Transformation of Pseudomonas putida and Escherichia coli with plasmid-linked drug-resistance factor DNA.

Abstract

Conditions optimal for the transformation of Pseudomonas putida and E. coli with a drug-resistance factor (RP 1) DNA, which specifies resistance to carbenicillin, tetracycline, kanamycin, and neomycin, are described. The transformants retain all the fertility, incompatibility, and drug-resistance characteristics present in the parent. Covalently-closed circular molecules of almost identical contour lengths have been isolated from the parent and the transformants. The harvested by centrifugation, and resuspended in 48 ml of cold 25% sucrose in 0.05 M Tris-HCI, pH 8.0. Lysozyme (7.9 ml), freshly prepared at 10 mg/ml in 0.25 M Tris-HCl buffer, pH 8.0, was added, and the mixture was kept on a slow shaker at 320 for 30 min. The cells were then placed in ice for 5 min, and 26 ml of 0.25 M disodium EDTA, pH 8.0, were added. The mixture was kept in the cold for 5 min; then 54 ml of a solution containing 2% Sarkosyl NL 97 in 0.05 M Tris-HCl (pH 8.0) and 62.5 mM disodium EDTA was added. The mixture was shaken gently for about 15 min.For removal of the bulk of chromosomal DNA, cold NaCl (5 M) was added to a final concentration of 1 M. The mixture was kept at 40 for at least 1 hr. The crude lysate was then centrifuged at 17,000 rpm in a Sorvall SS-34 rotor for 30 min at 4°. After centrifugation, 30 ml of the supernatant was layered over a 3-ml saturated CsCl cushion (61.7% w/w CsCl). The lysate was then centrifuged at 15,000 rpm for [16][17][18] hr in a Spinco type 30 rotor, and the lowest 8 ml from each tube was collected and pooled.The pooled lysate was then filtered through glass wool and ethidium bromide, 10 mg/ml in TES buffer (0.05 M Tris-HCI, 0.005 M EDTA, and 0.05 M NaCI, pH 8.0), was added to a final concentration of 250 jig/ml. The refractive index of the lysate was then adjusted to approximately 1.4000 with CsCl and TES buffer, and the lysate was centrifuged at 40,000 rpm for nearly 44 hr at 150 in a Spinco type 65 rotor.At the end of the centrifugation, the bottom of the tube was punctured and 10-drop fractions were collected. Fractions corresponding to the plasmid band were pooled and dialyzed against 0.05 M Tris-HCl buffer, pH 8.0, 0.8 M NaCl, and 0.01 M EDTA containing about 10-20 g of Dowex 50 resin (Na+ form) in the cold for 24 hr to remove ethidium bromide. The dialysate was further dialyzed against 0.01 M Tris-HCI, 0.001 M EDTA, pH 7.2 buffer for another 24 hr and stored in the cold.Electron Microscopy. Samples were spread according to Davis et al. (9), stained with uranyl acetate, and rotary shad-

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