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1 Transformation and Antibiotic Resistance

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

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