pglo transformation lab ap lab 7
TRANSCRIPT
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PGLO TRANSFORMATION LAB
(AP LAB 7)
BIO-RAD lab book
pGLOori
blaGFP
araC
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PURPOSE:
To observe gene expression in real time by performing
a genetic transformation procedure on E. coli bacteria
using a plasmid as a vector.
If successful, the plasmid will provide the E. coli with two
new traits:
• expression of a gene that codes for Green
Fluorescent Protein (GFP) bioluminescence under the
control of an operon
• resistance to the antibiotic Ampicillin
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BACKGROUND
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MECHANISMS OF GENE REGULATION
a wide range of mechanisms are used by cells to
increase or decrease the production of specific gene
products
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Prokaryotes regulate gene expression by controlling
the amount of transcription.
Eukaryotic gene expression is controlled at the levels of epigenetics,
transcription, post-transcription, translation, and post-translation.
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GENE REGULATION IN EUKARYOTES
The latest estimates are that a human cell, a eukaryotic cell,
contains some 21,000 genes.
• Some of these are expressed in all cells all the time. These
“housekeeping genes” are responsible for the routine
metabolic functions (e.g. respiration) common to all cells.
• Some are expressed as a cell enters a particular pathway of
differentiation.
• Some are expressed all the time in only those cells that have
differentiated in a particular way. For example, a plasma
cell expresses continuously the genes for the antibody it
synthesizes.
• Some are expressed only as conditions around and in the cell
change. For example, the arrival of a hormone may turn on (or
off) certain genes in that cell.
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Altering the rate of transcription of the gene is the
most important and widely-used strategy
There are several methods used by eukaryotes to regulate gene expression.
Promotors – sequence of DNA before the gene of interest where
transcription factors can bind and begin transcription
Enhancers – increase the rate of transcription of the gene
Silencers – can repress the transcription of the gene they control
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GENE REGULATION IN PROKARYOTES
Bacteria adapt to changes in their surroundings
by using regulatory proteins to turn groups of
genes on and off in response to various
environmental signals.
Prokaryotes are sensitive to their environment, and their genetic
activity is controlled by specific proteins that interact directly with their
DNA to quickly adjust to environmental changes.
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When the genes in an operon are transcribed, a single mRNA is produced for all
the genes in that operon.
An operon is a self-regulating series of genes that work in concert. An
operon includes a special segment of genes that are regulators of the
protein synthesis, but do not code for protein, called the promoter and
operator. These segments overlap, and their interaction determines
whether the process will start and when it will stop.
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The DNA of Escherichia coli can encode about 4000
proteins, but only a fraction of these are made at any one
time.
E. coli regulates the expression of many of its genes
according to the food sources that are available to it.
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The operator is a short region of DNA that lies partially
within the promoter and that interacts with a regulatory
protein that controls the transcription of the operon.
The regulatory gene lacI produces a mRNA that
produces a Lac repressor protein, which can bind to the
operator of the lac operon.
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The Lac regulatory protein is called a repressor because it
keeps RNA polymerase from transcribing the structural genes.
Thus the Lac repressor inhibits transcription of the lac operon.
In the absence of lactose, the Lac repressor binds to
the operator and keeps RNA polymerase from
transcribing the lac genes.
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When lactose is present, the lac genes are expressed
because allolactose binds to the Lac repressor protein
and keeps it from binding to the lac operator.
RNA polymerase can then bind to the promoter and
transcribe the lac genes.
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GENE REGULATION IN BACTERIA
pGlo lab
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WHAT IS A PLASMID?
A plasmid is a small
circular piece of DNA
(about 2,000 to 10,000
base pairs long) that
contains important
genetic information for
the growth of bacteria.
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WHAT IS TRANSFORMATION?
Uptake of foreign DNA plasmid – that
produces new traits in the bacteria
Bacterial
chromosomal DNA
pGLO plasmids
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WHY DO SCIENTISTS USE PLASMIDS?
