Download pGLO2011 Wilkes

Bacterial Transformation
Using the pGLO plasmid
PA Standards:
PA S. T. & E. Standards:
3.1.10.B1. Describe how genetic information is inherited and expressed.
3.1.10.B3. Describe the basic structure of DNA and its function in genetic inheritance.
3.1.B.B4. Explain how genetic technologies have impacted the fields of medicine, forensics, and
agriculture.
3.1.12.B3. Analyze gene expression at the molecular level.
Introduction:
In 1928, Fredrick Griffith witnessed a miraculous event, a transformation in the
literal sense of the word. During the course of an experiment, a living organism had
changed in physical form. The virulent bacteria, isolated from a mouse that had died from
a pneumonic infection, appeared different from the ones injected into a healthy animal
two days prior.
At the start of the experiment, Griffith had injected the mouse with a mixture of a
heat killed smooth (S) strain of pneumococcus bacteria and a living but nonvirulent rough
(R) strain. The smooth polysaccharide capsule of the S strain is essential for infection; the
R strain, which appears rough, lacks the polysaccharide capsule and thus is incapable of
infection. When injected alone, neither the heat-killed S strain nor the living R strain
caused infection in the mouse, but co-injection of the two strains killed it. Griffith had
isolated the S strain from the dead mouse.
Griffith hypothesized that some transforming principle was transferred from the
heat-killed S bacteria to the R bacteria that converted it to a virulent state.
Transformation appeared to be a genetic phenomenon. This association was strengthened
by the one gene-one enzyme hypothesis proposed by George Beadle and Edward Tatum
in 1940; according to this hypothesis, the transforming principle involved one or more
genes that produced enzymes needed to synthesize the polysaccharide coat. In 1944 a
team of researchers at the Rockefeller Institute, headed by Oswald Avery, purified the
transforming principle from pneumococcus. Biochemical tests revealed it to be
deoxyribonucleic acid (DNA). Taken together, all this evidence pointed to DNA as the
components of genes.
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The phenomenon of transformation, which provided a key clue to understanding
the molecular basis of the gene, also provided a tool for manipulating the genetic makeup
of living organisms. To a large extent, genetic engineering relies on adding relatively
short segments of DNA containing a foreign or modified gene to living cells.
Transformation- the uptake and expression of DNA by a living cell is the limiting factor
in the genetic engineering of any species. Genes can be cut from human, animal, or plant
DNA and placed inside bacteria. For example, a healthy human gene for the hormone
insulin can be put into bacteria. Under the right conditions, these bacteria can make
authentic human insulin. This insulin can then be used to treat patients with the genetic
disease diabetes, whose insulin genes do not function normally.
In this lab you will be transforming the bacteria Escherichia coil with a gene that codes
for Green Fluorescent Protein (GFP). The real-life source of this gene is the bioluminescent
jellyfish Acquorea victoria. The gene codes for Green Fluorescent Protein, which causes the
jellyfish to glow in the dark. Following the transformation procedure, the bacteria express their
newly acquired jellyfish gene and produce the fluorescent protein, which causes them to glow a
brilliant green color under UV light.
You will be moving the gene for GFP into the E. Coli with the plasmid pGLO. This
plasmid encodes the gene for GFP and a gene for resistance to the antibiotic ampicillin. The gene
for GFP can be turned on in transformed cells by adding the sugar arabinose, to the cells nutrient
medium. Selection for cells that have been transformed with the pGLO DNA is accomplished by
growth on antibiotic plates. Transformed cells will appear white (wild type phenotype) on plates
not containing arabinose and fluorescent green when arabinose is included in the nutrient agar.
Safety Notes:
1. Follow safety sterile techniques for use with microbes: clean table with
disinfectant solution at beginning and end of experiment. Take care to
carefully open and close Petri dishes, microtubes, etc. to minimize the
possibility of contamination.
2. Pipette tips or sterile loops should not come into contact with any
contaminating surfaces like table tops.
3. Wash hands carefully at the beginning and end of each lab period.
4. Wear safety glasses and gloves.
5. Dispose of all bacteria plates, inoculating loops, and materials that have come
into contact with bacteria in a biohazard bag.
6. Take care to not look directly at the long wave ultraviolet light. The lighted
part should be placed on the bottom directly over the bacterial plates with the
plastic unlit surface on top.
Vocabulary:
1. Transformation is the process that occurs when a cell takes up and expresses a
new piece of genetic material- DNA.
2. Transforming principle (DNA) is the master molecule that contains genetic
information that controls the traits of all living organisms and passes it from one
generation to the next.
3. A Plasmid is a small, circular piece of DNA that is naturally found in bacteria.
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4. Ampicillin is an antibiotic to which some bacteria are resistant due to genes they
possess.
5. The Phenotype is the appearance or expression of a gene, such as a green glow
under ultraviolet light.
6. Arabinose is a sugar that is a soruce of energy and carboin for bacteria. When
this sugar is present, certain genes are “turned on.”
7. Genetic engineering is the deliberate, controlled manipulation of the genes in an
organism, with the intent of making that organism better in some way or to make
useful products, such as human insulin.
8. Bioluminescence is the production and emission of visible light by a living
organism as the result of a chemical reaction during which chemical energy is
converted to light energy. It can also be seen as a green glow under ultraviolet
light.
