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. 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.

7. Gene expression controlled by administration of doxycicline

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

9. 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!

10. 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

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

12. 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

13. 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

14. 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)

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

17. 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