A plasmid is used as a vector*. The gene of interest is inserted into the vector plasmid and this newly constructed plasmid is then put into E. coli or some other target.
*Vector - Something that is used to transfer something else (a mosquito is a vector for the organism that causes malaria)
pGLOori
blaGFP
araCFor example: transformed bacteria can be used to make insulin, human growth hormone, and clotting factor cheaply and in great abundance.
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Plasmids can transfer
genes that occur
naturally within them, or
they can act as carriers
for introducing foreign
DNA from other sources
into recipient bacterial
cells.
Restriction
endonucleases can be
used to cut and insert
pieces of foreign DNA
into the plasmid vectors
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In nature, this information is often a gene that codes for a
protein that will make the bacteria resistant to an antibiotic.
Bacteria can exchange plasmids with one another.
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APPLICATIONS IN THE “REAL WORLD”
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WHAT IS THE GFP
GENE?
GFP is a green fluorescent protein that naturally occurs in some bioluminescent jellyfish.
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GLOWING IN NATURE
Many species have the ability to glow
Most are marine: jellyfish, dinoflagellates
Some live on land: firefly, glow worm
Purposes of glowing:
➢ Spook predators
➢ Lure prey
➢ Attract mates
➢ Communicate
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Fireflies
Glow worm and glow worm cave
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Jellyfish Gene put into Other Critters
http://www.technologyreview.com/files/21291/monkey_x600.jpg
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MOUSE UNDER BLUE LIGHT (LEFT) SAME MOUSE UNDER NORMAL LIGHT (RIGHT)
Mouse blood vessels (green-GFP) in tumor (red-DsRed). Mouse with brain tumor
expressing DsRed.
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ALBA – BUNNY CREATED FOR “ART”
•http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html
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Three kittens. Two have been genetically modified to make
red fluorescent protein. All three look similar under normal
light, but when irradiated with blue light only the two
genetically modified kittens glow red.
(Photo courtesy of Biology of Reproduction)
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In Brainbow mice, Harvard
researchers have introduced
genetic machinery that randomly
mixes green, cyan and yellow
fluorescent proteins in individual
neurons thereby creating a palette
of ninety distinctive hues and colors.
This is a photograph of the cerebral
cortex. In non-living preserved
brains the outer layers of this
portion of the brain are gray.
(Confocal image by Tamily Weissman.
Mouse by Jean Livet and Ryan Draft.)
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REAL-WORLD APPLICATION:
Fluorescence can be used to
“tag” specific cells – in this case
tumor cells. Scientists hope this
could be a noninvasive way to
identify the location of certain
tumors and cancers in humans.
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The World Health Organization estimates that 300-500 million cases of malaria
(transmitted by the female Anopheles mosquito’s bite) occur each year and
more than 1 million people die of malaria. A possible breakthrough in curtailing
the spread of malaria carrying mosquitoes was reported in October 2005 - the
creation of mosquitoes with green fluorescent testicles. Without green
fluorescent gonads it is impossible to separate mosquito larvae based on their
sex, and it is very difficult to separate the adults. Now male mosquito larvae
can easily be separated from female mosquito larvae.
Malaria is the
world's most
common and
deadly parasitic
disease.
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BACK TO OUR LAB…
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ARE WE GOING TO MAKE CATS GLOW
GREEN? NO, JUST BACTERIA.
In this lab, you will “transform” bacteria by making them take up a commercially prepared plasmid that contains three genes of interest:
amp, araC and GFP
Genetically modified organisms are called “transgenic”
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BACTERIAL TRANSFORMATION
Plasmids
Chromosomal
DNA
Bacterial Cell
The uptake of plasmid DNA
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The bacterium Escherichia coli or E. coli is an ideal organism for the
molecular geneticist to manipulate and has been used extensively in
recombinant DNA research. It is a common inhabitant of the human
colon and can easily be grown in suspension culture in a nutrient
medium such as Luria broth, or in a petri dish of Luria broth mixed
with agar (LB agar) or nutrient agar.
