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Preparation of E.coli Competent Cells

1. Objective:

Preparation of E.coli competent cells and transformation of these cells with a given plasmid.

2. Introduction:

During 1970, it was found that E.coli cells soaked in ice cold solution were more efficient in receiving foreign DNA than the treated normal cells. Hence, E.coli cells can be made more competent in laboratory by treating an ice-cold solution or CaCl₂, rubidium chloride or the other salts.

The process of receiving the foreign DNA in called transformation which was demonstrated first in 1928 by Griffith. Under normal conditions several bacteria like E. coli receives a limited amount of DNA.

Hence, in order to introduce foreign DNA efficiently into these cells, the cell should have to undergo a chemical treatment. Consequently, efficiency of receiving the foreign DNA is increased. Such chemically treated cells are called competent cells.

3. Principle:

By addition of CaCl₂ solution, Ca²⁺ ions destabilize the cell membrane or also form a complex with the foreign DNA which attaches to the cell surface. By gently raising the temperature to 42°C, the uptake of foreign DNA by E. coli cells is stimulated.

The transformed E. coli cells can be placed on an LB plate containing appropriate antibiotic (depending upon the antibiotic resistance gene present on the plasmid DNA used for transformation). Then cells growing on such medium are selected and purified.

4. Requirements :

i. E. coli host strain, Plasmid DNA, sodium chloride, yeast extract

ii. Agar, sodium hydroxide, calcium chloride

iii. Suitable antibiotic (e.g. streptomycin), tryptone, eppendorf tubes

iv. Tips, micropipettes, centrifuge tubes.

Lab Preparation:

The following preparation should be done in advance:

First Day:

Streak E. coli suspension onto the surface of fresh LB plate so that a single colony may be obtained. Incubate the plate at 37°C overnight.

Second Day:

In the evening pick a single colony and transfer into 5 ml LB broth. Incubate the tube at 37°C on a shaker (200-250 rpm) for about 16 to 18 hours.

On the third day:

Develop the competent E. coli cells as described below:

5. Procedure :

A. Development of Competent E. Coli Cells:

(i) Pour 1 ml of the pure culture obtained on 3^rd day as given in step (ii) into 100 ml LB medium and incubates at 37°C on a shaker (200-250 rpm). Grow the culture to get the 0.3-0.5 OD at 600 nm (A₆₀₀) (it takes 2-3 hours).

(ii) Then quickly keep the culture flask on ice in a refrigerator for 10-20 minutes.

(iii) Transfer the culture into sterile centrifuge tubes in a laminar air flow.

(iv) Centrifuge at 6,000 rpm for 8 minutes at 4°C (a refrigerated centrifuge is preferred).

(y) Discard the supernatant in a laminar air flow. Add 6 ml of ice-cold 0.1 M calcium chloride

(CaCl₂) to the cell pellet. Keeping the tubes on ice bucket, suspend the cell pellet gently in CaCl₂. Keep the centrifuge tubes on ice for 30 minutes.

(vi) Centrifuge cells at 6,000 rpm for 8 minutes at 4°C, if possible.

(vii) In a laminar air flow discard the supernatant and re-suspend the cell pellets gently in 0.5 ml of ice-cold 0.1M CaCl₂. The cells become competent.

(viii)Transfer 100 pi of the above competent cells into 5 eppendorf tube (care should be taken for all transfer work to carry out on ice and in the laminar air flow).

B. Transformation of Competent E. Coli Cells:

(All steps should be carried out inside the laminar airflow)

(i) Add 5 µl of the plasmid DNA (about 10-50 nanograms) to 100 pi of competent cells prepared. Gently tap and keep the eppendorf tube on ice for 20 minutes.

(ii) Maintain the temperature of a water-bath at 42°C. Keep the eppendorf tube in the water bath in such a way that the competent cells should be immersed for 2 minutes. During incubation in water-bath temperature should be maintained accurately.

(iii) After heat shock quickly remove the eppendorf tube and place it on ice for 2 minutes.

(iv) Pour 1ml of LB medium to the eppendorf tube and incubate the culture for 1 hour at 37°C. This helps the bacteria to recover from the heat shock and show antibiotic resistance.

(v) Prepare LB plates containing streptomycin or ampicillin or any other suitable antibiotic depending upon the plasmid used (melt the autoclaved LB-agar medium and allow it to cool. When the agar is around 44°C, add the required concentration of the antibiotic into it, for example, 100 µg/ml of ampicillin. Mix well and pour into Petri plates.

(vi) Add 50 µl, 100 (il and 200 µl of transformed E. coli cells separately [obtained after step (iv)] to three different plates. Properly spread the cells by a spreader. As a control, competent cells that have not been transformed with the plasmid DNA should also be plated onto a plate to rule out the contamination of cells. Similarly, transfer the non- transformed competent E. coli cells on antibiotic-containing LB medium as control to rule out the contamination.

(vii) Incubate the plates at 37°C overnight and observe them on the next day.

6. Results :

The control plates show no colonies on which competent cells containing no plasmid DNA were spread. The other plates show colonies on which competent cells transformed with the plasmid were spread.