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.