May 31, 2007
Genetic Engineering: Bacterial Transformation
What are bacteria?
Bacteria are single
celled microorganisms with a relatively simple cell structure. They do
not contain a nucleus with linear chromosomes. Instead, bacteria have a singl
e, large, circular
chromosome whose genes are generally essential to the life and reproduction of the bacterium.
contain smaller, circular pieces of DNA called
. The genes on plasmids
usually are not essential to the life and reprod
uction of the cell. Instead, the genes on plasmids
provide some advantage to the cells under specific environmental circumstances. Many strains of
bacteria pass plasmids from one bacterial cell to another. There are a variety of mechanisms by
ria exchange plasmids, one of which, transformation, will be performed in this
What is transformation?
Bacterial transformation is a process whereby cell
free DNA (found outside of bacterial
cell structures in the surrounding solution) is absor
bed by a living bacterial cell. When this DNA
uptake occurs, the genes may be expressed, i.e., new proteins synthesized. This will alter the
traits of the bacterium.
Transformation is rare in nature; most bacteria do not possess the natural ability to u
and express cell
free, 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 growing very rapidly, and 4) transforming with
these advances, the exact mechanism of DNA uptake is still unclear.
In this experiment, students will transform t
with a gene that codes for
Green Fluorescent Protein (GFP). The real
life source of this gene is the bioluminescent
GFP causes the jellyfish to fluoresce and glow in the dark.
Following the transformatio
n procedure, the bacteria express their newly acquired jellyfish
gene and produce the fluorescent protein causing them to glow a brilliant green color under
GLO plasmid will be utilized in this lab. Two genes with identifia
are on this plasmid. The
strain has neither of these genes.
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.
codes for the protein
lactamase, which provides resistance to
the antibiotic ampicillin.
A fluorescent green colony phenotype is easy to recognize, but how will we know if the
gene also was incorp
orated? Agar containing ampicillin will be our
transformed cells with the
gene, and, therefore,
, will grow. The
strain will not survive and form colonies on this type of medi
ABSOLUTELY NO FOOD OR DRINK IS ALLOWED IN THE LAB.
Gloves, aprons, and safety glasses must be worn at all times.
Dispose of pipets as instructed.
Dispose of all waste in biohazard bags.
Begin by cleaning the lab benches with the cleaning solution
and washing your hands.
per lab bench
1 tube containing 3
ml calcium chloride solution in a 500
ml beaker containing
culture on agar
1 vial of
GLO plasmid (may share among all groups)
1 vial or tube containing 3
2 sterile 8
ml test tubes with cap
ml pipets, pipet pump (blue)
Water bath at 42
C containing a test tube rack
5 disposable inoculating loops
Gloves, safety glasses
Ethyl alcohol in jars with lids and Bactispreaders (one/lab bench), Bunsen burners,
Students will suspend
cells in two tubes containing cold calcium
chloride solutions. One of these tubes will serv
e as our
; plasmid will not be added to the
control tube. In the other tube, the
GLO 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
cooled and broth added. Samples will be plated on three types of media: 1) plain
agar with ampicillin (Amp agar), and 3)
Amp agar that also contains arabinose
Amp/Ara agar). After incubation, bacterial growth and the appearance of
colonies under U.V. light will be checked.
1. Label one sterile 8
ml tube "+
". Label another sterile 8
ml tube "
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. IN THIS STEP, BE CAREFUL NOT TO TRANSFER ANY AGAR FROM THE PLATE TO
THE TUBE. AGAR CAN INHIBIT THE TRANSFORMATION PROCESS.
Do not puncture
the agar; carefully swipe the
of the agar to remove bacteri
Using an inoculating loop
transfer one or two large colonies of
containing calcium chloride.
IMMERSE THE LOOP
with the bacteria
IN THE CALCIUM CHLORIDE SOLUTION, AND
VIGOROUSLY TAP AGAINST WALL OF TUBE TO DISLODGE CELL MA
MAKE SURE THAT THE CELL MASS IS NO LONGER ON THE LOOP.
suspend cells in the
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
e up to the light to be sure the solution is uniform. No visible clumps of cells should
remain in the tube.
6. Return the
tube to the ice.
7. Again, use the loop to transfer another
from the plate to the
as described in step 5. Return the
tube to the ice.
8. Both tubes should be on ice.
9. If possible, keep the cell suspension in the ice while performing this step. Use a clean,
inoculating loop to transfer ONE
ion to the
The plasmid solution should form a "bubble" on the loop. Immerse the loop
cell suspension, and swish the loop
to disperse the plasmid.
When finished, discard the plastic inoculating loo
p 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.
. This is an
e containing transformed cells
. This is a
control plate. It will NOT contain transformed
, only the control cells (from the
GLO tube) that did not receive the plasmid
This is an
from the +
This is a
control plate. It will NOT contain
transformed cells, only the control cells (from the
GLO tube) that did not receive the
This is an
transformed cells from the +
This is a control plate. It will NO
only the control cells (fr
tube) that did not receive the
12. Following the 10 minute incubation on ice,
the cells. Carry the tubes STILL
ON ICE to the 42
C water bath. Remove both tubes
directly from the ice and immediately
them in the
water bath for
13. Return both tubes directly to the ice for two minutes.
14. Add 0.25
to each tube, using a new, sterile p
ipet with each tube. Gently tap the
bottom of the
tubes with finger to mix and set tubes in a test tube r
room temperature for
For Steps 15
(next page), follow the inoculation procedure in the illustration
IMPORTANT! Use different
for transferring +
E.coli + Plasmid (Transformed)
E. coli only (Untransformed)
ne for the
GLO tube and a different one for the
GLO tube. DO NOT MIX THEM.
. Observe the following precautions while transferring the bacteria to the plates.
Only lift the lids off of the plates when ready to trans
Do not set the plate
on the bench; hold them.
Do not puncture the agar with the
Replace the lid as soon as you have transferred the solution.
16. See illustration on previous page: Pipet 0.1
ml of +
bacteria to the
SURFACE of the
not to puncture the agar with the loop)
17. Repeat step 16 with plates
C and E
of bacteria from the same tube,
GLO, to plates C and E).
0.1 ml of
each of plates B, D, and F
19. Procedure for alcohol sterilization of glass bactispreaders :
a. Dip the glass bactispreader into the ethanol (in
). DO NOT ALLOW THE
ETHANOL TO DRIP DOWN THE HANDLE OF THE BACTISPREADER!!!
BRIEFLY pass the bactispreader through the flame.
Immediately move the
bactispreader away from the flame and the
d. Allow the flame to burn out. Cool for
e. Rub the bactispreader on an area of the agar
e bacteria to cool.
ead the cells over the agar of P
g. Flame the bactispreader again.
20. Repeat step 19
the rest of the plates. Flame and cool between each plate and after you are
. Allow the plates to set for a few
minutes, then wrap each group’s plates together with tape
. Place plates
in the 37
C incubator for
1) Plate A:
2) Plate B:
3) Plate C:
4) Plate D:
5) Plate E:
6) Plate F:
Observe the plates for growth under normal room lighting and under U.V. light
WEAR SAFETY GLASSES WHEN VIEWING UNDER U.V. LIGHT!
Genes: The Foundations of Life Laboratory
2. What are bacteria?
3. Define transformation.
4. List the two genes that were transferred in this lab AND their functions.
5. Which plates should be compared to determine if any genetic transformation has occurred?
6. In detail, explain why we included Plates E and Plate F in the experiment?