1. Transport Nucleus Cytoplasm Protein gene DNA mRNA The Cell:

2. Circular piece of autonomously replicating DNA What is a plasmid? Originally evolved by bacteria May express antibiotic resistance or be modified to express proteins of interest ori bla

3. pGLO ori bla GFP araC pGLO Plasmid –Beta Lactamase Ampicillin resistance –Green Fluorescent Protein Aequorea victoria jellyfish gene –araC regulator protein Regulates GFP transcription

4. Uptake of foreign DNA often a circular plasmid What is transformation? GFP Amp Resistance

5. pGLO Transformation GFP pGLO plasmids Beta lactamase (ampicillin resistance) Bacterial chromosomal DNA Cell wall

6. p GLO Regulation araC RNA Polymerase Effector (Arabinose) B B B A A A D D D araC ara Operon RNA Polymerase araC ara GFP Operon GFP Gene araC GFP Gene araC GFP Gene Effector (Arabinose)

7. Transformation Procedure Suspend colonies in Transformation Solution Add pGLO plasmid DNA to “+” tube Incubate tubes 10 minutes on ice Heat shock 50 seconds at 42 o C Incubate 2 minutes on ice Incubate 10 minutes with LB broth Plate

8. Why perform each transformation step? 1. Incubation on ice slows fluid cell membranes 2. Heat-shock increases permeability of cell membrane 3. LB recovery allows beta lactamase expression GFP Cell wall Beta lactamase (ampicillin resistance)