Transcript PITT pGLO Transformation Lab Protocol

Making Cells Glow:
Bacterial
Transformation with
pGLO Plasmid DNA
BACTERIAL TRANSFORMATION,
GENETIC ENGINEERING,
AND RECOMBINANT PROTEINS
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Essential Components of
Genetic Engineering
bacterial
A TRANSGENE
intended to give the host
cell or organism new or
altered traits.
A VECTOR
a plasmid or virus DNA used to
•assemble the recombinant
construct
•maintain it in its temporary and
permanent host cells
•introduce the transgene into
cells
•ensure expression of the
transgene in the new host cell
animal
HOST
CELLS
with their
own
genomic
DNA
plant
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Plasmids are frequently used as vectors.
Extrachromosomal bacterial DNAs
host
chromosome
plasmids
Small
4000 bp; compared to bacterial
chromosome of ~4 million base
pairs
Easy to work with
Can be removed, altered, then
returned to cells.
Replicate independently of
bacterial chromosome
Single or multiple copies per cell
Discovered in nature as a source
of antibiotic resistance
Some plasmids integrate
into the host genome
http://commons.wikimedia.org/wiki/File:Plasmid_episome.png
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Features of a Typical
Cloning Vector
Origin of Replication
reporter
gene
polylinker
ensures replication of DNA in a host cell
Selectable Marker
allows for selection of transformants;
usually confers antibiotic resistance
Reporter Gene
gene whose phenotype changes depending
on whether a foreign transgene has been
inserted into the plasmid
promoters
Promoter(s)
promotes transcription of selectable marker
and transgene/reporter gene
Polylinker or Multiple Cloning Site (MCS)
series of closely spaced, unique restriction
sites at which the plasmid can be cut
(linearized) to allow insertion (ligation) of
the transgene into the plasmid
selectable
marker
origin of
replication
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bla:
a common selectable marker
Ampicillin
Beta-Lactamase
An antibiotic
Prevents the growth of bacteria by
inhibiting an enzyme that is
needed for building new cell wall
peptidoglycan
An enzyme
Chemically breaks the beta-lactam
ring, inactivating the enzyme
Chemical structure based on a Betalactam ring
Beta-lactam antibiotics include
penicillins (amoxycillin) and
cephalosporins
The bla gene
Encodes the beta-lactamase
enzyme
Makes bacteria resistant to
ampicillin (ampr)
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Issues in Moving Transgenes
Between Hosts
Prokaryotes
Eukaryotes
Bacteria
Simple cell structures
Fungi, Protists, Plants, Animals
Complex cell structures
No nucleus; DNA spread throughout
the cell
Simple gene structures
Protein-coding DNA sequence (open
reading frame, or ORF) is contiguous
No machinery for RNA splicing
Simple promoters
Fewer transcription factor proteins
Internal membrane-enclosed
organelles, including a nucleus
Complex gene structures
Protein-coding DNA sequence (exons)
is interrupted by non-coding
sequences introns
Requires RNA splicing to convert premRNA (primary transcript; exons +
introns) into mRNA (exons)
Complex promoters
Many transcription factor proteins
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Eukaryotic Gene Structure
http://commons.wikimedia.org/wiki/File:DNA_exons_introns.gif
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cDNA Cloning of Eukaryotic Genes into
Prokaryotic Hosts
Since eukaryotic genes
have introns that
prokaryotic cells can’t
remove, a cDNA
transgene is created from
a DNA copy of the mRNA
with introns removed.
cDNA: complementary
DNA
Transgene must be
attached to a prokaryotic
promoter to ensure
transcription in new
bacterial host.
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Regulation of Transcription in Prokaryotes
at the Para and Plac Promoters
In the absence of inducer
(lactose or arabinose),
transcription is turned OFF
Repressor protein binds to the
operator, blocking the RNA
polymerase from the
promoter.
In the presence of inducer
(lactose or arabinose),
transcription is turned ON
Inducer binds to the repressor
protein, causing a change in its
shape. The repressor falls off
the operator, allowing RNA
polymerase to bind to the
promoter and transcribe the
gene.
