1. Bacterial Transformation Lab

3. 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

4. TS = Transformation Solution1 ml
LB = Luria Broth
250 µl .5
100 µl
TS = Transformation Solution

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

6. 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

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

8. 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!

9. 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.

10. Other Glowing Critters

13. 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

14. 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.

15. 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