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PGLO TRANSFORMATION LAB
(AP LAB 7)
BIO-RAD lab book
pGLO ori
bla
GFP
araC
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
BACKGROUND
OLD CENTRAL DOGMA
OF MOLECULAR
BIOLOGY
WHAT IS TRANSFORMATION?
Uptake of foreign DNA plasmid – that
produces new traits in the bacteria
Bacterial
chromosomal DNA
pGLO plasmids
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.
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.
WHAT IS THE GFP GENE?
GFP is a green fluorescent protein that naturally occurs in some bioluminescent jellyfish.
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
Fireflies
Glow worm and glow worm cave
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)
pGLO ori
bla
GFP
araC For example: transformed bacteria can be used to make insulin, human growth hormone, and clotting factor cheaply and in great abundance.
Jellyfish Gene put into Other Critters
http://www.technologyreview.com/files/21291/monkey_x600.jpg
ALBA – BUNNY CREATED FOR “ART”
•http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html
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)
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.
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.)
REAL-WORLD APPLICATION:
The World Health Organization estimates that 300-500 million cases of malaria
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.
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”
BACTERIAL TRANSFORMATION
Plasmids
Chromosomal
DNA
Bacterial Cell
The uptake of plasmid DNA
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.
Aequorea victoria Source of “glowing gene” for this experiment
source of GFP
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 system
pGLO ori
bla
GFP
araC Ori-
Plasmid
Replication
genes
PBAD arabinose promoter
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
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
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
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!
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
normal (have NOT
been transformed)
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.
WHAT SHOULD YOU EXPECT?
If your technique is good, you should expect to see green glowing bacteria in some plates and not others.
Read the transformation lab
procedure and answer
questions 1 – 4 for Lesson 1.
protocol
BACTERIAL TRANSFORMATION
MAKE OBSERVATIONS!
Be sure to make observation and answer questions on pg.
30 of your packet – 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.
Label Tubes
+ pGLO - pGLO
Gather Supplies - 10 groups
What do these
represent?
Use sterile pipette to
add 250µL transformation
solution to pGLO + and – tubes
Transformation
solution (CaCl2)
WHEN USING THE PIPETTE FOR
MEASUREMENTS TAKE INTO ACCOUNT THE
FOLLOWING GRADUATIONS
GET YOUR RACK ON ICE!
INNOCULATE TUBES WITH
E. COLI BACTERIA
Pick one colony
Twirl loop in +pGLO tube
USE SPECIAL
GARBAGE BAG FOR
DISPOSAL OF USED LOOPS
Get new loop
Pick one colony
Twirl loop in –pGLO tube
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
USED AS A GENETIC TOOL http://www.mshri.on.ca/nagy/GFP%20mice.jpg
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
GET YOUR RACK ON ICE!
10
minutes!
WHILE YOUR TUBES COOL
LABEL YOUR PLATES
UPSIDE DOWN AND WRITE LABELS ON BOTTOM
… NOT ON TOP!
SHOCKING INCREASES UPTAKE OF FOREIGN DNA
(PLASMID)
OSMOTIC SHOCK =Transforming solution
CaCl2
HEAT SHOCK
RAPID TEMPERATURE CHANGE is the key
50 SECONDS!! 2 MINUTES
•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
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
FLIP PLATES UPSIDE DOWN
STACK AND TAPE
LABEL WITH YOUR GROUP NAME
PLACE IN INCUBATOR
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++
O CH
2
O
P O
O
O Base
CH2
O
P
O
O
O
Base
OH
Sugar
Sugar
O Ca++
REASONS FOR EACH TRANSFORMATION STEP
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
SELECTION FOR PLASMID UPTAKE
Antibiotic becomes a selecting agent
only bacteria with the plasmid will grow on
antibiotic (ampicillin) plate
LB/amp plate LB plate
all bacteria grow
only transformed
bacteria grow
a
a
a a a
a
a a
a a
a a
a a
a
cloning
a a
Transformation Results
All cells grow since
there is no antibiotic
on the plate
LB PLATE
Luria Broth
+
- PGLO = NO Plasmid
→
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
→
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”?