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Recombinant Proteins
Proteins that are produced through genetic
engineering.
Encoded by the introduced transgene.
Produced upon the transgene’s transcription and
translation.
Can be purified from the transgenic cells or
organisms.
Can be produced in much higher quantities that
protein available from natural sources.
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Examples of Recombinant Proteins
Medicine
Human insulin (1982): Used to treat diabetes.
rHGH or human growth hormone (1985): Stimulates
growth (height) and development of muscle.
Cadaver-derived natural HGH transferred Creutzfeldt-Jacob
disease (1985)
rBST or bovine somatotropin (1994): Stimulates milk
production in cows
Required or permissible labeling of rBST milk or non-rBST milk is
debated
EPO or erythropoeitin: Stimulates creation of red blood
cells.
Used to treat anemia in cancer chemotherapy patients. Common
blood doping agent for athletes.
tPA or tissue plasminogen activator: Enzyme given to
heart attack patients to dissolve blood clots blocking
arteries.
Factor VIII: Blood clotting factor missing in hemophiliacs
Industry & Consumer
Products
In Laundry Detergents
Protease for proteins, lipases for greases, and amylases
for carbohydrates
Amylases and Maltases
For production of high fructose corn syrup from corn
starch
Cellulases and Ligninases
Enzymes that digest cellulose into sugars to be fermented
in ethanol for biofuels
Pectinases
Clarify fruit juices
Rennin
Used in cheese production
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SESSION 1:
TRANSFORMING THE BACTERIA
Mixing bacterial cells and DNA under
transformation conditions.
Introduces DNA into cells.
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The pGLO
Plasmid
Origin of
replication
bla gene:
a selectable
marker; encodes
beta-lactamase
enzyme; confers
ampr phenotype
araC gene:
encodes the
repressor protein
that blocks
transcription at
Para promoter in
absence of
arabinose
Para promoter:
allows
transcription of
the gfp gene
when cells are
treated with
arabinose
gfp gene:
a transgene;
encodes Green
Fluorescent
Protein (GFP);
confers glowing
phenotype 14
Label Your Transformation Culture Tubes
Use a lab marker to label two 15ml
round-bottom culture tubes:
+DNA initials
Place these tubes in your ice cup to
chill.
-DNA initials
-DNA & your initials
+DNA & your initials
It is very important that the
transformation reactions be keep cold.
Don’t handle these tubes or have them
out of the ice for more than a few
seconds at a time.
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Making the Transformation Mixtures
+DNA initials
To the +DNA tube, add 10µl of 5ng/µl pGLO plasmid
DNA directly to the drop of cells.
+DNA initials
To the –DNA tube, add 10µl of TE Buffer directly to the
drop of cells
-DNA initials
Provided in a microtube in your ice cup
Pipet slowly (the cells are fragile)
Carefully deposit the drop of cells to the very bottom of
the tube.
Keep tubes on ice.
Promptly replace the snap-on caps to avoid
contamination by bacteria and fungi in the air.
Don’t forget: always use a fresh pipet tip each time!
-DNA initials
To each tube, add 100µl of competent E. coli cells.
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Cold-Incubating the Transformation
Mixtures
Gently tap the bottom of each tube to gently
mix the cells and solutions.
Incubate on ice for 15 minutes.
During this time, DNA becomes attached to the
outer surface of the cells.
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Heat-Shocking the Transformation
Mixtures
Bring your ice cup with the two culture tubes to the
42°C heat block.
Quickly place your pair of tubes into the heat block.
Note the time.
After exactly 45 seconds, quickly remove your pair
of tubes and immediately place them back in your
ice cup for at least one minute.
This “heat-shock” step opens pores in the cell’s
membranes, allowing the DNA to enter some cells. The
heat shock requires instantaneous transitions between
cold to hot to cold.
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Initial Cell Culture:
Recovery and bla Gene Expression
Add 800µl of LB broth to each culture tube.
