Download Advanced Bacterial Conjugation Kit

Student Guide
Name
21-1127
Date
Advanced Bacterial Conjugation Kit
In this experiment you are introduced to a naturally occurring mechanism called conjugation, by which DNA
from one cell is transferred to another cell to produce a new recombinant cell. Sometimes the DNA that is
transferred codes for antibiotic resistance. The intercellular transfer of this bacterial DNA coding for
resistance to antibiotics is a type of genetic recombination that enables the new recombinant bacterial cell
to express resistance to an antibiotic to which it was formerly sensitive.
While bacterial chromosomes normally carry all the genes necessary for growth and reproduction, bacteria
also contain genes carried on extra-chromosomal DNA, called plasmids. Plasmids are double-stranded circular
pieces of DNA that carry anywhere from 3 to 25 genes. Numerous plasmids have been described in a variety
of bacteria. They contain specialized genes, can replicate independently of the bacterial chromosome, can move
from one bacterial cell to another, and may even be exchanged between cells of different bacterial species.
One of the first plasmids to be described was originally called the “F (fertility) Factor.” This plasmid, found
in the common colon bacterium Escherichia coli, contains about 25 genes, most of which regulate the
formation of pili, elongated appendages that extend from the surface of the cell. These pili can function as a
bridge between two bacteria to allow the transfer of DNA from the donor to the recipient. This process is
called conjugation.
In 1959, it was shown that resistance to antibiotics can be transferred between bacteria during conjugation,
and that this transfer involves plasmids. Plasmid-mediated drug resistance has created numerous problems
for physicians and patients because bacteria are able to transfer these “resistance genes” very rapidly.
Under optimal conditions, the rate at which a conjugative plasmid can spread through a population can be
exponential, showing resemblance to a bacterial growth curve.
In preparation for Lab Day 1, pure cultures of E. coli strains I (resistant to streptomycin; Strr) and II (resistant
to ampicillin; Ampr and naladixic acid, Nalr) are grown overnight or over multiple days in LB broth, an
enriched culture medium lacking antibiotics. On Lab Day 1, samples of each strain are transferred to LB agar
plates containing the antibiotics to which each strain is known to be resistant. This exercise confirms
resistance or sensitivity of these strains to the antibiotics. On Lab Day 2, after confirmation of the
appropriate growth response for each strain, samples of the two strains are mixed together on an
LB+amp+str “mating” plate where conjugation between strains I and II is expected to occur. This should
result in the transfer of DNA and the formation of recombinant cells.
After overnight growth, cells from the mating plate are tested for their growth response to the antibiotics
previously tested. The results of these tests should enable you to determine which strain is the donor and
whether plasmid or chromosomal DNA has been transferred.
Lab Day 1
Confirming Antibiotic Resistance in Each Bacterial Strain
1. On the bottom surface, draw a line down the middle of
each of the five different agar plates (shown in Figure 2).
Write “I” on one side of the line, and “II” on the other side.
Str r on chromosome
Strain I
Nal r on
chromosome
Strain II
2. Obtain the E. coli I and II broth cultures that will be shared
between your group and one other lab group. Strain I has
Amp r on plasmid
a gene for streptomycin resistance (Strr) on the
chromosome. Strain II has a gene for ampicillin resistance
Figure 1. Location of genes for antibiotic
Locationresistance
of Genes for
Resistance
in E. coli Stra
(Ampr) on a plasmid, plus a gene for naladixic acid
in Antibiotic
E. coli strains
I and II
resistance (Nalr) on its chromosome (see Figure 1).
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Transfer sample
of Strain I with a
new sterile loop to
region I on all
plates.
I
II
LB agar
(plain)
E. coli Strain I
I
II
str
I
II
amp
Transfer sample
of Strain II with a
new sterile loop to
region II on all
plates.