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
→
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
→
the operon
GENE EXPRESSION IN PROKARYOTES (DIFFERENT IN EUKARYOTES)
GENE REGULATION REVIEW
Organisms regulate expression of their genes and ultimately the amounts and kinds of proteins present within their cells for a myriad of reasons.
Gene regulation not only allows for adaptation to differing conditions, but also prevents wasteful overproduction of unneeded proteins which would put the organism at a competitive disadvantage.
The genes involved in the transport and breakdown (catabolism) of food are good examples of highly regulated genes. For example, the sugar arabinose is both a source of energy and a source of carbon.
E. coli bacteria produce three enzymes (proteins) needed to digest arabinose as a food source. The genes which code for these enzymes are not expressed when arabinose is absent, but they are expressed when arabinose is present in their environment.
Regulation of the expression of proteins often occurs at the
level of transcription from DNA into RNA.
This regulation takes place at a very specific location on the
DNA template, called a promoter, where RNA polymerase
sits down on the DNA and begins transcription of the gene.
In bacteria, groups of related genes are often clustered
together and transcribed into RNA from one promoter.
These clusters of genes controlled by a single promoter are
called operons.
The regulation of gene expression is a
complicated and varied process. One,
more well understood process is the
operon.
An operon is made up of several structural
genes3 arranged under a common
promoter1 and regulated by a common
operator2.
It is defined as “a set of adjacent structural
genes, plus the adjacent regulatory signals
that affect transcription of the structural genes.”
operon
3 1 2
Genes are transcribed together into a mRNA strand and
either translated together, or undergo trans-splicing to
create several strands of mRNA that each encode a single
gene product translated separately.
The result of this is that the genes contained in the
operon are either expressed together or not at all.
BASICS OF TRANSCRIPTION AND TRANSLATION
Transcription is the synthesis of RNA under the direction of DNA
Transcription produces messenger RNA (mRNA)
Translation is the synthesis of a polypeptide, which occurs under the direction of mRNA
Ribosomes are the sites of translation
THE LAC OPERON
The first operon to
be described was
the lac operon in
Escherichia coli.
Provides a typical example of operon function. It consists of
three adjacent structural genes, a promoter, a terminator,
and an operator.
The Lac operon - showing its genes and its binding sites.
The lac operon is required for the
transport and metabolism of lactose in
Escherichia coli and some other enteric
bacteria.
The lac operon is
regulated by several
factors including
availability of glucose and
lactose. (This is an example of the
negative inducible model.)
In the "repressed" state, the repressor IS bound to the operator.
RNA
polymerase
repressor
promoter operator
The gene is essentially turned off.
There is no lactose to inhibit the
repressor, so the repressor binds to
the operator, which obstructs the
RNA polymerase from binding to the
promoter and making lactase. gene sequence for lactase production
The gene is turned on. Lactose is
inhibiting the repressor, allowing
the RNA polymerase to bind with
the promoter, and express the
genes, which synthesize lactase.
Eventually, the lactase will digest
all of the lactose, until there is
none to bind to the repressor. The
repressor will then bind to the
operator, stopping the
manufacture of lactase
off
on
mRNA
ribosome polypeptide
Control of an operon is a type of gene regulation that
enables organisms to regulate the expression of various
genes depending on environmental conditions.
Operon regulation can be either negative or positive by
induction or repression.
APPLICATIONS IN THE “REAL WORLD”
THE PROMOTER
A nucleotide sequence that enables a gene to be
transcribed.
The promoter is recognized by RNA polymerase,
which then initiates transcription. In RNA
synthesis, promoters indicate which genes should
be used for mRNA creation – and, by extension,
control which proteins the cell manufactures.
