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EXERCISE 1
BACTERIAL CONJUGATION

Conjugation is a gene transfer mechanism that involves physical contact between

donor and recipient cells. It is involved in the lateral transfer of genetic material between
bacteria (Ochman et al., 2000), between bacteria and yeasts (Heinmann and Sprague,
1989), and between bacteria and plants (Lessl and Lanka, 1994). Typically, DNA is
transferred from a donor to a recipient strain by plasmids and by conjugative transposons.
Conjugation is responsible for widespread transfer of genes that confer antibiotic
resistance, metabolic functions, and virulence factors.

The physical contact between the donor and recipient cells is mediated by the F
pilus, a tubular structure that extrudes from the surface of the donor cell. Some workers
have suggested that F pilus is only needed to establish contact between the donor and
recipient cells, and DNA is transferred via a pore in the membrane (Panicker and Minkley,
1985). However, others have shown that the F pilus is the sole conduit for cell-to-cell
transfer of DNA (Harrington and Rogerson, 1990; Babic et al., 2008).

In this exercise, Escherichia coli isolates will be studied to evaluate their ability to
transfer their resistance genes to the antimicrobial susceptible E. coli strain J53-2 through
conjugation.

Strains, Growth Media, and Materials:

Escherichia coli J53-2 (recipient strain, rifr)

Escherichia coli (donor strains S6C2, S3P1, and S6P3; ampr)
Tryptic-soy broth (TSB)
Mueller-Hinton Agar (supplemented with 200 µg ml-1 rifampicin)
0.5 McFarland Turbidity Standard
sterile distilled water
0.85% sterile saline solution
Luria-Bertani agar + 200 µg ml-1 rifampicin (LAR)
Luria-Bertani agar + 32 µg ml-1 ampicillin (LAA)
Luria-Bertani agar + 200 µg ml-1 rifampicin + 32 µg ml-1 ampicillin (LARA)
Luria-Bertani Agar
Luria-Bertani broth
ethanol
20 ml screw-cap serial dilution tubes (up to 10-8) or test tubes with cotton plugs
sterile eppendorf tubes
bent glass rods
10 ml sterile screw-cap tubes
test tube rack
blue micropipette tips (sterile)
yellow micropipette tips (sterile)
micropipettors (P1000, P200 or P100)

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Experimental Procedure:

A. Preparation of E. coli strains (Jose and Cabrera, 2018; Jiao et al., 2007)

1. The donor and the recipient strains will be separately grown on 30 ml TSB at 37℃ for
16-18 hours, then subcultured in fresh 30 ml TSB for 4 hours at 37℃ to bring them to
logarithmic phase of growth.
2. Adjust the turbidity of cultures to that of 0.5 McFarland.
3. Spread plate 100 µl of each donor and recipient strains to separate MHA plates
supplemented with rifampicin using a flame-sterilized bent glass rod.
4. Incubate plates at 37℃ overnight.
5. Check the phenotypes of donor and recipient strains.

B. Liquid Mating

1. Streak parent strains on LAR, LAA, and LARA to check phenotypes.

2. Prepare an overnight culture of each E. coli donor and recipient strains in 30 ml LB
broth.
3. Transfer 7 ml of culture from LB on a sterile screw-cap tube. Centrifuge the culture at
5,000 rpm for 5 min. Discard the supernatant.
4. Suspend the cell pellet in 7 ml 0.85% NaCl.
5. Mix the two cultures in the following ratios v/v ratios: 1:1, 1:2, and 1:3 (recipient:donor).
Incubate for 30 min at ambient temperature.
6. Make serial dilutions and plate out 10-3 to 10-5 on LARA, and 10-6 to 10-8 on LB agar
using the spot plate method.
7. Separately plate out 0.1 ml of donor and recipient cultures on LARA as controls.
8. Incubate plates at 30℃ for 24 hours. Determine the frequency of conjugation as
follows:
CFU/ml of transconjugants
Frequency of Conjugation = ———————————— x 100
CFU/ml of total population

