pGLO Lab Purpose: To transform E. coli bacteria by adding plasmids that allow the bacteria to glow green under UV light in the presence of arabinose sugar and grow in the presence of the antibiotic, ampicillin.

Why are bacteria a good choice for genetic transformation? Single celled Use plasmid DNA Reproduce quickly

Gene isolated by restriction enzymes and placed into plasmid Source of “glowing gene” for this experiment Aequorea victoria: jellyfish Gene isolated by restriction enzymes and placed into plasmid

pGLO Plasmid – 3 genes Beta Lactamase Ampicillin resistance Green Fluorescent Protein jellyfish gene – glows green in prescence of arabinose sugar araC regulator protein Makes enzyme to digest arabinose – must switch on for GFP gene to turn on! pGLO ori bla GFP araC

Bacterial Transformation Plasmids enter bacterial cell and genes are expressed Cell wall GFP Bacterial chromosomal DNA Beta lactamase (ampicillin resistance) pGLO plasmids

What is in the agar? LB – nutrient broth for bacteria to feed on Ampicillin – antibiotic that kills bacteria Arabinose – Sugar necessary to switch on ara C gene and the GFP gene

Reasons for Each Transformation Step: CaCl2 treatment (TS) Positive charge of Ca 2+ neutralizes: • negative charge of DNA • negative charge of cell membrane

2.Incubation on ice slows cell membranes 3. Heat-shock Increases permeability of cell membrane These steps allow the plasmid to be taken in by the bacteria cells

4. Nutrient broth incubation allows beta lactamase gene to be expressed (for antibiotic resistance)

How will we know if bacteria is transformed? If bacteria grows in the presence of ampicillin If bacteria glows in the presence of arabinose