PHENOTYPIC AND GENOTYPIC
CHANGES IN BACTERIA
1993 Woodrow Wilson Biology Institute
The purpose of this series of lab activities is to demonstrate both phenotypic and genotypic changes in bacteria that have been transformed with a gene that codes for antibiotic resistance.
In the first activity, students use a rapid method to render the bacterium Escherichia coli "competent" to uptake plasmid DNA. Escherichia coli cells are then transformed with the pAMP plasmid, which carries the gene coding for resistance to ampicillin. The recipient E. coli strain, MM294, lacks this gene; thus only transformed cells acquire the ability to grow on ampicillin. Ampicillin then is the selective agent in this experiment. E. coli cells are scraped from two large colony starter plates and suspended in two tubes containing a solution of calcium chloride. pAMP plasmid is added to ONE cell suspension, and both tubes are incubated at 0 deg C for 15 minutes. Following a brief heat shock at 42deg C, cooling, and the addition of LB broth, samples of the cell suspension are plated on two types of media: plain LB agar and LB agar with ampicillin. The plates are incubated for 12-24 hours at 37deg C then checked for bacterial growth. Only cells that have been transformed by taking up the plasmid DNA with the ampicillin-resistant gene will grow on the LB/amp agar plates. Students will also have growth on the plain LB plates by the untransformed cells (controls). This portion of the lab demonstrates the phenotypic changes associated with transformation.
In the second activity, students will perform a cell resuspension from the transformed and untransformed colonies, followed by a plasmid minipreparation on those cells, to enable them to demonstrate that only transformed cells have the plasmid present.
In the final activity, the students will complete a restriction digest and gel electrophoresis which will allow them to compare the untransformed with the transformed E. coli cells (containing the pAMP gene).
2nd year biology students
STUDENT/CLASS TIME REQUIRED (in class periods):
Prelab....1 or 2
Colony transformation....1 or 2
overnight suspension culture....1
PART I: Rapid Colony Transformation
(for 24 students, divided into 8 groups of 3 students)
B. SPECIAL INSTRUCTIONS:
Follow sterile techniques as outlined in this module. Student culture plates and station set up. (1 hour)
- Use presterilized, ready-to-pour agar as a
convenience. It needs only to be melted in a
microwave (carefully!) or boiling water bath,
cooled to approximately 60deg C, and poured
into sterile culture plates.
- Label at least 16 plates "LB" and 16 plates
"LB/amp" on the bottom with a permanent
marker and date them. Pour the LB broth
into the LB plates to a thickness of about a
quarter of an inch each; repeat for the
LB/amp plates. Work as quickly as possible,
using as sterile a technique as possible. Close
each plate as soon as you have poured the
agar, and do not disturb them until they have
cooled and solidified. Once they have cooled,
turn them upside down (to avoid
condensation falling on the agar) and store
- Set up the student work stations with the
materials indicated above. You may choose
to aliquot the amount of pAMP each group
will use, or leave the solutions in the total
amount indicated above to be shared by the
- After step 10 in the student instructions, you
can have the students stop. Make sure to
store their tubes in the cold (on ice in a
refrigerator works fine) until the next step.
- Throughout these experiments, emphasize
the importance of sterile technique.
PART II: CELL SUSPENSION & PLASMID MINIPREPARATION
(for 24 students, 8 groups of 3) colony transformation
plates and control plates from part I
B. SPECIAL INSTRUCTIONS:
Each student group should work with the following plates:
i) LB agar plate with untransformed cells
ii) LB/amp agar plate with transformed cells
PART III: RESTRICTION ANALYSIS OF PAMP PLASMID
(for 24 students; combine into a total
of 1 to 8 groups, depending on supplies).
