Download ara Operon

pGLO™ & GFP
Central Framework of Molecular Biology
The Central Dogma as proposed by Francis
Crick
DNA
RNA
Protein
Trait
Links of this
Lab to Realworld
• GFP is a visual marker
• Study of biological processes
(example: synthesis of proteins)
• Localization and regulation of gene
expression
• Cell movement
• Cell fate during development
• Formation of different organs
• Screenable marker to identify transgenic
organisms
Transformation
Procedure
Day 1
Day 2
Bacterial
DNA Images
Bacterial cell
Plasmid DNA
Genomic DNA
•
What is
Transformation?
Uptake of foreign
DNA, often a circular
plasmid
•
The plasmid that we want
the bacteria to uptake in
this lab is called pGLO.
•
There are two important
genes found on this
operon:
a. One called AMP that
codes for the enzyme
beta-lactamase (the
enzyme that allows
the bacteria to be
resistant to
amoxicillin – an
antibiotic.
b. One called GFP that
codes for the green
flourescent protein
(allows the bacteria
to glow under certain
conditions.)
GFP
Beta-lactamase
Ampicillin
Resistance
Bacterial
Transformation
Cell wall
GFP
Bacterial
chromosomal
DNA
Beta lactamase
(ampicillin resistance)
pGLO plasmids
Volume
Measurement
What is
Nutrient
Broth?
•
Luria-Bertani (LB) broth
•
Medium (Food) that contains nutrients for
bacterial growth and gene expression
– Carbohydrates
– Amino acids
– Nucleotides
– Salts
– Vitamins
•
The petri dishes are made using LB broth.
For this lab you will be using three
different dishes:
a. Some of the dishes will just have the LB
b. Some will have arabinose added to the broth,
and
c. Some dishes will have both arabinose and
ampicillin added.
Transcriptional
Regulation:
Modification of the
normal Arabinose
Operon
• Lactose operon
• Arabinose operon
• pGLO plasmid
Transcriptional Regulation: The lac and ara
(Arabinose Operon) are both inducible. The
effector is another name for the INDUCER.
ara Operon
lac Operon
LacI
Z
Y A
ara
C
Z
Y A
araC
Y A
B A D
RNA Polymerase
RNA Polymerase
Z
A D
Effector (Arabinose)
Effector (Lactose)
LacI
B
araC
B A D
Gene Regulation: How the ara Operon has been
modified. The B-A-D structural genes have been
enzymatically removed from the operon and have been
replaced by the GFP Gene.
ara GFP Operon
ara Operon
ara
C
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
Methods of
Transformation
• Electroporation
– Electrical shock makes cell membranes
permeable to DNA
• Calcium Chloride/Heat-Shock
– Chemically-competent cells uptake DNA after
heat shock
Transformation
Procedure
• Suspend bacterial colonies in
Transformation solution
• Add pGLO plasmid DNA
• Place tubes in ice
• Heat-shock at 42°C and place on ice
• Incubate with nutrient broth
• Streak plates
Reasons for
Performing
Each
Transformation
Step?
Ca++
Ca++
O
O P O
O
CH2
Base
O
Sugar
1. Transformation
solution = CaCI2
Positive charge of
Ca++ ions shields
negative
charge of DNA
phosphates
O
Ca++
O P O
Base
O
CH2
O
Sugar
OH
Why Perform
Each
Transformation
Step?
Cell wall
GFP
2. Incubate on ice
slows fluid cell
membrane
3. Heat-shock
Increases permeability
of membranes to allow
for plasmid uptake
4. Nutrient broth
incubation
Allows beta-lactamase
expression
(transcription/transl. of
the gene will occur)
Beta-lactamase
(ampicillin
resistance)