A segment of DNA that a regulator binds to. It
is classically defined in the lac operon as a
segment between the promoter and the genes
of the operon.
THE OPERATOR
THE REPRESSOR
In the case of a repressor, the repressor
protein physically obstructs the RNA
polymerase from transcribing the genes.
ARABINOSE OPERON The three genes (araB, araA and araD) that code for three digestive
enzymes involved in the breakdown of arabinose are clustered together in what is known as the arabinose operon.3 These three proteins are dependent on initiation of transcription from a single promoter, PBAD.
Transcription of these three genes requires the simultaneous presence of the DNA template (promoter and operon), RNA polymerase, a DNA binding protein called araC and arabinose. araC binds to the DNA at the binding site for the RNA polymerase (the beginning of the arabinose operon).
When arabinose is present in the environment, bacteria take it up. Once inside, the arabinose interacts directly with araC which is bound to the DNA.
The interaction causes araC to change its shape which in turn promotes (actually helps) the binding of RNA polymerase and the three genes araB, A and D, are transcribed.
Three enzymes are produced, they break down arabinose, and eventually the arabinose runs out.
In the absence of arabinose the araC returns to its original shape and transcription is shut off.
PGLO RECOMBINANT DNA The DNA code of the pGLO plasmid has been engineered to
incorporate aspects of the arabinose operon. Both the promoter (PBAD) and the araC gene are present. However, the genes which code for arabinose catabolism, araB, A and D, have been replaced by the single gene which codes for GFP.
Therefore, in the presence of arabinose, araC protein promotes the binding of RNA polymerase and GFP is produced. Cells fluoresce brilliant green as they produce more and more GFP.
In the absence of arabinose, araC no longer facilitates the binding of RNA polymerase and the GFP gene is not transcribed. When GFP is not made, bacteria colonies will appear to have a wild-type (natural) phenotype—of white colonies with no fluorescence.
This is an excellent example of the central molecular framework of biology in action: DNA➜RNA➜PROTEIN➜TRAIT.
The pGLO plasmid, which contains the GFP gene, also contains the gene for beta-lactamase, which provides resistance to the antibiotic ampicillin, a member of the penicillin family. Beta-lactarmase inactivates the ampicillin present in the LB nutrient agar to allow bacterial growth. Only transformed bacteria that contain the plasmid and express beta-lactamase can grow on plates that contain ampicillin.
TRANSFERRING BACTERIAL COLONIES
FROM AGAR PLATES TO MICROTUBES
The process of scraping a single colony off the starter plate leads to the temptation to get more cells than needed.
A single colony that is approximately 1 mm in diameter contains millions of bacterial cells.
To increase transformation efficiency, students should select 2-4 colonies that are 1-1.5 mm in diameter. Selecting more than 4 colonies may decrease transformation efficiency.
Select individual colonies rather than a swab of bacteria from the dense portion of the plate, the bacteria must be actively growing for transformation to be successful.
PLASMID DNA TRANSFER
The transfer of plasmid DNA from its stock
tube to the transformation suspension is
crucial. When dipping the inoculating loop
into the container, you must look carefully at
the loop to see if there is a film of plasmid
solution across the ring, similar to film on
wand when blowing bubbles.
HEAT SHOCK
Since the heat shock increases the permeability of the cell membrane to DNA, it is very important to follow the directions regarding time in the warm bath and the rapid temperature change.
While the mechanism is not known, the duration of the heat shock is critical and has been optimized for the type of bacteria used and the transformation conditions employed.
For optimal results, the tubes containing the cell suspension must be taken directly from ice, placed into the water bath at 42°C for 50 sec and returned immediately to the ice.
RECOVERY
The 10 minutes incubation period following
the addition of LB nutrient broth allows the
cells to recover and to express the ampicillin
resistance protein beta-lactamase so that
the transformed cells survive on the
ampicillin selection plates.