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.

 

 

 

TRANSFORMATION

 

Transformation

AIM: To perform and demonstrate the transformation mechanism by gene transfer technique

Principles: Transformation is the uptake by a recipient bacterium of a naked DNA molecule or a fragment from a culture medium and the incorporation of this molecule (or fragment) into the recipient chromosome in a heritable form. In natural transformation, the DNA comes from a donor bacterium. The process is random, and any portion of the genome may be transferred between bacteria. When bacteria lyse, they release considerable amounts of DNA into the surrounding environment. These fragments may be relatively large and contain several genes. If a fragment contacts a competent bacterium, one able to take up DNA and be transformed, it can be bound by the cell and taken inside. Transformation may be an important route of genetic exchange in nature.

Introduction of recombinant plasmid into cells is achieved by the transformation of competent cells. Competent cells are prepared by treating the cell with a divalent cation like calcium chloride. Once the cells are made competent, the plasmid DNA is mixed with the cells. The competent cells are then subjected to heat shock, which allows the DNA to enter the cells. The cells are then plated onto a medium containing antibiotics to allow identification of recombinants.

Competent Cells Preparation

The competent cells were prepared by the following methods:

Calcium chloride method:Material calcium chloride (50 mM), E. coli cells, ice, 10% sterile glycerol, tips, LBAgar plate, LB broth, etc.

Procedure

1.      Bacteria in glycerol frozen stock were streaked on a LB agar plate using a sterile platinum loop,

2.      grown at 37°C for 16–20 hours.

3.      A single isolated colony from the plate was transferred into 25 mL of LB broth and grown for 16 hours at 37°C with shaking until the culture reached an A600 nm of 0.4.

4.      The culture was chilled on ice and the cells were harvested by centrifuging at 4000 rpm for 10 minutes, at 4°C in a refrigerated centrifuge.

5.      The cell pellet was resuspended in 25 mL ice-cold sterile 50 mM calcium chloride and kept on ice for 20 minutes.

6.      Cells were recovered by centrifugation at 4°C as described above.

7.      Finally, the pellet was gently resuspended in 2.5 mL of ice-cold 50 mM calcium chloride containing 10% sterile glycerol, aliquoted (200 mL each) in 1.5-mL centrifuge tubes, and stored at –70°C for further use.

Transformation of Competent Cells

1.      Mix 10 mL of ligation mix (or any other plasmid that is to be transformed) with 100 mL of competent cells and incubate on ice for 1 hour.

2.      Apply a heat shock at 42°C for 2 minutes.

3.      Chill the tube to 0°C on ice immediately.

4.      Add 800 mL of sac medium and incubate at 37°C for 60 minutes with slow shaking.

5.      Prepare LB-agar plates by adding 2 mL ampicillin (50 mg/mL), 10 mL IPTG, and 10 mL X-gal.

6.      Stock per mL of melted LB agar.

7.      Spread 100 mL of transformed cells onto the plates and incubate at 37°C overnight.

Screening of Recombinants

1. Selection of recombinants is based on the color of the colony.

2. For a bluecol plasmid, insertion of foreign DNA is monitored by the loss of 13-galactosidase activity upon transformation. Cells with the intact lacZ gene produce functional 13-galactosidase, which converts the colorless substrates X-gal to blue chromophor in presence of an inducer IPTG and therefore produce blue colonies.

3. Select blue colonies as clones.

Storage of Clones

4. Transfer the white colonies one by one onto a fresh LB agar plate containing ampicillin, IPTG, and X-Gal.

5. Incubate the plate at 37°C overnight. This is the master plate of the clones.

6. Inoculate the clones from the master plate to 1 mL of LB containing 100 mL of ampicillin individually.

7. Incubate the tubes at 37°C overnight with constant shaking.

8. Add 150 mL of 100% glycerol to the wells of the microtiter plate.

9. Add 850 mL of the overnight grown culture to glycerol in a microtiter plate. Use 1 well per clone. Mix well.

10. Freeze the plate, cover, and store at –70°C.

RESULT:

The plate IS containing blue color colonies which reveals that the plasmid bluecol has been transferred in to the recipient and induce the bêta galactosidase production results the blue color compound and galactoside from Xgal. It is attached on the cell wall of the recipient bacteria which yields the blue color colonies.