18a cloning


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18a cloning

  1. 1. Gene Cloning gene cloning - a method of inserting new DNA into a simple organism to make many copies of the gene and protein Clones are used to examine the structure and function of the gene as well as the products of specific genes. It is easier to study in a simple organism than in a complex one where there may be many different effects observed.
  2. 2. Gene Cloning Bacteria are grown on Petri plates containing agar. Nutrients are mixed within agar providing a food source.
  3. 3. Gene Cloning Bacteria contain circular DNA as: a requirement (chromosome) 2. extra material (plasmids) 1. The chromosome defines the type of bacterium. The plasmids provide additional functions. There is only one chromosome, but there can be many (up to 100s) of different plasmids that can be carried.
  4. 4. Gene Cloning A synthetic plasmid is used for cloning. The vector is a circular piece of DNA into which your gene of interested is inserted.
  5. 5. Gene Cloning Gene Cloning Activity
  6. 6. Gene Cloning Three steps for gene cloning: Creation of recombinant DNA plasmid. b) Bacterial transformation. c) Screening for successful clones. a)
  7. 7. a) Recombinant Plasmid PCR is used to make many copies of the gene of interest - insert. Restriction enzymes are used to cut complementary sticky ends on both the vector and the insert. A ligation is performed to produce the recombinant plasmid.
  8. 8. a) Recombinant Plasmid
  9. 9. a) Recombinant Plasmid What are the possible results of a ligation reaction? The reaction is successful with vector insertion. The reaction is unsuccessful where the insert is absent from the vector.
  10. 10. b) Bacterial Transformation Two main methods to help bacteria pick up plasmid: cold CaCl2 treatment followed by heat-shocking a)  makes cell membrane leaky and more permeable electroporation b)  electric current to increase cell permeability
  11. 11. Gene Cloning Think about the reality of each of the steps so far. Is each step going to work perfectly? How will you know which bacteria picks up the correct vector?
  12. 12. a) Recombinant Plasmid The vector contains an antibiotic resistance gene. Antibiotics added to the agar. Only bacteria that pick up the vector will grow.
  13. 13. a) Recombinant Plasmid Is the presence of a vector also an indication of a successful ligation? How can we determine which colonies contain a vector with an insert?
  14. 14. a) Recombinant Plasmid Some inserts are designed to disrupt genes on the vector. i.e. The lacZ gene codes for the -galactosidase enzyme. When X-gal reagent is added to the agar, this enzyme produces a bluecoloured product.
  15. 15. c) Screening i.e. (continued) Vector is designed such that an insert will interrupt the lacZ gene. What type of results can you expect on the Petri plate?
  16. 16. c) Screening i.e. (continued) Successful clones will be white-coloured colonies grown on antibiotic-containing agar. Clones are transferred into larger containers to be grown to greater concentrations where they can be studied.
  17. 17. Gene Cloning Application Although much more complex and problematic, the principle of cloning can be applied to larger organisms. Flavr Savr Tomatoes Bt toxin – natural herbicide
  18. 18. Gene Cloning Application
  19. 19. Gene Cloning Application What are the ethics involved?