6. Gene Cloning Page 368 – 374
Gene Cloning gene cloning – 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.
Gene Cloning Bacteria are grown on Petri plates containing agar. Nutrients are mixed within agar providing a food source.
Gene Cloning Bacteria contain circular DNA as: • (chromosome) • (plasmids) The chromosome: The plasmids: There is only one chromosome, but there can be many (up to 100s) of different plasmids that can be carried.
Gene Cloning Three steps for gene cloning: • Creation of recombinant DNA plasmid. • Bacterial transformation. • Screening for successful clones.
a) Recombinant Plasmid PCR is used: ___________________ insert. Restriction enzymes are used to: A ligation is performed to:
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.
b) Bacterial Transformation Two main methods to help bacteria pick up plasmid: • cold CaCl2 treatment followed by heat-shocking • electroporation
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?
a) Recombinant Plasmid Use of an antibiotic resistance gene on the plasmid?
a) Recombinant Plasmid Is the presence of a vector also an indication of a successful ligation? Which colonies contain a vector with an insert?
a) Recombinant Plasmid The lacZ gene codes for the b-galactosidase enzyme. What happens when X-gal is added to the media?.
c) Screening How do we get the different colored colonies when using this screening method?
Gene Cloning Application Although much more complex and problematic, the principle of cloning can be applied to larger organisms. FlavrSavr Tomatoes Bt toxin – natural herbicide
Gene Cloning Application What are the ethics involved?