Download PGLO Transformation LAB AP LAB 7

PGLO TRANSFORMATION LAB
(AP LAB 7)
araC
ori
pGLO
bla
BIO-RAD lab book
GFP
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.
For example:
transformed bacteria can
be used to make insulin,
human growth hormone,
and clotting factor
cheaply and in great
abundance.
araC
ori
pGLO
bla
GFP
Vector - Something that is used to transfer something else (a mosquito
is a vector for the organism that causes malaria)
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:
Malaria is the
world's most
common and
deadly parasitic
disease.
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.
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
The uptake of plasmid DNA
Bacterial Cell
Chromosomal
DNA
Plasmids
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.
source of GFP
Aequorea victoria
Source of “glowing gene” for this experiment
PGLO
PLASMID
araC
OriPlasmid
Replication
ori
genes
pGLO
bla
GFP
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
PBAD arabinose promoter
GFP-Green Fluorescent Protein
- Glows green in fluorescent light
bla (β-lactamase)
- “on” all the time
- makes protein that breaks down ampicillin
- provides ampicillin resistance
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
ara Operon
araC
B
A
D
Effector
(Arabinose)
araC
B
A
D
RNA Polymerase
araC
B
A
D
INDICIBLE OPERON:
The presence of
arabinsoe turns on
genes that make
enzymes (proteins) to
digest the sugar
arabinose
PGLO
REGULATION
ara Operon
araC
B
A
D
GFP Gene
Effector
(Arabinose)
araC
B
A
D
GFP Gene
RNA Polymerase
araC
B
A
D
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.
Gather Supplies - 10 groups
Label Tubes
+ pGLO
- pGLO
What do these
represent?
Transformation
solution (CaCl2)
Use sterile pipette to
add 250µL transformation
solution to pGLO + and – tubes

WHEN USING THE PIPETTE FOR
MEASUREMENTS TAKE INTO ACCOUNT THE
FOLLOWING GRADUATIONS
GET YOUR RACK ON ICE!
INNOCULATE TUBES WITH
E. COLI BACTERIA
Get new loop
Pick one colony
Twirl loop in –pGLO tube
Pick one colony
Twirl loop in +pGLO tube
USE SPECIAL
GARBAGE BAG FOR
DISPOSAL OF USED LOOPS
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
REASONS FOR EACH TRANSFORMATION STEP
Ca++


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++
O
O P O
O
CH2
Base
O
Sugar
O
Ca++
O P O
Base
O
CH2
O
Sugar
OH
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
SELECTION FOR PLASMID UPTAKE

Antibiotic becomes a selecting agent
 only
bacteria with the plasmid will grow on
antibiotic (ampicillin) plate
all bacteria grow
only transformed
bacteria grow
a
a
a
a
a
a
LB plate
a
a
a
a
a
a
a
a
a
a
a
LB/amp plate
cloning
Transformation Results
LB PLATE
Luria Broth
+
- PGLO = NO Plasmid
→
All cells grow since
there is no antibiotic
on the plate
Transformation Results
LB/AMP PLATE
Luria Broth with antibiotic
+
- PGLO = NO plasmid
→
NO GROWTH
Cells without plasmid don’t have
antibiotic resistance. Can’t grow
on media with antibiotic added.
How does ampicillin in the agar act as a “selective pressure”?

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.

Transformation Results
LB/AMP PLATE
Luria Broth with antibiotic
+
+ PGLO = Plasmid added
→
LAWN
Cells with plasmid have antibiotic
resistance gene so can grow on
media with antibiotic
Transformation Results
LB/AMP/ARA PLATE
Luria Broth
+ antibiotic|
+ arabinose
+
+ PGLO = Plasmid added
Cells with pGLO plasmid
GROW & GLOW
-can grow on media with
antibiotic
GLOW on media with
arabinose (turns on GFP gene)
→
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.
operon
1
3
2
It is defined as “a set of adjacent structural
genes, plus the adjacent regulatory signals
that affect transcription of the structural genes.”
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.
The Lac operon - showing its genes and its binding sites.
Provides a typical example of operon function. It consists of
three adjacent structural genes, a promoter, a terminator,
and an operator.
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.
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.
off
repressor
RNA
polymerase
promoter
operator
gene sequence for lactase production
on
mRNA
polypeptide
ribosome
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
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.
THE OPERATOR
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 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 betalactamase, 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 24 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.