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Transformation and Antibiotic Resistance

Transformation and Antibiotic Resistance. www.le.ac.uk/genie. Basic Cloning I . DNA to be inserted. join/ligate. t ransform host cell. Plasmid vector. Recombinant DNA molecule. Basic Cloning II. select for cells containing recombinant DNA by growth in presence of antibiotic.

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Transformation and Antibiotic Resistance

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  1. Transformation and Antibiotic Resistance www.le.ac.uk/genie

  2. Basic Cloning I DNA to be inserted join/ligate transform host cell Plasmid vector Recombinant DNA molecule

  3. Basic Cloning II select for cells containing recombinant DNA by growth in presence of antibiotic transform host cell ABR Recombinant DNA molecule Host cells are made “competent” to accept plasmids. This can be achieved either: Chemically (with heat shock) OR Electrically

  4. Gene cloning – Gene library X X X X X X X A A A A A A A C C C C C C C B B B B B B B C C C C A A A B B A B B X vector X X B B X C A A C X A C B

  5. Gene cloning – Gene library B A

  6. Transformation and Selection • Use ligated DNA to transform bacteria • Not all ligated DNA maintained in bacteria • Select for bacterial cells containing vector with insert (screen for recombinants)

  7. Screen for recombinants Ensure library predominantly recombinants Simple screen to differentiate recombinants and vector alone For instance, blue-white screening using the lacZ gene • Vector alone able to grow on antibiotic-containing medium • Screen for recombinants identify lack of insert • Recombinant grows on antibiotic-containing medium • Recombinant identified by screen

  8. Blue-White Screening lacZ encodes β-galactosidase β-galactosidase converts X-Gal (colourless) to blue compound X-Gal 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside Vector containing lacZ Insert DNA fragments into sequence encoding lacZ Insertional inactivation β-galactosidase no longer produced, X-Gal not converted SCREEN for recombinants insert EcoRI EcoRI lacZ vector recombinant insert lacZ No LacZ activity White! LacZ activity Blue!

  9. Insertional Inactivation

  10. Insertional Inactivation X X X X TetR AmpR KanR pBR322 cut with enzymeX enzyme X enzyme X Ligate KanR TetR DNA ligase KanR TetR transformation TetR, AmpS ,KanR

  11. Recombinant Identification pGLO Insertional inactivation Phenotype of clone Physical characteristics of DNA TetR, AmpS,KanR

  12. Clone that Gene! Rationale of the experiment Which is which? Make bacterial clones (transformation) Investigate phenotype of clones (transformants) Investigate physical characteristics of DNA recombinant DNA vector only

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