嗜热双歧杆菌(B. thermophilum)RBL67 电转化 步骤

All tested electroporation buffers were made in 1 mM citrate buffer at pH 8.0.

1. An overnight pre-culture of B. thermophilum RBL67 in cMRS supplemented with 16 % (w/v) FOS (Cosucra, Warcoing, Belgium) was used to inoculate 8 mL of the same medium at 20%. The culture was incubated anaerobically at 37 °C for 2 – 5 h until an OD600 of 0.7 – 0.9 was reached in the late exponential phase.

2. Thereafter, the cells were chilled on ice for 20 min and harvested by centrifugation (4’000 x g, 4 °C, 5 min). The pellet was washed twice in the appropriate tested electroporation solution (Table 3), then resuspended in 260 µL of the respective solution and used for electroporation. Cells (100 µL) and plasmid DNA were mixed in pre-cooled electroporation cuvettes with an interelectrode distance of 0.2 cm (EquiBio, Witec AG, Littau, Switzerland) and set on ice for 15 min.

3. The cuvettes were placed in a Gene PulserTM (Bio-Rad, Richmond USA) and an electric pulse was delivered using a Pulse ControllerTM apparatus (Bio-Rad). The pulse controller was set at 200 Ω parallel resistance and an electrical capacity of 25 µF.

4. Immediately after electroporation, 1 mL of cMRS with 16 % FOS was pipetted into the cuvettes followed by anaerobic incubation at 37 °C for 3 – 5 h to regenerate the cells. 

5. Thereafter, appropriate dilutions were plated on cMRS plates supplemented with 3 µg/mL chloramphenicol and incubated anaerobically at 37 °C for 5 to 7 days.