Transcript pGLO
Bacterial Transformation Lab Procedure Overview • • • • • • • • • • • • Observe starter plate Label blank tubes “+” and “-” Pipet 250 µl TS into each tube ice Sterile loop 1 colony from starter into each tube Sterile loop plasmid into “+” tube (Mr. Mohn) Ice for 10 min, label plates while waiting Heat shock 50 sec back to ice Pipet 250 µl LB into each tube Room temp for 10 min Inoculate plates with 100 µl of “+” or “-” Spread with sterile loop, stack and tape plates Incubate 48 hours at 37 C LB = Luria Broth 250 µl 100 µl TS = Transformation Solution What is Bacterial Transformation? • Bacterial cells taking up DNA from their environment. • Discovered by Frederick Griffith. 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 Plasmid DNA Chromosomal DNA Bacterial Transformation Lab 1. Bacterial Cells and plasmid DNA are mixed. 2. Cells take up plasmid. 3. Cell/DNA mix is plated on nutrient agar with antibiotic. 4. Only cells which obtained plasmid DNA will grow…and glow! pGLO • A fluorescent protein from the jellyfish, Aequorea victoria • The plasmid used in this lab contains the genes necessary for producing and expressing the pGLO protein in whatever organism it is found in. Other Glowing Critters 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 bla GFP 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. 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