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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

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 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.

Keywords

Lysozyme Cell Regeneration Intact Cell Transformation Efficiency Sensitive Cell 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523Google Scholar
  2. Chassy BM, Flickinger JL (1987) Transformation of Lactobacillus casei by electroporation. FEMS Microbiol Lett 44:172–177Google Scholar
  3. Eigen M, DeMaeyer L (1963) Relaxation Methods. In: Friess SL, Lewis ES, Weissberger A (eds) Techniques of Organic Chemistry, Vol. 8, Pt. 2, John Wiley, New York, pp 895–1054Google Scholar
  4. Fiedler S, Wirth R (1988) Transformation of Bacteria with Plasmid DNA by Electroporation. Analyt Biochem 170:38–44Google Scholar
  5. Jacob HE, Förster W, Berg H (1981) Microbiological implications of electric field effects, II. Inactivation of yeast cells and repair of their cell envelope. Z Allg Mikrobiologie 21:225–233Google Scholar
  6. Mac Neil DJ (1987) Introduction of plasmid DNA into Streptomyces lividans by electroporation. FEMS Microbiol Lett 42:239–244Google Scholar
  7. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: Laboratory manual. Cold Spring HarbourGoogle Scholar
  8. Martin JF, Santamaria R, Sandoval H, Del Real G, Mateos LM, Gil JA, Aguilar A (1987) Cloning systems in amino acid-producing Corynebacteria. Biotechnol 5:137–146Google Scholar
  9. Miller JF, Dower WJ, Tompkins LS (1988) High-voltage electroporation of bacteria: Genetic transformation of Campylobacter jejuni with plasmid DNA. Proc Natl Acad Sci USA 85:856–860Google Scholar
  10. Neumann E, Rosenheck K (1972) Permeability changes induced by electric impulses in vesicular membranes. J Membrane Biol 10:279–290Google Scholar
  11. Neumann E, Rosenheck K (1973) Potential difference across vesicular membranes. J Membrane Biol 14:194–196Google Scholar
  12. Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH (1982) Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1:841–845Google Scholar
  13. Powell IB, Achen MG, Hillier AJ, Davidson BE (1988) A simple and rapid method for genetic transformation of lactic Streptococci by electroporation. Appl Environ Microbiol 54:655–660Google Scholar
  14. Santamaria R, Gil JA, Martin JF (1985) High-frequency transformation of Brevibacterium lactofermentum protoplasts by plasmid DNA. J Bacteriol 162:463–467Google Scholar
  15. Shillito RD, Saul MW, Paszkowski J, Müller M, Potrykus I (1985) High efficiency direct gene transfer to plants. Biotechnol 3:1099–1103Google Scholar
  16. Shivarova N, Förster W, Jacob H-E, Grigorova R (1983) Microbiological implications of electric field effects VII. Stimulation of plamid transformation of Bacillus cereus protoplasts by electric field pulses. Z Allg Mikrobiol 23:595–599Google Scholar
  17. Somkuti GA, Steinberg DH (1987) Genetic transformation of Streptococcus thermophilus by electroporation. In: Neijssel OM, Van der Meer RR, Luyben KCHAM (eds) Proc 4th Eur Congr Biotechnol, Vol 1, Elsevier, Amsterdam, p 412Google Scholar
  18. Sugar IP, Förster W Neumann E (1987) Model of cell electrofusion, membrane electroporation, pore coalescence and percolation. Biophys Chem 26:321–335Google Scholar
  19. Taketo A (1988) DNA transfection of Escherichia coli by electroporation. BBA 949:318–324Google Scholar
  20. Teissie J, Rols MP (1986) Fusion of mammalian cells in culture is obtained by creating the contact between cells after their electropermeabilization. Biochem Biophys Res Commun 140:258–266Google Scholar
  21. Thierbach G, Schwarzer A, Pühler A (1988) Transformation of spheroplasts and protoplasts of Corynebacterium glutamicum. Appl Microbiol Biotechnol 29:356–362Google Scholar
  22. Yoshihama M, Higashiro K, Rao EA, Akedo M, Shanabruch WG, Follettie MT, Walker GC, Sinskey AJ (1985) Cloning vector system for Corynebacterium glutamicum. J Bacteriol 162:591–597Google Scholar

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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