CONJUGATION

 

CONJUGATION

AIM:

To study the process of bacterial conjugation through transfer of genes coding for antibiotic resistance.

PRINCIPLE:

Conjugation is the mode of gene transfer in many species of bacteria. In 1950 William Hayes, Francis Jacob and Elie L. Wollman established that conjugating bacteria are of two mating types. Certain “male” types (designated as F+) donate their DNA and other “female types” (designated as F-) receive the DNA. F- cells become F+ when they acquire a small amount of DNA. Hence the F factor is called as the Fertility factor. In contemporary microbiology, the donor’s F factors are known to be plasmids which are the extrachromosomal elements. The factors (plasmids) contain about 20-30 genes, most of which are associated with conjugation. These genes encode enzymes that replicate DNA during conjugation and structural proteins needed to synthesize special pili at the cell surface. Known as F pili or sex pili, these hair like fibres contact the recipient bacteria, and then retract so that the surfaces of donor and recipient are very close or touching one another. At the area of contact, a channel or conjugation bridge is formed. Once contact via sex pili has been made, the F factor (plasmid) begins replicating by the rolling circle mechanism. A single strand of the factor then passes over through the channel to the recipient. When it arrives, enzymes synthesize a complementary strand, and a double helix is formed. The double helix bends to a loop and reforms an F factor (plasmid), thereby completing the conversion of recipient from F- cell to F+ cell. Meanwhile, back in the donor cell a new strand of DNA forms, to complement the leftover strand of the F plasmid. The transfer of F factors involves no activity of the bacterial chromosome; therefore the recipient does not acquire new genes other than those on the F factor.

MATERIALS REQUIRED:

Glass wares: Conical flask, Measuring cylinder, Sterile test tubes, Petri plates

Reagents: Distilled water

Other requirements: Incubator, Shaker, Spectrophotometer, Micropipettes, Tips, Sterile loops and spreaders

PROCEDURE:

MATERIALS

1X PBS (Phosphate-buffered saline )

Lysogeny agar

Lysogeny

STEP MATERIALS

1X PBS (Phosphate-buffered saline )

1X PBS (Phosphate-buffered saline )

PROCEDURE:

Day 1:

1.      Open the vials containing Donor and Recipient cultures and resuspend the cells with 0.25 ml of LB broth respectively.

2.      Pick up a loopful of Donor culture and streak onto LB plates with Tetracycline (30 μg/ml).

3.      Pick up a loopful of Recipient culture and streak onto LB plates with Streptomycin (100 μg/ml). 4. Incubate overnight at 370 C.

Day 2:

1.      Pick up a single colony from Donor and Recipient Strain grown overnight on LB plates and inoculate a single colony in 6 ml of LB broth having respective antibiotics.

2.      Incubate the test tubes overnight at 37o C.

Day 3:

1.      Take 25 ml of LB broth and add 25 μl of tetracycline into it and inoculate 1 ml of overnight grown culture into it. Incubate at 37oC in a shaker.

2.        Take 25 ml of LB broth with streptomycin at a concentration of 100 μg/ml and inoculate 3 ml of overnight grown culture in it. Incubate at 37oC in a shaker.

3.      Grow the cultures till O.D of the donor culture reaches 0.8-0.9 at A600.

4.      Add 0.2 ml of each donor and recipient cultures in a sterile test tube labeled as conjugated sample. Mix by gentle pipetting and incubate at 37oC for 1-1.5 hours.

5.      Take 2 sterile test tubes and label them as donor and recipient. Add 0.2 ml of respective cultures to the test tubes and incubate at 37oC for 1-1.5 hours.

6.      Add 2 ml of LB broth into each tube after incubation. Incubate the tubes at 37oC for 1.5 hours.

7.      Plate 0.1 ml of each culture on the antibiotic plates as indicated in Table .

8.      Incubate the plates overnight at 37oC overnight.

Observation and Result:

 

	LB + (Streptomycin)+X gal	LB + (Tetracycline)+IPTG	LB + (Streptomycin, Tetracycline)+X gal+ IPTG
Donor Strain A
Recipient Strain B
Conjugated Sample

NOTE: Keep uninoculated LB plate as control.

Interpretation:

On observing colonies on different plates the following interpretation can be made:

1.        Donor strains will grow only on tetracycline plates, similarly recipient strains will grow only on streptomycin plates.

2.        Donor strain is sensitive to streptomycin and recipient strain is sensitive to tetracycline, hence no growth will be seen in these plates.

3.        The conjugated sample will grow on tetracycline and streptomycin plate. The reason being, transfer of gene has occurred by means of conjugation.

4.        The donor and recipient strain will not grow on tetracycline + streptomycin plate since each of the strain is sensitive to one antibiotic in the plate.