Place the tubes into the foam adapter
mounted on a vortex mixer.
+DNA initials
Tap the bottom of the tube to mix.
-DNA initials
Don’t forget: always use a fresh pipet tip
each time!
Replace the caps promptly to avoid
contamination.
Your samples will be agitated at room
temperature for about 45 minutes. This gives
the new genes (on the plasmid DNA)
introduced into the cells time to be
transcribed and translated into proteins.
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Teacher Note
After about a class period of incubation (40-50
minutes), transfer the tubes to a lab refrigerator
(without food!) for overnight storage.
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SESSION 2:
SPREAD-PLATING THE
TRANSFORMATION CULTURES
Growing the transformation cultures on
non-selective, selective, and indicator
plates.
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Culture Media
LB
Luria-Bertani medium: a rich medium that provides
a complete mixture of nutrients (sugars, amino
acids) and vitamins in which bacteria can grow.
agar
a substance added to media that makes it semi-solid
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Culture Media Additives
amp: ampicillin
an antibiotic that kills bacteria,
except those cells that contain
genes that provide resistance
(such as the beta-lactamase or
bla gene, sometimes called an
ampicillin-resistance or ampr
gene)
selective medium
a growth medium that causes the
death, or prevent the growth, of
some cells but not others
ara: arabinose
a sugar that induces transcription
of a gene by removing the
repressor protein from the
gene’s specific “ara” promoter
indicator medium
a growth medium that causes
some cells to appear differently
than other cells, indicating the
presence or absence of certain
traits
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Why Grow Transformation Cultures On
Selective Media?
The transformation process is very inefficient.
Only a tiny fraction of the cells actually take up the DNA.
We face a “finding a needle in a haystack” problem.
How do we detect, and obtain, only the cells that have
been successfully transformed (the “transformants”)?
“Burn down the haystack!”
Kill off all the non-transformants on selective media.
Cells lacking the pGLO plasmid will lack its bla gene, and
thus will be sensitive to ampicillin.
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To possess a trait, you need to both
possess the gene and express it.
The gfp gene encodes the green
fluorescent protein (GFP) from
the bioluminescent jellyfish
Aequorea victoria.
For a cell to have GFP, it must
transcribe and translate the gfp
gene.
http://en.wikipedia.org/wiki/File:Aequorea_victoria.jpg
Arabinose induces the
transcription of the gfp gene.
The gfp gene is expressed when
transformed cells are treated
with arabinose.
http://en.wikipedia.org/wiki/File:GFP_structure.png
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Labeling the Plates
Non-selective
plate
Selective
plate
Selective &
Indicator
plate
Team ___
or initials
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or initials
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or initials
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or initials
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or initials
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or initials
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Spread-Plating the
Transformation Cultures
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or initials
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or initials
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or initials
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or initials
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or initials
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200µl
+DNA initials
-DNA initials
200µl
Team ___
or initials
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Spread-Plating
Your foil packet contains two sterile yellow spreaders. Feel the foil
packet and find the end shaped like a triangle. Carefully open the foil
at the stick (not the triangle) end, keeping the triangle ends covered
with foil. Keep the spreader in the opened pack for now.
Turn your three –DNA plates over (agar side on bottom) and apply
200µl of your –DNA culture to each of the three –DNA plates: LB, LB
amp, and LB amp ara. Use a fresh pipet tip each time.
Remove one spreader from the pack (keep the other spreader
covered) and use it to gently spread the liquid across the entire
surface of each plate, turning the plate as you spread. Don’t press too
hard, or the agar will tear. Place the used spreader in the collection
bin.
Repeat using the other spreader to apply the +DNA culture to each of
the three +DNA plates.
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Incubating Your Cultures
Allow your plates to sit, agar side down, for a
few minutes to allow the liquid to absorb into
the agar.
Tape your set of six plates together using
colored lab tape.
Label the tape with your class period.
Place you set of six plates into the 37°C
incubator for an overnight incubation.