C. Plate Mating

1. Using the cultures in step 4 in Part B, mix recipient and donor cultures at 1:1, 1:2, 1:3 to
a total of 0.5 ml in an eppendorf tube. Mix by slowly inverting the tube a few times. Pour
the mixture on LB agar plate and slowly tilt the plate from side to side to evenly
distribute the mixture on the agar surface. Dry the plates under the laminar flow hood
and incubate overnight at 30℃. After incubation, refrigerate the plate if the subsequent
steps cannot be performed.
2. Add 5 ml of 0.85% saline solution on each plate and scrape growth.
3. Centrifuge the resulting suspension at 5,000 rpm for 5 min. Discard the supernatant.
4. Suspend the cell pellet in 5 ml 0.85% saline solution.
5. Make serial dilutions and plate out 10-3 to 10-5 on LARA and 10-6 to 10-8 on LB agar using
the spot plate method.
6. Incubate plates at 30℃ for 24 hours. Determine the frequency of conjugation as in step
7 of Part B.

Output: The experiment will be done by group. Each group should make their own
hypothesis, objectives, method and submit their results following the scientific method.
However, submission of scientific writing will be done individually.

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References:

Babic A, Lindner AB, Vulic M, Stewart EJ, Radman M. Direct visualization of horizontal
gene transfer. Science. 2008 Mar 14;319(5869):1533-6. doi: 10.1126/
science.1153498. PMID: 18339941.

Harrington LC, Rogerson AC. The F pilus of Escherichia coli appears to support stable
DNA transfer in the absence of wall-to-wall contact between cells. J Bacteriol. 1990
Dec;172(12):7263-4. doi: 10.1128/jb.172.12.7263-7264.1990. PMID: 1979324;
PMCID: PMC210852.

Heinemann JA, Sprague GF Jr. Bacterial conjugative plasmids mobilize DNA transfer
between bacteria and yeast. Nature. 1989 Jul 20;340(6230):205-9. doi:
10.1038/340205a0. PMID: 2666856.

Jiao SC, Fami RML, Pedernal VAD, Cabrera EC. 2007. Prevalence of multiple drug-
resistant Escherichia coli from chicken, pig and Nile tilapia (Oreochromis nilotica)
intestines sold in wet markets in Manila and the conjugative transferability of the
resistance. Philipp Agric Scientist. 90(1): 64– 70.

Jose MA, Cabrera E. 2018. Multiple Antimicrobial Resistance of Escherichia coli Isolates
from Nile Tilapia Sold in Wet Markets in Metro Manila and the Conjugative
Transferability of the Drug Resistance. Philipp Agric Scientist. 101 (3): 284-292.

Lessl M, Lanka E. Common mechanisms in bacterial conjugation and Ti-mediated T-DNA

transfer to plant cells. Cell. 1994 May 6;77(3):321-4. doi:
10.1016/0092-8674(94)90146-5. PMID: 8181052.

Ochman H, Lawrence JG, Groisman EA. Lateral gene transfer and the nature of bacterial
innovation. Nature. 2000 May 18;405(6784):299-304. doi: 10.1038/35012500. PMID:
10830951.

Panicker MM, Minkley EG Jr. DNA transfer occurs during a cell surface contact stage of F
sex factor-mediated bacterial conjugation. J Bacteriol. 1985 May;162(2):584-90. doi:
10.1128/jb.162.2.584-590.1985. PMID: 2859268; PMCID: PMC218888.

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Data and Answer Sheet
EXERCISE 1
BACTERIAL CONJUGATION

Names: _________________________ Date Performed: _______________

_________________________ Date Submitted: _______________
_________________________
_________________________
Lab Section: _________________________

RESULTS:

Recipient
Mating Transconjugants Total Population Conjugation
and Donor
Method (CFU/ml) (CFU/ml) Frequency
Ratio
Liquid 1:1
1:2
1:3
Plate 1:1
1:2
1:3

Show your calculations:

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STUDY QUESTIONS:

1. Why is the conjugation a type of sexual reproduction?

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2. How does the donor cell transfer the plasmid containing antibiotic resistance gene to
the recipient cell?
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3. Do the frequencies of conjugation differ in liquid and plating methods? Account for the
difference, if any.
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4. How do you know whether conjugation occurred? How should the characteristic of
donor cells differ from that of recipient cells?
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MCB103 Microbial Genetics Laboratory