bacterial conjugation through the transfer of genes, coding for antibiotic resistance

Aim: To learn the process of bacterial conjugation through the transfer of genes, coding for antibiotic resistance.

Principle: Transfer of genetic material from one bacterial strain (donor) to another strain (recipient) is a common event that occurs in nature with the objective of mixing the gene pool, in otherwise asexually reproducing organisms. DNA transfer among bacteria is mediated in three ways viz., transfection, transduction and conjugation. Conjugation is the most widespread process of transferring genetic material from one bacterial cell to another. It is a process in which unidirectional transfer of DNA is mediated by conjugal plasmids or conjugal transposons requiring cell-to-cell contact. It was discovered by
Lederberg and Tatum in 1946.

Conjugation is best understood by considering properties of the 'F'factor, which is a small circular plasmid DNA that can replicate autonomously in the cell or can integrate into the host chromosome and thus transfer host chromosomal markers. When individual cells with an integrated 'F' are isolated and allowed to form pure colonies, the resulting strain can transfer chromosomal markers at very high frequency and are termed Hfr strains.

Materials Provided:
The list below provides information about the materials supplied in the kit. The products should be stored as suggested. Use the kit within 6 months of arrival.

Materials Required:
Equipment: Incubator, Shaker (37°C), Spectrophotometer.
Glassware: Conical-Flasks, Petri plates, Pipettes, Spreader, Test tubes.
Reagent: Distilled water.
Other Requirements: Cuvette (of 1 cm path length)
Micropipette, Tips.

Note:
Read the entire procedure before-starting the experiment.
All microbiological operations should be done strictly under aseptic conditions.
Revive the strains as soon as the lyophilized vial is opened.
Carry out the experiments within 2 weeks of reviving the strains.
For preparation of media and antibiotic, refer appendix. Bacterial Conjugation


Distinguishing characteristics of conjugation:
ONA transfer requires cell-cell contact.
DNA transfer occurs via a conjugal pore – resistant to DNase.
DNA transfer occurs in one direction - from donor to recipient and not vice versa.
DNA transfer does not require protein synthesis in donor cell.
DNA transfer requires energy in donor cell – primarily ATP.

Appendix:
Preparation of LB Broth/Agar (1 litre): Dissolve 25 g of media in 800 ml of distilled water. Adjust the pH to 7.0 with 5N NaOH (if necessary) and make up the volume to 1000 ml. Sterilize by autoclaving.
For LB agar, add 1.5% agar and autoclave.

Preparation of antibiotic:
Tetracycline: Dissolve 45 mg of tetracycline supplied in 1.5 ml of 70% ethanol, vortex if necessary. This gives a stock concentration of 30 mg/ml. Cover the vial with aluminium foil and store at 4°C. Use the antibiotic solution within 2 weeks. Add this antibiotic solution to LB media at a concentration of 30 mg/ml.

Streptomycin: Dissolve 150 mg of streptomycin supplied in 1.5 ml of sterile water, vortex if necessary. This gives a stock concentration of 100 mg/ml. Cover the vial with
aluminium foil and store at 4°C. Use the antibiotic solution within 2 weeks. Add this antibiotic solution to LB media at a concentration of 100 mg/ml.

Following aliquots of media are required for single conjugation experiment. (Excludes preparation of media for revival of strain).

• LB Broth 6 ml

• LB Broth + Tetracycline 6 ml + 25 ml

• LB Broth + Streptomycin 6 ml + 25 ml

• LB Agar + Tetracycline 60 ml

• LB Agar + Streptomycin 60 ml

• LB Agar + Tet. + Strep. 60 ml

Note: Prepare 25 ml LB Broth in a 100 ml conical flask.

Procedure:
Day 1: Revival of parental strains.
1. Break open one set of lyophilized vials (donor & recipient E. coli strains). Rehydrate each vial with 0.1 ml of sterile LB broth.
2. Streak the donor strain on LB plate with tetracycline (concentration 30 //g/ml),and the recipient strain on LB with streptomycin (concentration 100 //g/ml). (Streak in duplicates).
3. Incubate the plates at 37°C overnight.
Note: Store the revived plates at 4°C,and use within 2 weeks to carry out 5 experiments
in case of KT45.
Day 2:
4. Pick single colony each from the donor and recipient plate and inoculate into 6 ml LB broth containing the respective antibiotic.
5. Incubate at 37°C in a shaker overnight.
Day 3: Conjugation
6. Inoculate 1 ml of overnight donor culture into 25 ml LB broth (in 250 ml conical flask) with tetracycline at a concentration of 30 //g/ml.
7. Incubate at 37°C in a shaker.
8. Inoculate 3 ml of overnight recipient culture into 25 ml LB broth (in 250 ml conical flask) with streptomycin at a concentration of 100//g/ml.
9. Incubate at 37°C in a shaker.
10. Grow recipient and donor cultures till the O.D. of the donor culture reaches 0.8 - 0.9 at A600.
11. Take 0.2 ml each of donor and recipient cultures in a sterile cotton plugged test tube for conjugation. Label this as conjugated sample.
12. Gently mix and incubate in an incubator at 37°C for 11/2hr-2hrs.
13. Pipette 0.2 ml each of donor and recipient cultures into two different test tubes and Incubate at 37°C for 11/2 hrs.
14. Add 2 ml of sterile LB into each tube after 11/2, hours of incubation.
15. Incubate the tubes at 37°C for another 11/2 hrs.

Note: Do not place the tubes in a shaker during conjugation or subsequent incubation
Period.
16. Spread plate 0.1 ml of each of the samples (donor, recipient, conjugated sample) on antibiotic plates as indicated in the table.
17. Incubate the plates at 37°C overnight.

Observation: Tabulate your observations as follows for each experiment:

Note: Denote positive on observing bacterial growth and negative on seeing no growth.

Interpretation:
From the observations, one can interpret that:

Donor (Tef) and the recipient (Strep') grow only on those antibiotic plates to which they are resistant.
Donor and Recipient being sensitive to Streptomycin and Tetracycline respectively will not grow on those antibiotic plates.
Conjugated sample grow on Tetracycline and Streptomycin LB plate. This is because there is gene transfer of antibiotic resistance from F factor of donor to recipient via the process of conjugation. On the other hand, both the parental strains do not grow on the double antibiotic plate as it contains one or the other antibiotic to which they are sensitive.Bacterial lawn is also seen on plating conjugated sample on the individual antibiotic plates.