Download Beta lactamase

pGLO™ Transformation and Purification of
Green Fluorescent Protein (GFP)
Lab Timeline
• Introduction /Transformation
• Transform bacteria with pGLO plasmid
Central
Framework of
Molecular
Biology: The
Central Dogma
DNA
RNA
Protein
Trait
Context
• Genetic engineering of organism
• Use of experimental controls
• Interpretation of experimental results
• Calculate transformation efficiency
• GMO
• Cell biology
• Evolution: antibiotic resistance, selection
mechanism, adaptation
• Genetics: Central Dogma, gene regulation,
lac operon
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
Using GFP as a
biological tracer
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html
With permission from Marc Zimmer
What is
Transformation?
• Uptake of foreign
DNA, often a circular
plasmid
GFP
Beta-lactamase
Ampicillin
Resistance
What is a
plasmid?
• A circular piece of
autonomously
replicating DNA
• Originally evolved
by bacteria
• May express
antibiotic
resistance gene
or be modified to
express proteins of
interest
Bacterial DNA
Bacterial cell
Plasmid DNA
Genomic DNA
The Many
Faces of
Plasmids
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
Bacterial
Transformation
Cell wall
GFP
Bacterial
chromosomal
DNA
Beta lactamase
(ampicillin resistance)
pGLO plasmids
Transcriptional
Regulation
• Lactose operon
• Arabinose operon
• pGLO plasmid
Transcriptional
Regulation
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
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
Overview
Day 1
Day 2
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
Grow?
Glow?
• Follow protocol
• On which plates will colonies grow?
• Which colonies will glow?
Lab Safety
• The E. coli is not pathogenic
• Safety Procedure:
– Decontaminate work surface each day
– All liquid or solid waste needs to be
decontaminated (using bleach!)
– Wash hands after handling bacteria and before
leaving lab
– Carefully following all protocol / procedure.
– If you are allergic to ampicillin…let me know!
– UV lamp do not stare into the light or shine
on your own skin!
Volume
Measurement
Lab
technique
• Sterile technique!
– Do not introduce contaminating bacteria
– The inoculation loops, pipets, agar plates
should NOT touch or be placed onto
contaminating surfaces!
– Wash your Hands!!
– Be careful with timing…it’s the most important
part of the lab!!