Applied Microbiology and Biotechnology

, Volume 94, Issue 2, pp 487–493 | Cite as

An efficient transformation method for Bacillus subtilis DB104

  • Ljubica Vojcic
  • Dragana Despotovic
  • Ronny Martinez
  • Karl-Heinz Maurer
  • Ulrich SchwanebergEmail author
Methods and protocols


Bacillus subtilis strains are used for extracellular expression of enzymes (i.e., proteases, lipases, and cellulases) which are often engineered by directed evolution for industrial applications. B. subtilis DB104 represents an attractive directed evolution host since it has a low proteolytic activity and efficient secretion. B. subtilis DB104 is hampered like many other Bacillus strains by insufficient transformation efficiencies (≤103 transformants/μg DNA). After investigating five physical and chemical transformation protocols, a novel natural competent transformation protocol was established for B. subtilis DB104 by optimizing growth conditions and histidine concentration during competence development, implementing an additional incubation step in the competence development phase and a recovery step during the transformation procedure. In addition, the influence of the amount and size of the transformed plasmid DNA on transformation efficiency was investigated. The natural competence protocol is “easy” in handling and allows for the first time to generate large libraries (1.5 × 105 transformants/μg plasmid DNA) in B. subtilis DB104 without requiring microgram amounts of DNA.


B. subtilis DB104 Directed evolution Natural competence Transformation protocol 



This work was supported by the German Government through the Bundesministerium für Bildung and Forschung (Bioindustrie-2021, FKZ0315250) and Henkel AG & Co. KGaA.

Supplementary material

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ESM 1 (DOCX 7 kb)
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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Ljubica Vojcic
    • 1
  • Dragana Despotovic
    • 1
  • Ronny Martinez
    • 1
  • Karl-Heinz Maurer
    • 2
    • 3
  • Ulrich Schwaneberg
    • 1
    Email author
  1. 1.Lehrstuhl für BiotechnologieRWTH Aachen UniversityAachenGermany
  2. 2.International Research Laundry & Home Care, BiotechnologyHenkel AG & Co. KGaADüsseldorfGermany
  3. 3.AB Enzymes GmbHDarmstadtGermany

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