pGLO Bacterial
Transformation
DNA
RNA
Protein
Trait
SAFETY FIRST!
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WEAR GLOVES
DO NOT OPEN PLATES
CLOROX LAB STATION WHEN DONE
BRING PLATES TO BISHOP TO BE DESTROYED.
WASH HANDS WITH SOAP – HAPPY BIRTHDAY
JUMP TO SLIDE 29
What was the point of this lab?
What was alive?
Escherichia coli, a bacterium
Glass E. coli by Luke Jerram
Escherichia coli (E. coli)
• Found in human large intestines
• Model organism for molecular biology
• model organism – species that has been extensively
studied to better understand biological phenomena and
give insight to workings of other organisms
• “White rat” of molecular biology
scanning electron microscope
light microscope
What was the source of “new genes”?
A plasmid, genetically engineered to carry
specific genes
Symbol Bacterial structure
Plasmid containing a few genes
Circular bacterial chromosome
Illustration of E. coli K-12
Plasmid
• commonly found as small circular, double-stranded DNA molecules
in bacteria; a NORMAL component of prokaryotes (and a few
eukaryotes!)
• physically separate from, and replicate independently of,
chromosomal DNA within a cell
• carry genes that may benefit survival of the organism (e.g.
antibiotic resistance)
• may be modified to express proteins of interest; these “manmade” plasmids are widely used as vectors in molecular cloning
Bacterial DNA
DNA in nucleoid
region – NO nucleus
Bacteria have a
single circular
chromosome
Bacterial cell
Bacteria have
accessory DNA,
small circular pieces
called plasmids
Plasmid DNA
Genomic DNA
pGLO, a recombinant plasmid & vector
• Recombinant plasmid – plasmid into which
DNA fragments or genes have been inserted
• Vector – plasmid used experimentally as tool
to clone, transfer, and manipulate genes
• Because bacteria divide rapidly, they can be
used as “factories” to copy DNA fragments in
large quantities
How did you get the plasmid into
the E. coli?
TRANSFORMATION - Process by which bacteria
take up plasmids from the environment and
express the plasmid genes
EXPRESS – make proteins
TRANSFORMATION IS A NATURAL PROCESS BUT SOME BACTERIA ARE “BETTER” AT IT
THAN OTHERS. Our procedure was developed to improve the likelihood E. coli will
transform.
Bacterial Transformation
Process by which bacteria take up plasmids from the
environment and express the plasmid genes
Cell wall
GFP
Bacterial
chromosomal
DNA
Beta lactamase
(ampicillin resistance)
pGLO plasmids
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:
LB
LB/amp/+
LB/amp/LB/amp/ara/+
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
How do you know which cells
transformed (took in plasmid)?
pGLO has reporter genes
• bla (ampr)
• GFP
• araC
3 Genes of
interest in
pGLO plasmid
• Gene for Beta
Lactamase (bla)
– Protein that
inactivates the
antibiotic ampicillin
• Gene for Green
Fluorescent Protein
(GFP)
– Aequorea victoria
jellyfish gene
– Protein fluoresces
green after absorbing
UV or blue light
• Gene for araC
regulator protein
– Protein that regulates
GFP transcription –
“turns GFP gene on”
This is
DNA!
ANY QUESTIONS?
STUDENT MANUAL
• Lesson 1 page 33
• 1, 2, 4 – you should be able to answer
• 3 safety ideas?
• Does not produce toxic compounds which
make people sick
• Grows well in lab but can’t survive outside
lab
• Not able to infect animals and plants
STUDENT MANUAL
• Lesson 1 page 34
• Phenotype – physical appearance
• Starter Plate:
Let’s answer the questions
together.
STUDENT MANUAL
Number of colonies?
What is a
colony?
Descendants
of a single
cell!
STUDENT MANUAL
Size of of colonies?
STUDENT MANUAL
Color of colonies?
STUDENT MANUAL
Distribution of colonies?
STUDENT MANUAL
Appearance in UV light?
STUDENT MANUAL
Ability to reproduce in presence of antibiotic?
STUDENT MANUAL
You should be able to answer questions 1 and 2
STUDENT MANUAL
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Lesson 2 page 42
1? You Explain
2? You Explain
3? You Explain
4? You have 2 Control plates – You Explain
STUDENT MANUAL
• Lesson 3 page 43
• 1, 2, 3, 4 You do.
