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Nucleic Acids Research, 2003, Vol. 31, No. 21 e133
© 2003 Oxford University Press

One step assembly of multiple DNA fragments with a designed order and orientation in Bacillus subtilis plasmid

Kenji Tsuge, Kuniko Matsui and Mitsuhiro Itaya*

Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooya, Machida-shi, Tokyo 194–8511, Japan

*To whom correspondence should be addressed. Tel: +81 42 724 6352; Fax: +81 42 724 6316; Email: ita{at}libra.ls.m-kagaku.co.jp

A universal method to reconstitute sets of genes was developed. Owing to the intrinsic nature of the plasmid establishment mechanism in Bacillus subtilis, the assembly of five antibiotic resistance genes with a defined order and orientation was achieved. These five fragments and the plasmid have three-base protruding sequences at both ends. The protruding sequences are designed so that each fragment is ligated once in a row according to the pairing. Ligation by T4 DNA ligase in the presence of 150 mM NaCl and 10% polyethylene glycol at 37°C yielded high molecular tandem repeat linear form DNA. This multimeric form of DNA was preferentially used for plasmid establishment in B.subtilis. The method, referred to as Ordered Gene Assembly in B.subtilis (OGAB), allows for the design of multiple fragments with very high efficiency and great fidelity.


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