Bacterial Transformation Lab “pGLO”

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.

Other Glowing Critters

Green Fluorescent Protein (GFP) Useful in research to track the fate of specific cells during development. Other fluorescent proteins are now also available for use in genetic research.

Gene expression controlled by administration of doxycicline

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

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!

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 ori bla

Bacterial Cells and DNA Chromosomal Bacterial cell Chromosomal DNA Plasmid DNA

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

Reasons for Each Transformation Step Ca++ O CH2 P Base OH Sugar CaCl2 treatment Positive charge of Ca2+ ions neutralizes: negative charge of DNA phosphates negative charge of membrane phospholipids

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)

TS = Transformation Solution 1 ml LB = Luria Broth 250 µl .5 100 µl TS = Transformation Solution

Transformation Results LB LB/amp LB/amp LB/amp/ara All cells grow since there is no antibiotic on the plate Without pGLO plasmid, nothing can grow Only cells getting pGLO plasmid grow Only cells getting pGLO plasmid grow and glow