Bacterial Transformation Lab “ pGLO ”

1. Bacterial Transformation Lab “pGLO”

2. pGLO • A fluorescent protein from the jellyfish, Aequorea victoria • The pGLO plasmid contains several genes that are necessary for producing and expressing the pGLO protein in whatever organism it is found in.

3. Other Glowing Critters

6. What is Bacterial Transformation? • Bacterial cells taking up DNA from their environment. • Discovered by Frederick Griffith.

7. Bacterial Transformation Lab Bacterial Cells and plasmid DNA are mixed. Cells take up plasmid. Cell/DNA mix is plated on nutrient agar with antibiotic. Only cells which obtained plasmid DNA will grow…and glow!

8. ori bla What is a plasmid? • Small circular DNA molecule. • Replicates autonomously • Originally evolved in bacteria • May contain antibiotic resistance gene or be modified to contain other genes. • bla is an ampicillin resistance gene

9. Chromosomal Bacterial cell Plasmid DNA Chromosomal DNA Bacterial Cells and DNA

10. pGLO Plasmid: Most Important Components • bla gene • Bacteria with this gene produce beta lactamase, an enzyme that allows them to grow in the presence of ampicillin • GFP gene • Bacteria with this gene glow under UV light pGLO GFP bla

11. Ca++ O Ca++ O P O Base O O CH2 Sugar O Ca++ O O P Base O O CH2 Sugar OH Reasons for Each Transformation Step • CaCl2treatment Positive charge of Ca2+ ions neutralizes: • negative charge of DNA phosphates • negative charge of membrane phospholipids

12. Reasons for Each Transformation Step • Incubation on ice slows fluid cell membranes • Heat-shock increases permeability of cell membrane • Nutrient broth incubation allows beta lactamase expression prior to exposure • Growth on agar containing arabinose initiates transcription of GFP genes (modified operon)

13. Transformation Results Only cells getting pGLO plasmid grow Only cells getting pGLO plasmid grow and glow Without pGLO plasmid, nothing can grow All cells grow since there is no antibiotic on the plate