Materials listed are per individual group
B. SPECIAL INSTRUCTIONS:
- You may wish to have the students practice
pipetting before they attempt to load their
samples into the wells. Carolina Biological has
kits for practicing. Having the students use two
hands on the pipette will help steady it. The
contents should be expelled slowly out, making
sure the tip is not poking through the bottom of
- In order to visualize the DNA bands on the gel,
the gel needs to be stained in the buffer
containing either ethidium bromide or
methylene blue. Ethidium bromide gives better
results, but it is a carcinogen, and should not be
used by students. Also, the equipment used to
see the bands is much more expensive than that
for the methylene blue method (which does not
give as good a resolution of bands). Both
methods are described in great detail in DNA
- At the end of the laboratory series, all bacterial
cultures, tubes, pipettes, and other instruments
that have come in contact with the cultures
should be collected. Materials and glassware
should be disinfected with a 10% bleach solution
and placed in bio bag for disposal.
RAPID COLONY TRANSFORMATION (Adapted from DNA Science)
- Use a permanent marker to label one sterile
15ml tube +pAMP and the other tube -pAMP.
Plasmid DNA will be added to the +pAMP tube;
none will be added to the -pAMP tube. Put your
name on the tubes as well.
- Use a sterile transfer pipette to add 250 ul of
cold CaCl2 solution to each tube; place both tubes
- Use a sterile inoculating loop to transfer one or
two large colonies (3mm) from the starter plate
to the +pAMP tube:
- Immediately resuspend cells in +pAMP tube by
repeatedly pipetting in and out using a sterile
transfer pipette. Be careful not to suction any
cells up into the bulb!
- Return +pAMP to ice.
- Transfer a second mass of cells to the -pAMP
tube as described in steps 3 through 5 above.
Use a new transfer pipet to resuspend cells.
- While both tubes are on ice, use a new sterile
transfer pipette to add 10.0 ul of 0.005 ug/ul
pAMP solution directly into cell suspension in
the +pAMP tube. Tap tube with finger to mix.
Avoid making bubbles in the suspension or
splashing suspension up the sides of the tubes.
- Return +pAMP tube to ice. Incubate both tubes
on ice for an additional 15 minutes.
- While your tubes are incubating, use a
permanent marker to label two LB plates and
two LB/amp plates with your name and the
date. Take the time to predict what your results
- Following a 15 minute incubation, heat shock
the cells in both tubes:
NOTE: At this point, you may need to give your tubes to your teacher for overnight storage. Make sure your tubes are clearly labelled.
- Place +pAMP and -pAMP tubes in test tube
rack at room temperature.
- Use a sterile pipette to add 250 ul of LB broth
to each tube. Gently tap tubes with finger to
- Use the matrix below as a checklist for
spreading +pAMP and -pAMP cells on the four
Agar Used +pAMP -pAMP
* * *
LB/amp 100 ul 100 ul
LB 100 ul 100 ul
- Spread cells over the surface of each plate.
- Let plates set for several minutes to allow
suspension to become absorbed into the agar.
Wrap the plates together with tape, place them
upside down in the incubator, and incubate for
- After initial incubation, store plates at 4deg C to
arrest E. coli growth.
- Save all plates with colonies on them for the
- Take time for responsible cleanup:
- Observe the plates, and count the number of
colonies on each. Observe colonies through the
bottom of the culture plate, using a marker to
mark each colony as it is counted. If cells are
too dense, record as "lawn". Were results as
- Compare and contrast the growth on each of the
following pairs of plates. What does each pair of
results tell you about the experiment?
CELL SUSPENSION & PLASMID MINIPREPARATION OF pAMP
- Label 2 sterile 50 ml tubes with name, date, and
which colony you will be suspending (i.e., tube
#1 = untransformed, tube #2 = transformed).
- Use 10 ml pipette to sterilely transfer 5 ml of LB
broth into tube #1. Use a second pipette to
transfer 5 ml of LB/amp broth into tube #2.
- Locate a well-defined colony on your LB plate.
- Sterilize inoculating loop and scrape up a cell
mass from your plate.
- Sterilely transfer colony into culture tube #1.
