| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 3395032 | Anaerobe | 2014 | 5 Pages |
•An electrotransformation protocol for Weissella paramesenteroides DX is described.•A number of parameters that influence the efficiency of transformation were studied.•A high-density electric field pulse of 2.5 kV, 25 μF and 200 Ω was applied.•The obtained transformation efficiencies provide an 8.75-fold increase for pVS44.•Plasmid stability is ensured for 120 generations grown in non-selective medium.
The present investigation sought to provide a reliable and highly efficient electrotransformation method for the lactic acid bacterium Weissella paramesenteroides DX. Experiments were carried out with the shuttle vectors pVS44 (2910 bps), pTRKH3 (7766 bps) and its derivative pTRKH3-1 (4855 bps). Several parameters, including the concentration of transforming plasmid DNA, plasmid size, electric field strength, age of the culture, cell density, and the pretreatment of cells with dl-threonine, lysozyme, and combined treatment with lithium acetate and dithiothreitol, were investigated and proved to influence the efficiency of transformation. Electrocompetence was found to peak in the early stationary phase (OD600 1.2). Other optimized conditions included: the concentration of 10 μg/ml transforming DNA, the cell density of 1010 cells/ml, a high-density electric field pulse of 2.5 kV, 25 μF and 200 Ω, pretreatment of cells with 40 mM dl-threonine and 2000 U/ml lysozyme, and yielded 3.5 × 104 transformants/μg DNA for pVS44 while 1.2 × 104 transformants/μg DNA for the large plasmid TRKH3. Compared to previously reported data, the obtained transformation efficiencies provided an 8.75-fold increase for pVS44 and ensured plasmid stability for 120 generations in non-selective medium.
