Electrotransformation of intact and osmotically sensitive cells of Corynebacterium glutamicum |
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Authors: | Hendrik Wolf Alfred Pühler Eberhard Neumann |
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Affiliation: | (1) Fakultät für Chemie, Universität Bielefeld, Postfach 86 40, D-4800 Bielefeld 1, Federal Republic of Germany;(2) Fakultät für Biologie, Universität Bielefeld, D-4800 Bielefeld, Federal Republic of Germany |
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Abstract: | 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
with time constants
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/g 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 g/ml up to 2 g/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 temperatures20°C. |
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