Download Transformation

Name:
Section:
Introductory Microbiology
Transformation
Prelab Assignment
1. (1pt.) Define transformation.
2. (0.5pt.) Who first discovered transformation?
3. (2pt.) List four factors that greatly increase the efficiency of transformation in the lab.
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4. (1pt.) List the two genes we will be examining in this lab AND the functions of the proteins
they code for.
5. (0.5pt.) What is the name of the plasmid which carries the two genes above (#4). We will use
this plasmid to transform our E. coli strain.
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Lab Exercise
Transformation
Oct. 28, 2014
Bacterial transformation is the process whereby naked (cell-free or free) DNA from a
donor cell is absorbed by a recipient cell. If the absorbed DNA is from a chromosome, it is
incorporated into the recipient's chromosome (recombination). If the absorbed DNA is a
plasmid, it will remain in the cytoplasm. When this DNA uptake occurs, the donor's genes are
expressed, and donor polypeptides are synthesized.
Transformation was discovered in 1928 by the British scientist Frederick Griffith. While
working with encapsulated and unencapsulated strains of Streptococcus pneumoniae, he noticed
that when heat-killed encapsulated S. pneumoniae and living unencapsulated strains were
injected into mice, the mice died. When he isolated living bacteria from the dead mice, they had
a capsule. The unencapsulated strain had been transformed! In 1944, a research team led by
Oswald Avery at the Rockefeller Institute in New York isolated the transforming substanceDNA. In today's genetic engineering labs, transformation is a common research procedure.
Transformation is rare in nature; most bacteria do not possess the natural ability to uptake
and express naked, foreign DNA. However, in 1970, M. Mandel and A. Higa discovered certain
factors which greatly increase the efficiency of transformation in the lab: 1) treatment with a cold
solution of calcium chloride, 2) a brief "heat shock" at 42C, 3) performing the experiment when
the cells are at the log phase of growth, and 4) using small, circular plasmids. However, the
exact mechanism of DNA uptake is still unclear.
In this experiment, students will transform the bacterium E. coli with a gene that codes
for Green Fluorescent Protein (GFP). The real-life source of this gene is the bioluminescent
jellyfish Aequorea victoria. GFP causes the jellyfish to fluoresce and 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
ultraviolet light.
The unique pGLO plasmid will be utilized in this lab. Two genes with identifiable
phenotypes are on this plasmid. The recipient E. coli strain has neither of these genes.
1) GFP gene – codes for Green Fluorescent Protein. When bacteria that
have been transformed are grown in the presence of the sugar arabinose,
the GFP gene is “turned on” and the bacteria glow under U.V. light.
2) bla gene – codes for the protein beta-lactamase, which provides resistance to
the antibiotic ampicillin.
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A fluorescent green colony phenotype is easy to recognize, but how will we know if the
bla gene also was incorporated? Agar containing ampicillin will be our selective medium. Only
transformed cells with the bla gene, and, therefore, ampicillin resistant, will grow. The
untransformed original E. coli strain will not survive and form colonies on this type of media.
Materials: per group of 4
1 vial or tube containing 3 ml calcium chloride solution in a 500ml beaker containing
chipped ice.
E. coli culture on LB (Luria) agar
1 vial of pGLO plasmid (may share among all groups)
1 vial or tube containing 3 ml LB broth
2 sterile 8 ml test tubes with cap
Five 1ml pipets, pipet pump (blue)
100 ml 95% ethanol in jar with screw top lid
Glass bactispreaders
Burners and striker
Water bath at 42C containing a test tube rack
Wire inoculating loop
Plastic, disposable inoculating loop
2 plain LB agar plates
2 LB/Amp agar plates
2 LB/Amp/Ara agar plates
Gloves, safety glasses
Biohazard bag
Students will suspend untransformed E. coli cells in two tubes containing cold calcium
chloride solutions. One of these tubes will serve as our control; plasmid will not be added to the
control tube. In the other tube, the experimental tube, pGLO plasmid will be added. Both tubes
will then be incubated on ice for 10 minutes. A brief "heat shock" at 42C will follow. The cells
are then cooled and broth added. Samples will be plated on three types of media: 1) plain LB
agar, 2) LB agar with ampicillin (LB/Amp agar), and 3) LB/Amp agar that also contains
arabinose (LB/Amp/Ara agar). After incubation, bacterial growth and the appearance of
fluorescent green colonies under U.V. light will be checked.
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Day 1
Procedure:
1. Label one sterile 8 ml tube "+ pGLO". Label another sterile 8 ml tube "- pGLO".
2. Using a sterile pipet, add 0.25 ml of ice-cold calcium chloride to each tube.
3. Immediately, place both tubes on ice.
4. After flaming the wire loop, transfer one or two large colonies of E. coli to the + pGLO tube
containing calcium chloride.
-BE CAREFUL NOT TO TRANSFER ANY AGAR FROM THE PLATE TO THE TUBE.
AGAR CAN INHIBIT THE TRANSFORMATION PROCESS.
