Download Biotechnology Explorer Program Serious About Science Education

Biotechnology
Explorer Program
Serious About Science Education
Aequorea victoria
Central Framework of
Molecular Biology
DNA
RNA Protein Trait
Links to real-world research
 study of biological processes :
 localization of gene expression
 cell movement
 cell fate during development
 formation of different organs
 ** screenable marker **to
identify transgenic organisms
(glowing trait and antibiotic
resistance)
BioRad Kit Protocol
• Transform bacteria with pGLO plasmid
• Select for ampicillin resistance
• Induce expression of GFP with arabinose
• Grow transformed colony
• Create glowing art with E. coli “paint”
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 (AP Bio only)
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 loop of 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)
Volume Measurement
(know these units and measuring points)