Molecular Biology. Practical Guide - Velikov V.A. 2013

Bacterial Transformation
Preparation of Competent E. coli Cells

Трансформацию бактерий очищенной ДНК впервые осуществили Мандел и Хига (Mandel, Higa, 1970). При инкубации ДНК фага лямбда λ с клетками E.coli в растворе СаСl₂ при температуре 0°С произошло проникновение ДНК фага внутрь бактериальных клеток, что в дальнейшем привело к размножению фаговых частиц и лизису клеток бактериальной культуры. То есть произошло то же самое, что и при инфекции природным фагом. Однако в норме «голую» ДНК бактериальные Клетки поглощать не способны (кроме гемофильных и ряда грам-положительных).

Further Development of the method proceeded empirically. The modern Procedure involves the preparation of so-called "competent" E. coli Cells capable of taking up foreign DNA, a "heat Shock" that facilitates DNA entry into The Cell, and the Selection of transformed cells by cultivating the bacterial culture under selective conditions.

During E. coli transformation, Plasmids enter only a very small fraction (0.01–5%) of the cells. To select for transformants carrying recombinant DNA, selective markers are used, such as Antibiotic Resistance genes. Many vectors contain the ampicillin resistance Gene bla. It encodes the enzyme β-lactamase, which cleaves the lactam ring of ampicillin (App. 3, 4). Following the transformation procedure, the Bacteria are plated onto nutrient Agar containing ampicillin. As a result, only those cells that have taken up the plasmid and synthesized active β-lactamase survive and form colonies. These E. coli clones are referred to as transformants. Other vectors carry resistance genes for kanamycin and tetracycline (see App. 1).

Thus, cells that have received the recombinant plasmid—that is, transformed or genetically modified cells—acquire The ability to proliferate on nutrient media containing Antibiotics, whereas antibiotics exert a bacteriostatic or bactericidal effect on the rest of the bacterial culture.

Electrotransformation Methods developed later exhibit a significantly higher efficiency compared to chemical or Ca²⁺-mediated transformation. These techniques require specialized equipment for cell membrane electroporation—electroporators (Works 8.3–8.4).

The described procedure involves generating competent E. coli cells by incubating them at a low Temperature in a solution containing Ca²⁺ cations (Inoue, Nojima, Okayama, 1996). To enhance transformation efficiency, various substances are added to the solution alongside CaCl₂. The resulting calcium-competent cells should be used immediately (Work 8.2). If this is not feasible for any reason, they can be stored in a refrigerator at +4°С for a maximum of 1–2 days until they completely lose their competence. Therefore, it is preferable to snap-freeze them in liquid nitrogen and store them at -70°С. All Procedures involving competent Cells must be performed on ice, using trays filled with finely crushed ice or snow.

Materials and Equipment

E. coli XL1-Blue strain, dimethyl sulfoxide (DMSO).

Solutions

- SOB medium. 2% tryptone; 0.5% Yeast extract; 10 mM NaCl; 2.5 mM KCl; 10 mM MgCl₂; 10 mM MgSO₄.

- TB buffer. 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, PIPES or BES can be used); 15 mM CaCl₂; 55 mM MnCl₂; 250 mM KCl.

Procedure

1. Streak bacteria onto a selective SOB agar plate and incubate overnight at 37°C.

2. Inoculate 40 ml of SOB medium in a 0.5–1 L flask with 10–12 single E. coli colonies (approximately 2–3 mm in diameter). Grow with vigorous shaking on an orbital shaker at 200–300 rpm until reaching

an optical density OD₆₀₀ ⁼ 0.6 at 37°C ^a. This typically takes 2–2.5 hours. Perform all subsequent procedures on ice.

3. Transfer 30 ml of the cell culture into a 50 ml centrifuge tube and incubate on ice for 10–15 minutes.

4. Centrifuge for 10 minutes at 3000 rpm and +4°C. Discard the supernatant.

5. Centrifuge for 20 seconds at 3000 rpm and +4°C. Remove the remaining liquid using a pipette.

6. Resuspend the cells in 10 ml of TB buffer and incubate on ice for 10–15 minutes.

7. Repeat steps 4 and 5.

8. Resuspend the cell pellet in 2 ml of TB buffer.

9. Add the cryoprotectant DMSO to a final concentration of 3.5% (70 µl) and incubate on ice for 10–15 minutes.

10. Repeat step 4. Resuspend the cells in the residual liquid.

11. Aliquot 100 µl into pre-chilled 1.7 ml tubes. Use the "calcium competent cells" immediately or freeze them in liquid nitrogen.

Note

^a Culturing bacteria at 37°C yields a moderate level of competence. When E. coli cultures are grown at 18°C, cell competence is enhanced, but the time required to reach the desired density increases to several days.