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

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

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

4. Gene cloning – Gene library A B C X

5. 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 EcoRI insert lacZ recombinant vector No LacZ activity White! LacZ activity Blue!

9. Insertional Inactivation

10. Tet R Amp R pBR322 Tet R cut with enzyme X DNA ligase Ligate transformation X enzyme X X Tet R, Amp S enzyme X X X Kan R,Kan R

11. Recombinant Identification Insertional inactivation Phenotype of clone Physical characteristics of DNA Tet R, Amp S,Kan R pGLO

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 vector only recombinant DNA