Transcript Beta lactamase

pGLO™ Transformation and Purification of
Green Fluorescent Protein (GFP)
Discovery of GFP
Naturally produced in Jellyfish–
Aequorea victoria
Discovered in 1960’s
Osamu Shimomura first
isolated GFP from the jellyfish
Aequorea victoria,
Martin Chalfie demonstrated
the value of GFP as a luminous
genetic tag for various biological
phenomena.
Roger Y. Tsien contributed to
our general understanding of
how GFP fluoresces. He also
extended the colour palette
beyond green allowing
researchers to give various
proteins and cells different
colours.
Osamu Shimomura
Martin Chalfie
Roger Y. Tsien
Osamu Shimomura:
first isolated GFP from
the jellyfish Aequorea
victoria,
Martin Chalfie:
C. elegans glowing with
green fluorescent protein
Roger Tsien:
range of fluorescent proteins
have brought some colour to
laboratory work.
The barrel structure of GFP
The tertiary structure of
GFP is barrel-like, consisting
of 11 beta sheets depicted as
the green ribbons and an
internal chromophore of
three adjacent amino acids,
depicted as green spheres
Cyclization of the tripeptide
Ser-Tyr-Gly: The active
chromophore of GFP is
comprised of three adjacent
amino acids in the primary
amino acid chain. The three
amino acids are enzymatically
converted to an active cyclic
chromophore in vivo
Excitation and emission profiles of the GFP chromophore
Using GFP as a
biological tracer
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html
With permission from Marc Zimmer
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
Microtubule
dynamics in pombe
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
Transformation
Cell wall
GFP
Bacterial
chromosomal
DNA
Beta lactamase
(ampicillin resistance)
pGLO plasmids
Gene
Expression
• Beta Lactamase
– Ampicillin resistance
• Green Fluorescent
Protein (GFP)
– Aequorea victoria
jellyfish gene
• araC regulator
protein
– Regulates GFP
transcription
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
Now, prepare 3 kinds of plates
1. LB
2. LB + Amp+ (3 plates)
3. LB + Amp+ + Arabinose (2 plates)
1: LB plates
2、3、4: LB +Amp+plates
5、6、7: LB+ Amp++Arabinose plates
Store concentration
Working concentration
Amp+
10 mg/ml
50mg/ml
Arabinose
200mg/ml
50mg/ml
50ml LB medium+250ml Amp+
50ml LB medium+250ml Amp++12.5ml Arabinose
Ampicillin will be degraded in high temperature!
Central
Framework of
Molecular
Biology
DNA
RNA
Protein
Trait
What is
Transformation?
• Uptake of foreign
DNA, often a circular
plasmid
GFP
Beta-lactamase
Ampicillin
Resistance
Methods of
Transformation
• Electroporation
– Electrical shock makes cell membranes
permeable to DNA
• Calcium Chloride/Heat-Shock
– Chemically-competent cells uptake DNA after
heat shock
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)
Transformation
Procedure
Extra competent cells:
Add 1ml of pGLO plasmid
• Prepare 2 tubes, add 250ml of 50mMCacl2
• Suspend 2-4 bacterial colonies in Cacl2
• add 1ml of 0.2mg/ml pGLO plasmid DNA to one
tube, the other one leave as control
•Place tubes on ice（10mins）
•Heat-shock at 42°C for 50sec~1min and place on ice（1min）
•Add 250ml LB medium and Incubate at 37oC for10 mins
•Centrifuge ,get rid of 300-350ml supernatant, re-suspend,
•Streak plates
•Incubate LB plates in incubator o/n
Transformation
Procedure
Overview
Day 1
Day 2
Make sure to streak to correct plates!
extra competent cells
Grow?
Glow?
• Follow protocol
• On which plates will colonies grow?
• Which colonies will glow?