• But first let’s predict!
PREDICTION
Did bacteria get plasmid?
Is bacteria ampicillin resistant?
Will bacteria glow in the dark?
RESULTS
PREDICTION
Did bacteria get plasmid?
Is bacteria ampicillin resistant?
Will bacteria glow in the dark?
RESULTS
STUDENT MANUAL
• Lesson 3 page 44
• 1, 2 You do
• 3 Badly worded. Infer cells are expressing the
bla gene – making a protein that inactivates
ampicillin
• 4 Hint what plates should you compare and
why?
STUDENT MANUAL
• Lesson 3 page 45
• 1 The sample did not flouresce
• 2, 3 You do
• 4 You must answer based on you results
If successful, state evidence from plates
If unsuccessful, state evidence from plates
STUDENT MANUAL
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Lesson 3 page 46
Yes, E. coli grow on the LB plate
1, 2 you answer
3 a. What are the 2 factors?
3 b. What does arabinose do? What does UV
light do
• 3 c. You answer
STUDENT MANUAL
• Lesson 4 follow the instructions and do the
math.
• Page 48 #1. Some of you have no colonies on
the LB/amp/ara/+pGLO plate
USE THIS AS NUMBER OF COLONIES: 50
PAGE 51: REPORT TO BISHOP AS SOON AS YOU
HAVE NUMBERS. WE WILL SHARE NUMBERS
LATER.
Grow?
Glow?
• Follow protocol
• On which plates will colonies grow?
• Which colonies will glow?
From Wikipedia, the free encyclopedia
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Left: pGLO under ambient light
Right: pGLO visualized under ultraviolet light
The pGLO plasmid is an engineered plasmid used in biotechnology as a vector for creating genetically modified organisms. The plasmid contains several reporter
genes, most notably for the green fluorescent protein (GFP) and the ampicillin resistance gene. GFP was isolated from the jelly fish Aequorea victoria. Because it
shares a bidirectional promoter with a gene for metabolizing arabinose, the GFP gene is expressed in the presence of arabinose, which makes the transgenic
organism fluoresce under UV light. GFP can be induced in bacteria containing the pGLO plasmid by growing them on +arabinose plates.
pGLO is made up of three genes that are joined together using recombinant DNA technology. They are as follows:
-Bla, which codes for the enzyme beta-lactamase giving the transformed bacteria resistance to the beta-lactam family of antibiotics (such as of the penicillin
family)
-araC, a promoter region that regulates the expression of GFP (specifically, the GFP gene will be expressed only in the presence of arabinose)
-GFP, the green fluorescent protein
Like most other circular plasmids, the pGLO plasmid contains an origin (ori), which is a region of the plasmid where replication will originate. The pGLO plasmid
was made famous by researchers in France who used it to produce a green fluorescent rabbit named Alba.
Other features on pGLO, like most other plasmids, include: a selectable marker, Ori (origin of replication), and an MCS (multiple cloning site) located at the end of
the GFP gene. The plasmid is 5371 base pairs long. In supercoiled form, it runs on an agarose gel in the 4200-4500 range.[1][2][3]
The green fluorescent protein GFP consists of 238 amino
acids, linked together in a long chain. This chain folds up
into the shape of a beer can. Inside the beer can structure
the amino acids 65, 66 and 67 form the chemical group that
absorbs UV and blue light, and fluoresces green. (Credit:
Image courtesy of Nobel Foundation)
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
Transformation
Procedure
Overview
Day 1
Day 2
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
What is
Transformation?
• Uptake of foreign
DNA, often a circular
plasmid
GFP
Beta-lactamase
Ampicillin
Resistance
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
The Many
Faces of
Plasmids
Graphic representation
Scanning electron micrograph of
supercoiled plasmid
How do you know which cells
transformed (took in plasmid)?
pGLO has reporter genes
• bla (ampr) – transformed cells will grow on
agar containing ampicillin, an antibiotic
– Codes for enzyme called beta lactamase
• GFP – transformed cells will glow in UV light
– Codes for green fluorescent protein
• araC
– regulates expression of GFP; GFP expressed only
in presences of arabinose, a sugar
Methods of
Transformation
• Electroporation
– Electrical shock makes cell membranes
permeable to DNA
• Calcium Chloride/Heat-Shock
– Chemically-competent cells uptake DNA after
heat shock