Electrotransformation of intact and osmotically sensitive cells of Corynebacterium glutamicum

Summary

Intact and osmotically sensitive cells of Corynebacterium glutamicum can be efficiently transformed by electroporation. This was shown by using the plasmid vector pUL-330 (5.2 kb), containing the kanamycin resistance gene of transposon Tn5. The following electric parameters yielded efficient transformation. For intact cells: one exponentially decaying field pulse $E = E_0 exp( - t/\tau _E )$ with time constants $\tau _E = 450 - 500$ and with initial field intensities of E ₀=35–40 kV cm^-1; prepulse temperature 20°C. Cell regeneration (survival) was 100%–80%. Transformation efficiency can be increased by an additional freeze and thaw cycle of the cells, prior to electroporation. Lysozyme treated cells (osmotically sensitive) were transformed with three successive pulses of E ₀=25–30 kV cm^-1. Cell regeneration under these conditions was found to be 20–30%. The optimum yield of transformants/μg plasmid-DNA was 3×10³ for intact cells, 2×10⁴ for intact cells which were frozen and thawed twice and 7×10⁴ for osmotically sensitive cells if the cell suspension was pulsed at a cell density of 1–3×10⁸/ml and at a DNA concentration of 0.2 μg/ml up to ≤2 μg/ml. The data obtained for osmotically sensitive cells suggest that the temperature increase accompanying the electric field pulse enhances colony formation and transformation efficiency if the initial prepulse temperature is ≥20°C, although regeneration of electroporated C. glutamicum cells starts to decrease at temperatures≥20°C.