I
II
str+amp
I
II
nal
Incubate:
Overnight at 37 C
or
36–48 hrs at room temperature
E. coli Strain II
Figure 2. Preparation of “confirmation” plates to confirm the resistance (growth) or sensitivity (lack of growth)
of E. coli strains I and II to streptomycin (str) ampicillin (amp), and naladixic acid (nal)
3. Open the handle end of a loop’s wrapper and remove the loop, being careful not to touch anything.
Using the sterile technique described by your teacher, remove and hold the cap (open surface down)
from the Strain I broth culture bottle. Dip the sterile loop into the Strain I culture while holding the cap
slightly away and not touching anything. Withdraw the loop and replace the cap immediately. Be
careful not to touch the loop to anything before placing it in the bottle or after withdrawing it from the
bottle. If you suspect something has been touched, discard the loop and obtain a fresh one. (One
person may want to maneuver the cap or steady the bottle for another person so that the work may be
accomplished quickly.) Touch the loop lightly to the agar in the middle of each section marked “I” on
each of the five plates (see Figure 2). Use the flip-side of the loop to touch the fourth and fifth plates to
ensure that some bacteria remain. If necessary, refrigerate the cultures for up to one week to preserve
them for reuse. Discard the loop into the disposal cup or biohazard bag or replace it into its wrapper for
later disposal.
4. Using a new loop, repeat Step 3 with the second culture to inoculate Region II of each plate. Discard the
loop into the disposal cup or biohazard bag. or replace it into its wrapper for later disposal. Allow the
plates to sit briefly so the liquid can soak in before inverting. Tape the five plates together and mark
them clearly with your group’s initials. Incubate them overnight at 37°C, or for 36–48 hours at room
temperature. Refrigerate them after viewing.
5. In Table 1 on the next page, list the results you “expect” to obtain following incubation. Use (+) for
bacterial growth (i.e., resistance to the antibiotic) and (–) for lack of growth (i.e., sensitivity to the
antibiotic). Give your reasons for your “expected” results in the space below the table.
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Contents of Petri Plates
I
Expected
II
Observed
Expected
Observed
LB agar
LB agar+str
LB agar+amp
LB agar+str+amp
LB agar+nal
Table 1. Expected and observed results of the growth of E. coli strains I and II on LB agar and the antibiotic
“confirmation” plates containing LB agar plus streptomycin (str), ampicillin (amp), streptomycin plus ampicillin
(str+amp), or naladixic acid (nal).
Lab Day 2
Observation of “Confirmation” Plates and Preparation of “Mating Plates”
1. Examine the “confirmation” plates prepared during the first lab session and record the “observed”
results in Table 1. Compare the “expected” and “observed” results and account for any differences
between these results and those of other students and with those provided by the instructor.
2. Prepare an LB+amp+str “mating” plate as shown in Figure 3. Remember that this plate is used to bring
the cells of Strains I and II into close proximity so that conjugation (the transfer of DNA between cells)
can occur. Draw lines on the bottom of the plate to divide the agar into the three regions indicated in
Figure 3. Use a sterile loop to place a drop of Strain I into the region marked Region I and into the
Sterile loop
Sterile inoculating
loop
Strain I
I
I
Small
drop
I
II
Strain II
II
II
III
Mix strain I and II
together in region III,
then spread over
surface of "mating"
plate.
In region III of "mating"
plate apply drops of
I and II about 1/2
inch apart.
Incubate
overnight at
37 C or
24–48 hours at
room temperature.
Figure 3. Preparation of the mating plate to enable conjugation and the subsequent formation of recombinant cells. The
mating plate contains streptomycin and ampicillin.
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mating region of Region III. Use a separate loop to place a drop of Strain II into the region marked II and
into Region III one half inch from the drop of Strain I. Use a separate sterile loop to mix only the cells in
Region III. Quickly spread the mixed cells over the surface of the plate as shown in Figure 3.
(Note: Drops from pipets are too large. Use loops.) Open the lid of the plate just enough to slip the loop
under, being careful not to touch anything. Do not lay the lid down on the lab bench. Allow the liquid
on the plate to soak in completely before inverting the plate, so that the bacteria in the separate areas
will not run together. The loops should be disposed of in a disposal cup or biohazard bag, or replaced
in the wrapper for later disposal.
3. Record the “expected” results of growth on the mating plate in Table 2. Explain your reasoning in the
space below the table.
