In the pGLO lab, we will: Use recombinant DNA Genetically engineer E. coli bacteria by inserting a plasmid Plate and grow bacteria Determine if the proteins are expressed by genes

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

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

pGLO ori bla GFP araC pGLO Plasmid – 3 genes –Beta Lactamase Ampicillin resistance –Green Fluorescent Protein jellyfish gene – glows green in prescence of arabinose sugar –araC regulator protein Control gene (switches on in the presence of arabinose) Creates a protein that turns on GFP gene!

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

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: 1.CaCl 2 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

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.