14.14.2 Calcium Chloride (CaCl2) induced transformation in E. coli.

A genetic alteration in a cell resulting from the introduction of free DNA from the environment

across the cell membrane is known as transformation. Genetic transformation is a process by

which a cell takes up naked DNA from the surrounding medium and incorporates it to acquire an

altered genotype that is heritable. Bacteria are the only organisms (except yeast) known to

transform in nature although cells of higher organisms including that of mammals are

transformable by artificial techniques. The overall process in naturally transformable organisms

may be divided into five common steps: (i) development of competence (ii) binding DNA (iii)

integration of DNA (iv) formation of pre-integration complex and (v) integration of DNA into

the recipient cell chromosomes or exist as an extra-chromosomal DNA (plasmid).

The competence of a bacterial cell for transformation could be artificially induced by exposing

cells to calcium chloride prior to the addition of DNA. It involves the exposure of growing

bacterial cells to a hypotonic solution of calcium chloride at 00C, causing cells to swell

(spheroplast formation). DNA added to the transformation mixture forms a DNase resistant

complex of hydroxyl-calcium phosphate that adheres to the cell surface. This complex can be

taken up by the cell during a brief 420C heat pulse. After a few hours of growth in rich medium

to allow the spheroplast to recover and the transformed genes to be expressed, transformants can

be isolated by plating on selective medium. For example, cells transformed by pBR322 DNA can

be selected on medium containing the antibiotics ampicillin and tetracycline. With minor

modifications, this basic transformation protocol has been made to work for a variety of bacteria

that do not take up exogenous DNA normally. It is suggested further that magnesium ions may

play an important role in the stability of the DNA during uptake, and many transformation

methods now include MgCl2 during the treatment of the bacteria (Figure 14.36).
14.14.2.1 MATERIALS
Conical flasks, test tubes, centrifuge, pipettes, incubator, Petri-plates, autoclave, eppendorf tubes,

colorimeter, tryptone, yeast extract, sodium chloride, NaOH, distilled water, CaCl2, DNA, Tris

EDTA buffer, ampicillin, and tetracycline, distilled water, E. coli, plasmid DNA (pBR 322).

LB medium g/L in distilled water: Casein enzymic hydrolysate 10, Yeast extract 5, Sodium

chloride 5, Final pH ( at 25C) 7.00.2

Tris-EDTA buffer 10 mM Tris 1 mM EDTA dissolved in distilled water.

14.14.2.2 PROTOCOL

1. From the overnight grown culture take 0.1 mL and inoculate into the 25 mL of fresh LB

medium in 100 mL flask.

2. Allow it to grow till the O.D reaches 0.5 0.6 at 540 nm.

3. Chill the culture by keeping it in ice for 30 min.

4. Spin the culture at 5000 rpm for 10 min and discard the supernatant.
5. Suspend the pellet in 5 mL of 50 mM or 10 mM CaCl2.

6. Keep it in the ice (0C) for 30-60 min.

7. Spin down the culture and discard the supernatant.

8. Suspend the pellet in 1 mL of 10 mM CaC12, take 0.2 mL of culture and add 50 mg to 500 mg

of DNA in Tris-EDTA buffer and keep in the ice for 30 min.

9. In the control experiment, to 0.2 mL of the culture, add only buffer without DNA.

10. Give a heat shock at 42C for 2 min and transfer it to ice and keep again for 5 min.

11. Now add 0.8 mL of sterile LB broth and incubate at 37C for 45 min. For the cells to

multiply and express the marker.

12. Take an aliquot of 0.1 mL each of these cells and plate on LB agar containing Ampicillin (50
 mg/mL) or Tetracycline (15 g/mL) or both and incubate the plates at 37C for overnight.

13. Also, plate 0.1 mL of the control culture (step 9) on the selective plate and incubate at 37C

along with plates of step 12 (after incubation no colonies should appear in the control plates).