Objective:
1. To demonstrate transformation with an antibiotic-resistant gene.
Materials: (per group)
1 starter plate of E. Coli K-12 strain: HB1O1 or HB 294
1 vial pGLO plasmid (on ice), 1 stock tube per class
1 LB agar plate
2 LB/amp. agar plates
1 LB/amp/ara agar plate
1 vial sterile transformation buffer (CaCI. soln)
1 vial LB broth
2 sterile microtubes
7 sterile inoculation loops
roll of tape
1 stop watch per group
5 sterile pipettes
Biohazard bag, 1 per class
1 foam microtube holder
1 styrofoam cup of crushed ice
1 circular floating dish with holes for microtubes for water bath.
1 marking pen or wax pencil
UV lamp, 1 per class
water bath, 1 per class
incubator, 1 per class
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Procedure: (day 1)
1. Label one closed microtube + DNA and another DNA. Label both tubes with your
group’s name. Place them in the foam tube rack.
2. Open the tubes and, using a sterile transfer pipette, transfer 250 µL of transformation
solution into each tube.
3. Place the tubes on ice.
4. Use a sterile loop to pick up one single colony (spot) of bacteria from your starter
plate. Be sure you can actually see that you have picked up a colony. Pick up the + DNA
tube and immerse the loop into the transformation solution at the bottom of the tube. Be
sure that you have actually transferred the colony to the tube. Spin the loop between your
index finger and thumb until the entire colony is dispersed in the transformation solution
(no floating chunks). Place the tube back in the tube rack in the ice. Using a new sterile
loop, repeat for the DNA tube.
5. Examine the pGLO DNA solution with a UV lamp. Note your observations. Immerse a
new sterile loop into the plasmid DNA stock tube. Withdraw a loop full. There should
be a film of plasmid solution across the ring. This is similar to seeing a soapy film
across a ring for blowing soap bubbles. Mix the loop full into the cell suspension of the
+ DNA tube. Do not add plasmid DNA to the - DNA tube. Why not?
6. Incubate the tubes on ice for 10 minutes. Make sure to push the tubes all the way down
in the rack so the bottoms of the tubes stick out and make contact with the ice.
7. While the tubes are sitting on ice, label your four agar plates on the bottom (not the lid)
as follows:
label one LB/amp plate:
+ DNA
label the LB/amp/ara plate:
+ DNA
label the other LB/amp plate:
- DNA
label the LB plate:
- DNA
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8. Heat shock. Using the foam circle as a holder, transfer both the (+) and (-) tubes into
the water bath set at 42°C for exactly 50 seconds. Use a stop watch to time this process.
Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick
out and make contact with the warm water.
When the 50 seconds are done, place both tubes back on ice. The change from ice to
42°C and then back to ice must be rapid and abrupt. Incubate tubes on ice for 2
minutes.
9. Remove the rack containing the tubes from the ice and place on the bench top. Open a
tube and, using a new sterile pipette, add 250 µL of LB broth to the tube and close it.
Repeat with a new sterile pipette for the other tube. Incubate the tubes for 10 minutes at
room temperature.
10. Tap the tubes with your finger to mix. Using a new sterile pipette for each tube,
pipette 100 µL of the transformation and control suspensions onto the plates as follows:
100 µL from the + DNA tube to each of the two + DNA plates
100 µL from the - DNA tube to each of the two - DNA plates
11. Use a new sterile loop for each plate. Spread the suspensions evenly around the
surface of the agar by quickly skating the flat surface of a new sterile loop back and forth
across the plate surface.
12. Stack the plates and tape them together. Put your group name and class period on the
bottom of the stack and place it upside down in the 37°C incubator until the next day.
Day 1 review questions:
1. On which plate would you expect to find non- transformed bacteria? Explain your
predictions.
2. If there are any genetically transformed bacterial cells, on which plate(s) would they be
most likely located? Explain your predictions.
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3. Which plates should be compared to determine if genetic transformation has occurred?
Why?
Procedure: (day 2)
1. Observe the results you obtained from the transformation lab under normal room
lighting. Then turn out the lights and hold the UV lamp over the plates.
2. In the space below construct a data table of your results and carefully draw what you
see on each of the four plates. Include a description of the bacterial growth, colony color
(room light and UV), and number of bacterial colonies.
Day 2 review questions:
1. From the results that you obtained, how could you prove that these changes that
occurred were due to the procedure that you performed?
2. What factors might influence transformation efficiency?
3. Very often an organism’s traits are caused by a combination of its genes and its
environment. In this case what two factors must be present in the bacteria’s
environment for you to see the green color?
4. What advantage would there be for an organism to be able to turn on or off particular
genes in response to certain conditions?
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Calculation of transformation efficiency:
transformation efficiency = total number of cells growing on the agar plate / amount
of DNA spread on the agar plate
Use the number of colonies on your LB/amp/ara plate as your number of cells. Why does
each colony represent a single cell in this example?
The amount of DNA is determined from the following information: you used 10µL of
pGLO at a concentration of 0.03 µg/µL. You therefore used 0.3mg of DNA. You spread
lOOµL of cells containing DNA from a test tube containing 510 µL total solution. Use
the fraction of DNA actually spread on the LB/amp/ara plate to calculate the
transformation efficiency. Express this number as transformants/µg.
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