1. Transport
Nucleus
Cytoplasm
Protein
gene
DNA
mRNA
The Cell:

2. What is a plasmid? Circular piece of autonomously replicating DNA
ori
bla
Originally evolved by bacteria
May express antibiotic resistance
or be modified to express proteins of interest

3. pGLO Plasmid Beta Lactamase Green Fluorescent Protein
Ampicillin resistance
Green Fluorescent Protein
Aequorea victoria jellyfish gene
araC regulator protein
Regulates GFP transcription
pGLO
ori
bla
GFP
araC

4. What is transformation?
Uptake of foreign DNA often a circular plasmid
GFP
Amp Resistance

5. pGLO Transformation Cell wall Bacterial chromosomal DNA Beta lactamase
GFP
Bacterial
chromosomal DNA
Beta lactamase
(ampicillin resistance)
pGLO plasmids

6. pGLO Regulation RNA Polymerase araC ara GFP Operon GFP Gene ara OperonB A D
Effector (Arabinose)
Effector (Arabinose)
araC B A D
RNA Polymerase
araC B A D

7. Transformation Procedure
Suspend colonies in Transformation Solution
Add pGLO plasmid DNA to “+” tube
Incubate tubes 10 minutes on ice
Heat shock 50 seconds at 42oC
Incubate 2 minutes on ice
Incubate 10 minutes with LB broth
Plate

8. Why perform each transformation step?
1. Incubation on ice slows fluid cell membranes
2. Heat-shock increases permeability of cell membrane
3. LB recovery allows beta lactamase expression
Cell wall
GFP
Beta lactamase
(ampicillin resistance)