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Aequorea victoria(Crystal Jelly) Source of “glowing gene” for this experiment
source of GFP
The Crystal jelly (Aequorea victoria) is a jellyfish that is found off the west coast of North America. The species is best known as the source of two proteins involved in bioluminescence; aequorin and green fluorescent protein which led to their discoverers winning the Nobel Prize in Chemistry.
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PGLO PLASMID
bla (β-lactamase)
- “on” all the time
- makes protein that breaks down ampicillin
- provides ampicillin resistance
GFP-Green Fluorescent Protein
- Glows green in fluorescent light
Arabinose Operon (inducible)
turns on (makes a protein)
when arabinose sugar is present
Allows bacteria to “turn on” the
Fluorescence because it has been
linked into the same operon systempGLOori
blaGFP
araCOri-
Plasmid
Replication
genes
PBAD arabinose promoter
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GENES OF INTEREST: AMP, ARAC, GFP
amp – this gene will give our transgenic bacteria
resistance to the antibiotic ampicillin
araC – this gene will produce a protein in the
presence of arabinose (a sugar that is added to agar)
that will allow the bacteria to turn on the GFP gene
GFP – in the presence of arabinose, this gene will
“turn on” and cause the transformed (transgenic)
bacteria to glow green
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ARABINOSE OPERON REGULATION
araC
RNA Polymerase
Effector
(Arabinose)
B
B
B
A
A
A
D
D
D
araC
araC
ara Operon
INDICIBLE OPERON:
The presence of
arabinsoe turns on
genes that make
enzymes (proteins) to
digest the sugar
arabinose
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PGLO REGULATION
araC
RNA Polymerase
Effector
(Arabinose)
B
B
B
A
A
A
D
D
D
araC
araC
ara Operon
GFP Gene
GFP Gene
GFP Gene
GFP GENE HAS
BEEN ADDED TO
ara OPERON
WHEN ARABINOSE
IS PRESENT,
OPERON IS
TURNED ON and
GFP GENE
IS EXPRESSED
TOO!
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GETTING STARTED
E. coli starter plate
This plate has the
bacteria we will use in
the lab growing in a
luria broth (LB) agar
plate.
These bacteria are wild
type (“normal”, have
NOT been transformed)
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EXPLANATION OF AGAR PLATES
LB/amp/
This plate will have E. coli bacteria on LB agar to which ampicillin has been added.
LB/amp/ara
This plate will have bacteria growing on agar that has both ampicillin and arabinose added to it.
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WHAT SHOULD YOU EXPECT?
If your technique is good, you should expect to see green glowing bacteria in some plates and not others.
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protocol
BACTERIAL TRANSFORMATION
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MAKE OBSERVATIONS!
Be sure to make observation and fill in the observations
you can make today – before you start the experiment.
The liquid (broth) and solid (agar) nutrient media are made from an extract
of yeast and an enzymatic digest of meat byproducts, which provide a
mixture of carbohydrates, amino acids, nucleotides, salts, and vitamins, as
nutrients for bacterial growth. The foundation, agar, is derived from
seaweed. It melts when heated, forms a solid gel when cooled and
functions to provide a solid support on which to culture bacteria.
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Label Tubes
+ pGLO - pGLO
Gather Supplies
What do these
represent?
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Use sterile pipette to
add 250µL transformation
solution to pGLO + and – tubes
Transformation
solution (CaCl2)
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WHEN USING THE PIPETTE FOR
MEASUREMENTS TAKE INTO ACCOUNT THE
FOLLOWING GRADUATIONS
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GET YOUR RACK ON ICE!
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INNOCULATE TUBES WITH
E. COLI BACTERIA
Pick one colony
Twirl loop in +pGLO tube
Get new loop
Pick one colony
Twirl loop in –pGLO tube
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EXAMINE PGLO PLASMID DNA
Use UV light to examine pGLO plasmid vial
UV light can be harmful to your eyes!Wear your goggles.Do not shine in eyes.
