Disposable cuvette

To obtain sufficient quantities of the constructed plasmids (pNZ8148/BLF, pNZ8148/HLF, and pNZ8148/PLF) for electroporation, they were introduced into E. coli TG1 using the CaCl₂ method in combination with the heat shock approach [26 ]. Then, a large quantity of plasmid DNA was extracted using the Viogene DNA extraction kit. For the preparation of electrocompetent cells, probiotic strains were grown in MRS broth at 37 °C to an optimal optical density at 600 nm (OD₆₀₀) of 0.3 to 0.5, after which the bacteria were ice bathed for at least 10 min. Bacterial cells were harvested by centrifugation (5000× g, 15 min, 4 °C), and subsequently washed three times with cold distilled water. Finally, the cells were resuspended in 100 μL of 10% glycerol. Electroporation was carried out by mixing 100 μL of resuspended cells with 0.3 to1 μg of plasmid DNA. The suspension was transferred to a disposable cuvette (Bio-Rad Laboratory, Richmond, CA, USA) with a 0.2 cm electrode gap and subjected to an electric pulse using a MicroPulser (Bio-Rad Laboratory, Richmond, CA, USA). Transformed cells were diluted in 1 mL of recovery medium and incubated at 37 °C for 3 h. Finally, transformed cells were cultured on MRS plates or in MRS broth supplemented with 2.5 to 20 µg/mL chloramphenicol for the selection of transformants.