bifidum PRL2010 cells, which were cultivated in the presence of different complex carbohydrates such as FOS or GOS. Interestingly, PRL2010 transformants were isolated when cells were grown in MRS supplemented

with FOS at a final content of 16% as well as with MRS enriched by 10% GOS with a transformation efficiency of 103 CFU μg−1 DNA (Table 2). Such findings may be explained by the effects that these oligosaccharides have on the composition of the cell wall as well as on other cell envelope constituents (e.g. decreased thickness of capsular polysaccharide layers and/or reduction of the cell wall/capsular complexity). Furthermore, the presence of a high amount of complex carbohydrates in the growth medium may exert a protective action against the stressful conditions encountered by bifidobacterial cells during transformation (Guglielmetti et al., 2008). Previous studies Protein Tyrosine Kinase inhibitor have reported that the composition of the bacterial cell wall, and consequently the efficiency of DNA uptake, seems to be significantly influenced by the growth phase of the bacterial cells (Rossi et al., 1996). Thus, based on the growth curve of B. bifidum PRL2010 cells cultivated on MRS, we harvested PRL2010 cells at different

time points corresponding to early (OD value of 0.4) and late exponential phase (OD value of 0.7) (Fig. 1). Subsequently, such cells were submitted to the electroporation procedure, and corresponding transformation Selleckchem CH5424802 efficiency was evaluated (Table 2). Notably, the maximal transformation efficiency

was observed when PRL2010 cells were collected at late log phase (Table 2). Incubation of the cells in an electroporation buffer was found to be crucial for Bifidobacterium transformation (Argnani et al., 1996). We observed that storage Decitabine cell line of bacterial cells for two hours before electroporation at 4 °C in an electroporation buffer composed of 16% FOS or 10% GOS and 1 mM citrate buffer (pH 6.0) significantly improved their transformation efficiency, increasing from < 102 to 104 CFU per μg DNA. Under these conditions, we assume that the low molarity of ammonium citrate acts as an osmotic stabilizer that supports controlled cell envelope removal/degradation without affecting cell viability, which may then result in improved cell wall permeability for exogenous DNA. Resistances of 100 or 200 Ω and voltages between 7.5 and 12.5 kV cm−1 were tested. Optimal results were obtained when the voltage applied to the cuvette was 12.5 kV cm−1 and the resistance was set at 200 Ω. When the resistance was set at 100 Ω, no transformants was observed. The transformation efficiency achieved with a voltage of 7.5 kV cm−1 and a resistance of 200 Ω was low (Table 2). After incubation, the transformants were selected on MRS supplemented with chloramphenicol and incubated at 37 °C. The presumptive transformants were verified by colony PCR using primers based on the DNA sequence of pNZ8048.