Improved Electro-transformation Protocol for Bacillus subtilis DB104

枯草桿菌Bacillus subtilis DB104電轉型法之改進

葉娟美(Chuan-Mei Yeh) ； 王志鵬(Jyh-Perng Wang) ； 蘇芳仙(Fang-Sian Su)

《臺灣農業化學與食品科學》 43卷5期 (2005/10) Pp. 368-375

https://doi.org/10.6578/TJACFS.2005.046

摘要

目前已發表的電轉型法應用於枯草桿菌Bacillus subtillis DB104時，轉型效率均低於10^3轉型株/μg DNA，無法應用於黏合質轉的直接轉型。本研究中，改進酥胺酸弱化細胞壁之勝任細胞電轉型法，以提高電轉效率。以8.75kV/cm，500Ω電擊強度及SHMYPT電轉緩衝液，可改進電轉效率至10^4轉型株/μg DNA。電轉後立刻冰浴2.5分鐘，並於37℃，120rpm條件下，於SB再生培養基中震盪培養2小時，可提高電轉效率至10^5轉型株/μg DNA。本改進可應用於黏合質體於枯草桿菌Bacillus subtilis DB104之直接電轉型，並可避免轉型過程中基因發生壓抑、重排或刪除。

關鍵字

電轉型 ； 枯草桿菌 ； 黏合質體

並列摘要

The most up-to-date electro-transformation protocols achieve electro-transformation efficiencies of no higher than 10^3 transformants/μgDNA in Bacillus subtilis DB104. These protocols are not applicable in direct transfer of ligated plasmids. In this study, an improved protocol was developed. Several factors were examined in threonine cultured competent cells, with the aim to increase the electro-transformation efficiency. A pulsation period of 8.75kV/cm, 500Ω pulse strength, in SHMYPT buffer improved the efficiency to 10^4 transformants/μg DNA. Immediate ice incubation for 2.5 minutes followed by regeneration in super broth recovery medium at 37°C, 2 hours shaking at 120 rpm increased the efficiency to 10^5 transformants/μg DNA. This improved protocol enables the direct transformation of ligated mixtures in B. subtilis DB104 and avoids genetic changes such as suppression, rearrangement or deletion.

並列關鍵字

Electro-transformation ； Bacillus subtilis ； Ligated plasmid