An efficient transformation method for Bacillus subtilis DB104

被引：60

作者：

Vojcic, Ljubica ^{[1
]}

Despotovic, Dragana ^{[1
]}

Martinez, Ronny ^{[1
]}

Maurer, Karl-Heinz ^{[2
,3
]}

Schwaneberg, Ulrich ^{[1
]}

机构：

[1] Rhein Westfal TH Aachen, Lehrstuhl Biotechnol, D-52074 Aachen, Germany

[2] Henkel AG & Co KGaA, Int Res Laundry & Home Care, Biotechnol, D-40191 Dusseldorf, Germany

[3] AB Enzymes GmbH, D-64293 Darmstadt, Germany

来源：

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY | 2012年 / 94卷 / 02期

关键词：

B. subtilis DB104; Directed evolution; Natural competence; Transformation protocol; DIRECTED EVOLUTION; SCREENING SYSTEM; SOLID MEDIA; DNA; CONSTRUCTION; COMPETENCE; PROTEASES;

D O I：

10.1007/s00253-012-3987-2

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

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 (a parts per thousand currency sign10(3) transformants/mu g 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 x 10(5) transformants/mu g plasmid DNA) in B. subtilis DB104 without requiring microgram amounts of DNA.

引用

页码：487 / 493

页数：7

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