B. longum 105-A and B. infantis JCM 1222T and their derivatives were cultured in 8 ml of GAM. Cells at the exponential phase (OD600 = 0.4 to 0.6) were harvested by centrifugation (19,000g, 5 min, 4°C for B. longum and 5000g, 3 min, 4°C for B. infantis), washed twice with the same volume of ice-cold 1 mM ammonium citrate (pH 6.0) buffer containing 50 mM sucrose, and resuspended in 400 μl (for B. longum) or 200 μl (for B. infantis) of the same solution. Then, the cells (50 μl) were electroporated with 0.1 to 3.0 μg of plasmid DNA (<5 μl) that was isolated from E. coli DH5α with the Wizard Plus SV Minipreps DNA Purification System (Promega, Madison, WI). Gene Pulser Xcell (Bio-Rad Laboratories, Hercules, CA, USA) was used for electroporation under the condition of 10 kV/cm, 25 μF, and 200 ohms (0.2-cm cuvette). The pulsed cells were immediately mixed with 1 ml of anaerobically stored GAM (Nissui). After 3 hours of anaerobic incubation, the cells were spread onto GAM agar plate (Nissui) containing appropriate antibiotics. The plates were incubated anaerobically until colonies were visible (typically 2 to 4 days).

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