Applied Microbiology and Biotechnology

, Volume 30, Issue 3, pp 283–289 | Cite as

Electrotransformation of intact and osmotically sensitive cells of Corynebacterium glutamicum

  • Hendrik Wolf
  • Alfred Pühler
  • Eberhard Neumann
Applied Genetics and Regulation


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 E0=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 E0=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.


Lysozyme Cell Regeneration Intact Cell Transformation Efficiency Sensitive Cell 
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Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • Hendrik Wolf
    • 1
  • Alfred Pühler
    • 2
  • Eberhard Neumann
    • 1
  1. 1.Fakultät für ChemieUniversität BielefeldBielefeld 1Federal Republic of Germany
  2. 2.Fakultät für BiologieUniversität BielefeldBielefeldFederal Republic of Germany

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