-IMMERSE THE LOOP IN THE CALCIUM CHLORIDE SOLUTION, AND VIGOROUSLY
TAP AGAINST WALL OF TUBE TO DISLODGE CELL MASS.
-MAKE SURE THAT THE CELL MASS IS NO LONGER ON THE LOOP.
5. IMMEDIATELY suspend cells in the + pGLO tube by CAREFULLY stirring the solution with
the loop. Do not produce bubbles or splash the solution up against the walls of the tube. Hold
the tube up to the light to be sure the solution is uniform. No visible clumps of cells should
remain in the tube.
6. Return the + pGLO tube to the ice.
7. Using a flamed loop, transfer a second bacterial mass from the plate to the - pGLO tube and
suspend as described in step 5. Return the - pGLO tube to the ice.
8. Both tubes should be on ice.
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9. If possible, keep the cell suspension in the ice while performing this step. Use a plastic,
disposable inoculating loop to transfer ONE loopful (~10μl) of the pGLO plasmid solution to
the + pGLO tube.
The plasmid solution should form a "bubble" on the loop. Immerse the loop directly into the E.
coli cell suspension, and swish the loop to disperse the plasmid.
When finished, discard the plastic inoculating loop in the biohazard bag.
10. Incubate both tubes on ice for an additional 10 minutes. Go to Step 11 while the tubes are
incubating in the ice.
11. Observe the following plates and label if necessary.
a) Plate A. "Amp +". This is an experimental plate containing transformed cells.
b) Plate B. “Amp -". This is a negative control plate. It will NOT contain transformed
cells.
c) Plate C. “Amp/Ara +". This is an experimental plate. It will contain transformed cells.
d) Plate D. “Amp/Ara -". This is a negative control plate. It will NOT contain
transformed cells.
e) Plate E. “LB experimental control +". This is a positive, experimental control plate. It
will contain transformed cells.
f) Plate F. “LB negative control -". It will NOT contain transformed cells.
12. Following the 10 minute incubation on ice, "heat shock" the cells. Carry the tubes STILL
ON ICE to the 42C water bath. Remove both tubes directly from the ice and immediately
immerse them in the water bath for 50 seconds.
13. Return both tubes directly to the ice for two minutes.
14. Add 0.25 ml (250 μl) of LB broth to each tube, using a new sterile pipet with each tube.
Gently tap tubes with finger to mix and set tubes in a test tube rack at room temperature for 10
minutes.
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For Steps 15-20 (next page), follow the inoculation procedure in the illustration
below.
EXPERIMENTAL
CONTROL
+ pGLO tube
E.coli + Plasmid (Transformed)
Plate A
LB/Amp
Plate C
LB/Amp/Ara
- pGLO tube
E. coli only (Untransformed)
Plate E
LB
Plate B
LB/Amp
Plate D
LB/Amp/Ara
Plate F
LB
0.1ml into each plate
0.1ml into each plate
IMPORTANT! Use two different pipetsOne for the + pGLO tube and a different one for the – pGLO tube. DO NOT MIX
THEM.
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15. Use a sterile pipet to transfer 0.1 ml (100μl) from the + pGLO tube to
each of plates A, C, and E.
16. Procedure for alcohol sterilization of glass bactispreaders :
a. Dip the glass bactispreader into the ethanol (in beaker). DO NOT ALLOW THE
ETHANOL TO DRIP DOWN THE HANDLE OF THE BACTISPREADER!!!
b. BRIEFLY pass the bactispreader through the flame.
c. Immediately move the flaming bactispreader away from the flame and the
ethanol.
d. Allow the flame to burn out.
e. Rub the bactispreader on an area of the agar away from the bacteria to cool.
f. Spread the cells over the agar of Plate A.
g. Flame the bactispreader again.
17. Repeat step 16 for Plates C and E.
18. Use a different sterile pipet to add 0.1 ml (100 μl) from - pGLO tube to
each of Plates B, D, and F.
19. Repeat Step 16 for Plates B, D, and F
20. Allow the plates to set for a few minutes; then wrap each group’s plates together with tape.
21. Place plates inverted in the 37C incubator for 48 hours.
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Day 2
Observe the plates for growth under normal room lighting and under U.V. light (Long Wave).
WEAR SAFETY GLASSES WHEN VIEWING UNDER U.V. LIGHT!
Expected Results:
1) Plate A:
2) Plate B:
3) Plate C:
4) Plate D:
5) Plate E:
6) Plate F:
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Name:
Section:
Introductory Microbiology
Lab Report Transformation
1. (1pt.) Expected Results:
Plate A:
Plate B:
Plate C:
Plate D:
Plate E:
Plate F:
-Actual Experimental Results:
Plate A:
Plate B:
Plate C:
Plate D:
Plate E:
Plate F:
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2. (1pt.) Some of the colonies are expected to be fluorescent green under U.V. light. In detail,
explain why.
3. (1pt.) Describe the ingredients in the selective medium used in this lab. In what way is it
"selective"?
4. (1pt.) Which plates should be compared to determine if any genetic transformation has
occurred? Why?
5. (1pt.) In detail, explain why we included Plates E and Plate F in the experiment?
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