Download From a Gel to a Cell:

The Cell:
DNA
gene
Nucleus
mRNA
Transport
Cytoplasm
Protein
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

pGLO Plasmid
–Beta Lactamase
• Ampicillin resistance
–Green Fluorescent
Protein
• Aequorea victoria
jellyfish gene
–araC regulator protein
• Regulates GFP
transcription
araC
ori
pGLO
bla
GFP
What is transformation?
• Uptake of foreign DNA often
a circular plasmid
GFP
Amp Resistance
pGLO Transformation
Cell wall
GFP
Bacterial
chromosomal DNA
pGLO plasmids
Beta lactamase
(ampicillin resistance)
pGLO
Regulation
ara GFP Operon
ara Operon
araC
B
A
D
araC
Effector
(Arabinose)
Effector
(Arabinose)
araC
B
A
D
araC
RNA Polymerase
araC
B
A
D
GFP Gene
GFP Gene
RNA Polymerase
araC
GFP Gene
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
Why perform each transformation
step?
1. Incubation on ice
slows fluid cell
membranes
Cell wall
GFP
2. Heat-shock increases
permeability of cell
membrane
3. LB recovery allows
beta lactamase expression
Beta lactamase
(ampicillin resistance)