- Repeat steps 4 & 5 for the LB/amp plate.
- Reflame loop before placing it on the lab bench.
- Loosely replace caps to allow for air flow.
- Incubate 1 or more days at 37deg C until ready for
- Shake culture tubes to resuspend E. coli cells.
- Label two 1.5 ml tubes with your initials and
indicate "untransformed" (#1) and
"transformed" (#2). Use sterile transfer pipette
to transfer 1 ml of your overnight LB
suspension into the tube labeled
"untransformed". Use a second pipette to
transfer 1 ml of your overnight LB/amp broth
into the tube labeled "transformed".
- Close caps, and place tubes in microcentrifuge
rotor. Make sure you place the tubes opposite
for balance. Spin for 1 minute to pellet cells.
- Pour off supernatant from tubes into waste
beaker for later disinfection. Do not disturb cell
pellets. Invert tubes, and tap gently on surface
of clean paper towel to drain thoroughly.
- Add 0.35 ml of lysis buffer solution to tubes.
Resuspend pellets by pipetting solution in and
out several times. Check to make sure the
solution is homogeneous before continuing.
- Close the caps and place in 42deg C water bath for
3 minutes. Immediately cool on ice for 5
- Spin for 5 minutes with tubes BALANCED in the
- Pour the supernatant into two clean 1.5 ml
- Add 400 ul of isopropanol to supernatant.
Close caps and mix vigorously by rapidly
inverting several times. Stand at room
temperature for 2 minutes.
- Place tubes in a balanced configuration in
microfuge and spin for 5 minutes.
- Pour off supernatant.
- Add 1.0ml iced 70% ethanol to the tubes and
invert the tubes gently a few times. Do NOT
resuspend! This is a cleaning step only.
Centrifuge again in the microfuge for 2 minutes.
- Again, carefully pour off the supernatant. Stand
the tubes upside down on a paper towel to
allow all of the liquid to drain off, making sure
the pellets don't slide down the tube.
- Let dry 5-10 minutes until all the alcohol smell
is gone. Resuspend the pellets by vortexing
(vigorous mixing) in 50 ul of TE buffer. Give to
your teacher in labeled tubes to be stored in the
III. RESTRICTION ANALYSIS OF PLASMID DNA
- Set up the following mixtures in different
microfuge tubes and incubate for 30 min
at 37deg C. Make sure to add the solutions in the
- While the DNA is being digested by the EcoRI
restriction enzyme, pour the gel. (This can also
be done ahead of time, just make sure the gel is
completely covered by buffer so it doesn't dry
- Allow the gel to solidify for approximately 20
- When the gel has solidified, pour approximately
150 ml TAE electrophoresis buffer into the
chamber to completely cover the gel. Wait a
couple of minutes, then carefully and slowly pull
the comb straight up so the gel will not break.
- After the digest incubation has ended, heat
the restriction enzymes by putting the digests at
65deg C for 5 minutes. Then remove 5 ul of each
digest to a new microtube (don't forget to label
them)and add 3 ul of loading dye solution to
- Load the samples into the wells in the order
below. If you mix up the order, record your
actual order in your notes.
- When all the samples are loaded, close the lid on
the gel box and attach the electrical leads, taking
care not to jostle the box. Connect the red
(positive) leads to the red jack, and the black
(negative) leads to the black jack.
- Turn on the power and adjust to the appropriate
voltage as indicated by your power supply (in
general, lower voltages result in a longer running
time). Electrophorese your samples until the dye
has reached the middle of the gel.
- Follow the staining procedures as given to you by
Remember that the migration of DNA molecules in agarose gels is roughly proportional to the inverse of the log of their molecular weights (sizes). Sketch the banding patterns present on the gel and compare the sizes to those found on the 1 KB Ladder Marker. What do the results indicate? Plot their sizes (y-axis) against the distances migrated (mm, x-axis) onto semi-log paper or using computer graphics.