pGLO Transformation
What is transformation?
• Uptake of foreign DNA
-- often a circular plasmid
A plasmid used as a vector must have:
a. An origin of replication
Origin of plasmid
replication
pGLO
b. A gene conferring resistance
to an antibiotic
for survival of the transformed
organism.
Beta-lactamase
to break down
ampicillin
Transformation with a plasmid
• Uptake of foreign DNA … that
a. self replicates
b. codes for a protein that
confers antibiotic resistance
to the host organism
Amp Resistance
A plasmid used as a vector must have:
a. An origin of replication
b. A gene conferring resistance
to an antibiotic
for survival of the transformed
organism.
Origin of plasmid
replication
In addition …
pGLO contains
pGL
O
c. The gene to produce Green
Fluorescent Protein - the protein of
interest
d. An ON/OFF switch that controls
synthesis of GFP by the presence
or absence of the sugar,
arabinose.
ON/OFF
switch
Beta-lactamase
to break down
ampicillin
Green
Fluorescent
Protein
What is pGLO
• Plasmid that contains an extra gene from a
jellyfish -- gene codes for GFP
…in the presence of arabinose
GFP
Amp Resistance
pGLO
Regulation
ara “BAD” Operon
araC
B
A
D
ara GFP Operon
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
However, after synthesis,
the GFP requires UV light in order to fluoresce ...
GFP
Amp Resistance
Transformation Procedure
• Suspend colony in CaCl2 Transformation Solution
• Add pGLO plasmid DNA to “+” tube
• Water to “ - ” control tube
• Incubate tubes 10-15 minutes on ice
• Heat shock 50 seconds at 42oC
• Incubate 2 minutes on ice
• Add LB broth for “recovery” period
• Plate on appropriate “flavored” media
Why cold - heat shock - cold?
• To affect membrane so cells take in plasmid
Why add LB broth?
• So cells can start replicating plasmid
and begin expressing new proteins
Amp Resistance
What do you expect to grow on the various plates?
- pGLO
Plain LB agar
LB agar + ampicillin
(before arabinose)
LB agar + ampicillin
(after arabinose)
+ pGLO
What color to you expect the colonies to be, in room light?
- pGLO
Plain LB agar
LB agar + ampicillin
(before arabinose)
LB agar + ampicillin
(after arabinose)
+ pGLO
What color to you expect the colonies to be, in UV light?
- pGLO
Plain LB agar
LB agar + ampicillin
(before arabinose)
LB agar + ampicillin
(after arabinose)
+ pGLO
Let’s test your hypothesis !