Region
Expected
Observed
I (Strain I)
II (Strain II)
III (mating mix Strains I + II)
Table 2. Expected and observed growth on mating plate consisting of LB agar plus streptomycin and ampicillin. Strain I
cells added to Region I. Strain II cells added to Region II. Strain I plus Strain II cells added to Region III, mixed together,
and streaked over the surface of the agar.
Lab Day 3
Observation of Recombinant Growth and Donor Determination
Examine the “mating” plate prepared during the second lab session. Record the “observed” results in
Table 2. Whether the growth observed represents “recombinant” cells (i.e., those in which conjugation has
occurred) should be evident. No growth should occur in regions I and II since each strain is sensitive to one
of the antibiotics. Samples of cells from the “mating” plate will now be transferred onto plates containing
the antibiotics previously tested. Prepare four plates as demonstrated in Figure 4, marking them first on the
bottom, then transferring random cells with sterile loops. In doing this, lift the lids only enough to allow
access for the loop. Do not lay the lids on the lab bench, but keep them almost in place as a “shield” for the
cells from any contamination. The details of the bacterial transfer are as follows:
1. Remove one sterile yellow transfer loop from its wrapper by the handle end, not touching the loop to
anything. Slightly lift the lid of the agar “mating” plate containing the “suspected” recombinant cells,
and pick up some bacteria in the streaked area. Close the lid. Touch some of these bacteria to each “#1”
spot on each new plate. Then repeat this process of transfer for an additional four separate random
samples of the conjugated bacteria to each of the numbered sections, in sequence. Use a new loop for
taking each new sample (#2, #3, etc.) Discard the loops in the disposal cup or biohazard bag or replace
into the wrapper for later disposal. Allow any fluid to soak in before inverting the plates to prevent
inadvertent mixing of the samples.
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Using 5 separate sterile loops, touch 5 random
colonies (1, 2, 3, etc.) and transfer bacteria by touching
to corresponding area on each plate.
1
2
3
4
5
str
1
2
3
4
5
1
2
3
4
5
2
3
4
5
nal
amp
str+amp
1
Incubate overnight
at 37 C or 24–48 hours at
room temperature.
Figure 4. Procedure for transferring prospective recombinant cells to determine their growth response to different
antibiotics. Cells from randomly selected colonies are transferred to a corresponding area on antibiotic plates (i.e.,
Colony 1 to Region 1 on each plate, and so forth).
2. Record the “expected” growth results for the recombinant cell growth only, in Table 3 below.
Contents of Petri Plates
Expected, if donor is strain:
I (chromosome)
II (plasmid)
II (Chromosome)
Observed
LB agar+str
LB agar+amp
LB agar+ str+amp
LB agar+nal
Table 3. Expected and observed growth response of recombinant cells to the antibiotics streptomycin, ampicillin,
naladixic acid, or a combination of streptomycin plus ampicillin. The “expected” results will vary depending on whether
strain I is the donor or whether strain II is the donor and transfers plasmid genes only, or chromosomal genes only.
3. Invert and tape together the four plates and clearly label them with the group’s initials.
4. Incubate the plates overnight at 37ºC, or for 24–48 hours at room temperature.
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Lab Day 4
Observation of Recombinant Cell Growth and Determination of Donor Strain
In Table 3, record the recombinant cell’s growth on the various antibiotic plates. You will try to determine
from observed growth if conjugation (recombination) has actually occurred. There are several
recombination possibilities based on whether Strain I or Strain II is the donor. Furthermore, if Strain II is the
donor, different growth would be expected, depending upon whether the plasmid genes, chromosomal
genes, or both were transferred.
Results and Discussion
1. Why did Strain I not grow on the LB+amp plate?
2. Why did Strain II not grow on the LB+str plate?
3. Why did neither strain grow on the media containing both antibiotics?
4. What type of growth was expected on the “mating” plate?
5. If growth is present on the plate containing ampicillin plus streptomycin, does this support the
prediction of recombination of antibiotic-resistant genes into one strain? Explain.
6. Based on the results of this study, can one determine whether the str gene from Strain I was
transferred to Strain II or if the amp gene on the plasmid was transferred from Strain II into Strain I?
Explain.
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