cuts are blunt, some are sticky- one strand is a few bases longer than the other, leaving exposed nucleotides, makes it easier to insert desired gene into different organism
  • another way to isolate is isolating mRNA for the gene and using reverse transcriptase to convert it to cDNA- easier to identify desired gene as some cells make specific types of mRNA
• state the main stages of GE

  1. isolation of gene
  2. formation of recombinant DNA (the vectors, plasmids)
  3. transformation- transferring vector
• state the steps involved in the formation of recombinant DNA

  • bacterial plasmids used as vectors to transfer gene to host cell
  • plasmid combines with DNA to form recombinant DNA
  • plasmids contain marker gene- gene eg which has antibiotic resistance
  • same restriction endonuclease used to isolate DNA cuts open plasmid and leaves complementary sticky ends
  • DNA ligase forms phosphodiester and sugar bonds and joins together backbone
  • second marker gene might be inserted to show plasmid is recombinant- has fluorescence/enzyme that causes colour change, if no fluorescence, engineering = successful
• state the process of transferring the vector

  process called transformation- transferring the plasmids into the host cell. 2 methods:

  
  

  culture bacterial cells and plasmids in calcium rich solution and increase the temperature

  • causes the bacterial membrane to become more permeable so plasmids can enter

  electroporation

  • small electric current applied to bacteria- makes membranes porous so plasmids move into cells.
  • Can be used to get plasmids directly into eukaryotic cells. New DNA will fuse with nuclear DNA.
  • electric current has to be controlled carefully otherwise whole cell damaged
• what other technique can be used to produced GE cells?

  Electrofusion

  • tiny electric current applied to the membranes of two different cells
  • cells fuse together to form hybrid/polyploid cell
  • contains DNA from both- used to produce GM plants
  • in animals used to form monoclonal antibodies as polyploid cells don't last long in organism
• what can engineering prokaryotes be useful for?

  • insulin
  • antibiotics
  • vaccines
• what can engineering plants be useful for?

  • herbicide resistance
  • pesticide production
  • higher yield
• what can engineering animal cells be useful for?

  • for medically important proteins
  • cure genetic diseases eg CF