Bacterial Transformation

Real image of a bacterial plasmid (Scanning electron micrograph) pGLO Plasmid GFP araC GFP: gene for Green Fluorescent Protein araC: gene for Beta- lactamase (Ampicillin resistance) Make plasmid Real image of a bacterial plasmid (Scanning electron micrograph)

Bacterial Plasmids

Streak bacteria onto LB plates

Add transformation solution CaCl2 Tubes

Transformation solution: CaCl2 Positive charge of Ca++ ions shields negative charge of DNA O CH2 P Base OH Sugar Put bacterium, plasmid, and in tube, remove negative signs from bottle. Positive charge of Ca++ ions shields negative charge of DNA phosphates

Inoculate 2 tubes with bacteria CaCl2 Tubes

Add pGLO plasmid to one tube only CaCl2 Tube

Incubate tubes on ice Slows movement of cell membrane Cell membrane pGLO plasmids Bacterial chromosomal DNA Cell membrane Remove cap

Heat-shock tubes@ 42°C for 50 seconds Increases permeability of membrane Bacterial chromosomal DNA Cell membrane Put tube in the “hot water” bath and push the plasmid into the cell Increases permeability of membrane pGLO plasmids

Incubate tubes on ice for 2 minutes Returns bacteria to temperature necessary for survival pGLO plasmids Bacterial chromosomal DNA Cell membrane Put tube in the “hot water” bath and push the plasmid into the cell

Add nutrient broth (LB), RT for 10 minutes Provides nutrition for growth and gene expression Bacterial chromosomal DNA GFP Beta lactamase (ampicillin resistance) pGLO plasmids

Streak Plates with bacteria

What is an operon?

Arabinose operon

Arabinose operon and GFP

What do you expect to happen?