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Predict Whether Cells Will Grow On Each
Plate, and What They Will Look Like
- DNA
Grow?
Lawn or
Colonies?
+DNA
Glow
under UV
light?
Grow?
Lawn or
Colonies?
Glow
under UV
light?
LB
LB
amp
LB
amp ara
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Teacher Note
After overnight incubation, if the students will
not be observing their results the following day,
wrap the plates in parafilm and store them in a
refrigerator (with no food!).
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SESSION 3:
INTERPRETING RESULTS
Examining for evidence of transformation
and recombinant gene expression.
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Use Your “Two Minds”
Imagining Mind
Thinking Mind
Imagines what’s possible
Decides what’s real
Finds all alternatives
true or false
Eliminates alternatives
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Scientific Thinking is Critical Thinking
E + A = C
Evidence
Assumptions Conclusions
“Facts we SEE”
“Things we THINK”
•Observations
•Data
•Results
•Materials
•Procedures
•Experimental
Design
•CONTROLS
“Claims we MAKE”
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Words of Wisdom from Sherlock Holmes
“It is an old maxim of mine that when you have
excluded the impossible, whatever remains,
however improbable, must be the truth.”
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DESIGNING AN EXPERIMENT:
EXPERIMENTAL VARIABLES
•Manipulated Variables
•Controlled Variables
•Responding Variables
•Uncontrolled Variables
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Manipulated Variables
Also called the Independent Variable.
The condition or treatment that is changed or
manipulated during the experiment.
Each sample is subjected to different conditions
for the manipulated variable: treatment,
amount, time, duration, etc.
The manipulated variable is the "cause" for
which we wish to identify an "effect".
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Controlled Variables
Conditions and treatments that are identical for
all samples within the experiment
Conditions that are to be ruled out as affecting
the outcome.
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Responding Variables
Also called the Dependent Variable.
The properties to be observed or measured.
The "effect(s)" associated with changes in the
manipulated variable.
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Uncontrolled Variables
Factors which may impact experimental samples
or subjects differently, resulting in effects that
are not due to the manipulated variable.
Experimenter error
Bias
Environmental conditions
Non-random sampling
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ANALYSIS OF RESULTS
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Arrange your plates like this.
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or initials
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or initials
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or initials
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or initials
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or initials
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What do you conclude from THIS plate ALONE?
What ELSE might you conclude?
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or initials
Critical
Thinking
involves
identifying
and
considering
ALL
alternatives!
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or initials
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or initials
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or initials
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or initials
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or initials
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Negative
results have
no meaning
Period __
EXCEPT in
comparison
to a POSITIVE
CONTROL.
Period __
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What do you conclude from THIS plate ALONE?
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or initials
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or initials
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or initials
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What is the Manipulated Variable? Controlled
Variables? Responding Variable? Conclusion?
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or initials
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or initials
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or initials
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or initials
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What is the Manipulated Variable? Controlled
Variables? Responding Variable? Conclusion?
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or initials
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or initials
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or initials
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or initials
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or initials
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or initials
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What is the Manipulated Variable? Controlled
Variables? Responding Variable? Conclusion?
Team ___
or initials
Period __
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or initials
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or initials
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or initials
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or initials
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or initials
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Examining for Production of
Green Fluorescent Protein
Turn out the room lights.
Hold the UV lamp over your plates.
Do not look directly into the UV lamp.
Record which of your plates have colonies that
glow green.
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What is the Manipulated Variable? Controlled
Variables? Responding Variable? Conclusion?
Team ___
or initials
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or initials
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or initials
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or initials
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or initials
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or initials
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GOOD EXPERIMENTAL DESIGN
•Seeks to control variables.
•Confirms all of our assumptions about
materials and procedures.
•Allows us to conclude a clear
CAUSE-and-EFFECT relationship between
the MANIPULATED VARIABLE and the
RESPONDING VARIABLE.
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Sample Results
Natural Light
UV Light
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What Questions Do YOU Have?
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