Transcript Chapter 20 DNA Transformation

Chapter 20
DNA Transformation
A. P. Biology
Mr. Knowles
Liberty Senior High School
pGLO™ Transformation and Purification of
Green Fluorescent Protein (GFP)
Central
Framework of
Molecular
Biology
DNA
RNA
Protein
Trait
The Central Dogma
Trait
(phenotype)
Using GFP as a
biological tracer
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html
With permission from Marc Zimmer
Real-World Applications
Links to
Real-world
• 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
GFP Transformed into
Bacteria
Applications of GFP
GFP in a Plant, Arabadopsis
GFP in a Mouse
Genetically Engineered Fish
Bacterial DNA
Bacterial cell
Plasmid DNA
Genomic DNA
What is a
plasmid?
• An
extrachromosomal,
circular piece of
autonomously
replicating DNA.
• Originally evolved
by bacteria.
• May express an
antibiotic
resistance gene
or be modified to
express proteins of
interest.
The Many
Faces of
Plasmids
•Plasmids act as
vectors –
carriers for
foreign DNA
fragments
(genes) that have
been inserted
into them and
then propagated.
•Usually put into
cells to make the
desired protein.
Graphic representation
Scanning electron micrograph of
supercoiled plasmid
Gene
Expression
• Beta Lactamase
– Ampicillin resistance
• Green Fluorescent
Protein (GFP)
– Aequorea victoria
jellyfish gene
• araC regulator
protein
– Regulates GFP
transcription
pBAD
pGLO
Bacterial
Transformation
Cell wall
GFP
Flagellum
Bacterial
chromosomal
DNA
Beta lactamase
(ampicillin resistance)
pGLO plasmids
What is
Transformation?
• Uptake of foreign
DNA, often a circular
plasmid.
GFP
• May involve bacteria or
many eukaryotic types of
cells.
• Process of copying
genes of interest and
making proteins from
them.
Beta-lactamase
Ampicillin
Resistance
Transformation
Procedure
Overview
Day 1
Day 2
Transcriptional
Regulation
• Lactose operon
• Arabinose operon
• pGLO plasmid
Transcriptional
Regulation
ara Operon
lac Operon
LacI
Z
Y A
araC
B
Effector (Arabinose)
Effector (Lactose)
LacI
Z
Y A
araC
Y A
B A D
RNA Polymerase
RNA Polymerase
Z
A D
araC
pBAD
B A D
Gene
Regulation
ara GFP Operon
ara Operon
ara
C
B
A D
araC
GFP Gene
Effector (Arabinose)
Effector (Arabinose)
araC
B A D
araC
RNA Polymerase
araC
B A D
GFP Gene
RNA Polymerase
araC
pBAD
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 on 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
4. Nutrient broth
incubation
Allows beta-lactamase
expression
Beta-lactamase
(ampicillin
resistance)
What is
Nutrient
Broth?
• Luria-Bertani (LB) broth
• Medium that contains nutrients for
bacterial growth and gene expression
– Carbohydrates
– Amino acids
– Nucleotides
– Salts
– Vitamins
• We will have THREE Types of Agar Plates:
- LB only
- LB + Amp
- LB + Amp + Arabinose
Grow?
Glow?
• Follow protocol
• On which plates will colonies grow?
• Which colonies will glow?
Laboratory
Quick Guide
Volume
Measurement
Aequorea victoria- a jellyfish