GFP =Green Fluorescent Protein
isolated from jellyfish
http://www.mshri.on.ca/nagy/GFP%20mice.jpg
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PLASMID DNA TRANSFER
THIS STEP IS CRUCIAL!
Look closely to make sure you have a film of
solution across the ring.
(Similar to soapy film when you blow bubbles)
ADD PLASMID TO + TUBE
DO NOT ADD PLASMID
TO - TUBE
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GET YOUR RACK ON ICE!
10
minutes!
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WHILE YOUR TUBES COOL
LABEL YOUR PLATES
UPSIDE DOWN AND WRITE LABELS ON BOTTOM
… NOT ON TOP!
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SHOCKING INCREASES UPTAKE OF FOREIGN DNA
(PLASMID)
OSMOTIC SHOCK =Transforming solution
CaCl2
HEAT SHOCK
RAPID TEMPERATURE CHANGE is the key
50 SECONDS!! 2 MINUTES
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•Place foam rack with + and – tubes on desktop
•Use new sterile pipette to add 250 µL LB (broth) to + tube
•Use new sterile pipette to add 250 µL LB (broth) to – tube
• Incubate at ROOM TEMPERATURE 10 min
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TAP WITH FINGER TO
MIX!
Use NEW STERILE
pipette for each vial
to add 100 µL
bacterial suspension
to CORRECT DISH
(CHECK LABELS!)
Use a NEW STERILE
LOOP FOR EACH PLATE
to spread suspension
evenly on surface of plate
QUICKLY REPLACE LIDS
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FLIP PLATES UPSIDE DOWN
STACK AND TAPE
LABEL WITH YOUR GROUP NAME
PLACE IN INCUBATOR
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The transformation solution
CaCl2
It is thought that the Ca2+
cation neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane to allow the DNA to enter the cells.
Ca++
Ca++
OCH2
O
P O
O
OBase
CH2
O
P
O
O
O
Base
OH
Sugar
Sugar
OCa++
REASONS FOR EACH TRANSFORMATION STEP
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Incubation on ice slows fluid cell
membranes
Heat-shock increases permeability of
cell membrane
Nutrient broth incubation allows
beta lactamase expression
Reasons for Each Transformation Step
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SELECTION FOR PLASMID UPTAKE
Antibiotic becomes a selecting agent
only bacteria with the plasmid will grow on
antibiotic (ampicillin) plate
LB/amp plateLB plate
all bacteria grow
only transformed
bacteria grow
a
a
a aa
a
aa
aa
aa
aa
a
cloning
a a
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Transformation Results
All cells grow since
there is no antibiotic
on the plate
LB PLATE
Luria Broth
+
- PGLO = NO Plasmid
→
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Transformation Results
NO GROWTH
Cells without plasmid don’t have
antibiotic resistance. Can’t grow
on media with antibiotic added.
LB/AMP PLATE
Luria Broth with antibiotic
+
- PGLO = NO plasmid
→
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only bacteria that have acquired the plasmid can grow on the plate. Therefore, as long as you grow the bacteria in ampicillin, it will need the plasmid to survive and it will continually replicate it, along with your gene of interest that has been inserted to the plasmid.
Selective Pressure - The same as
in evolution - only the organisms
that have a particular trait
(in this case antibiotic resistance)
will survive.
How does ampicillin in the agar act as a “selective pressure”?
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Transformation Results
LAWN
Cells with plasmid have antibiotic
resistance gene so can grow on
media with antibiotic
LB/AMP PLATE
Luria Broth with antibiotic
+
+ PGLO = Plasmid added
→
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Transformation Results
Cells with pGLO plasmid
GROW & GLOW
-can grow on media with
antibiotic
GLOW on media with
arabinose (turns on GFP gene)
LB/AMP/ARA PLATE
Luria Broth
+ antibiotic|
+ arabinose
+
+ PGLO = Plasmid added
→