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This was the first type of transfer of genetic information discovered in bacteria. The first transformation was done by Fredrick Griffith in 1928. In this method a foreign plasmid DNA is introduced into a bacterium and uses this to multiply plasmids in order to obtain large amounts of it.
Principle:
The E. coli (DH5α) used in this experiment lacks plasmids and is sensitive to ampicillin. It also lacks a functional gene (lac Z) for the production of β galactosidase. This enzyme is capable of hydrolysing an analogue of lactose known as X-Gal (5-bromo-4-chloro-3indolyl-beta-D-galactopyranoside). The PBR322 plasmid, on the other hand, has the gene for production of β-lactamase (an enzyme which destroys ampicillin) and the gene for β galactosidase production.
This analogue of lactose, X-Gal is colourless, but the product formed as a result of hydrolysis by β galactosidase is blue in colour. If transformation of this plasmid (pBR322) into the host cell is successful and if the host cells takes up the plasmid, the transformed cells will be resistant to ampicillin and will convert colourless X-Gal into a blue coloured product.
Requirements:
1. IPTG solution:
Dissolve IPTG in sterile distilled water to get a 0.1 M solution. Spread 40 µl on L.B. plus Ampicillin plate.
2. X-Gal Solution:
Dissolve 20 mg/ml of X-Gal in Dimethyl form-amide (DMF) Spread 40 µl on L.B plus Ampicillin plate.
3. L.B. Medium (pH 7.0 – 7.3):
Autoclave at 15 lb (121°C) for 15 minutes, cool the medium to 45° and add 100 mg/1 ampicillin (or as required) and keep the medium at room temperature.
4. CaCl2 – 0.1M.
5. Plasmid DNA (pUC 18).
6. Ampicillin 100 mg/ml.
7. E.coli cultures (DH5a).
8. Glycerol 20%.
9. Laminar clean air flow hood.
10. Centrifuge (refrigerated).
11. Refrigerator.
12. Ice in ice bucket.
13. Eppendorf vials.
14. Pipettes.
15. Petri plates and tubes.
16. Incubator.
17. Bunsen flame.
18. Spreader.
19. Inoculation Loop.
20. Water bath 42°C.
Experiment should strictly be carried out under aseptic conditions.
Procedure:
a. Preparation of competent cells of E. coli:
1. Streak DH α on L.B. agar plate and incubate at 37°C for 12 hours.
2. Pick up single colonies and inoculate into 100 ml L.B. broth and incubate at 37°C. Transfer 0.4 ml of the overnight grown culture to 9.6 ml of L.B. broth and incubate at 37°C for 2-3 hr at 220 rpm.
3. Chill the culture flask quickly on ice and leave it on ice in a refrigerator for 10-20 minutes. Following steps should be carried out at 4°C.
4. Transfer the culture, aseptically into sterile centrifuge tubes and centrifuge at 6000 rpm for 8 minutes at 4°C.
5. Discard supernatant, add 5 ml of ice cold 0.1 (M) CaCl2 and suspend the cell pellet gently and keep the tube in an ice bucket for 30-40 minutes.
6. Centrifuge at 6000 rpm for 6—7 minutes at 4°C.
7. Discard the supernatant and resuspend the pellet gently in 0.5 to 1.0 ml of 0.1 (M) ice cold CaCl2 with 20% glycerol.
8. Transfer aseptically 100 pi of this suspension of competent cells into five eppendorf vials kept on ice.
b. Transformation:
9. Add 5 µl of ligated DNA to 100 µl of competent cells, gently mix using pipette tip and keep the vial on ice for 20-30 minutes.
10. Immerse the vial in a water bath at 42°C for two minutes. Temperature should be accurately maintained during this period.
11. After this heat shock, quickly remove the vial and chill on ice for 20-25 minutes.
12. Aseptically add 1 ml of L.B. broth to the vial and incubate for one hour at 37°C with 220 rpm. This is done for the bacteria to recover and express antibiotic resistance.
13. Prepare L.B. ampicillin in plates: When L.B. agar medium is cooled to 45°C add 100 µg/ml ampicillin. Spread 40 µl of X-Gal and IPTG to each of L.B. agar plates, using sterile spreader. Concentrate the transformed cells at 3000 rpm for 3 minutes. Add 200 µl of fresh L.B. broth to the pellet.
14. Plating is done by spreading 100 µl of transformed bacterial cells on LB + ampicillin + IPTG + X-Gal plates. Control plates without DNA also should be prepared.
Observation:
i. There will not be any growth in plates where no DNA was added whereas plates with DNA had growth of E.coli colonies.
ii. Efficiency of competent cells will be the number of transformants/µg of DNA. e.g. if 100 ng of plasmid DNA transformed in 1 ml of culture upon plating 100 µl (10 ng) on A100 plate has around 1000 colonies then, the efficiency of competent cells is 1 x 100,000/µg.