Random mutagenesis by recombinational capture of PCR products in Bacillus subtilis and Acinetobacter calcoaceticus
Random mutagenesis by recombinational capture of PCR products in Bacillus subtilis and Acinetobacter calcoaceticus
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DOI：
10.1093/nar/27.4.1056
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发表时间：
1999-02-15
影响因子：
14.9
通讯作者：
Youngman, PJ
中科院分区：
文献类型：
--
作者：
Melnikov, A;Youngman, PJ
我们描述了一种通过易错PCR或DNA改组对克隆基因进行随机诱变的一般方法，该方法消除了每个诱变循环后扩增亚克隆的需要。该方法利用了革兰氏阳性细菌枯草芽孢杆菌和革兰氏阴性细菌醋酸钙不动杆菌在自然转化过程中DNA摄取的高效和重组性。设计质粒系统，其允许通过与存在于转化受体中的结构相似的辅助质粒的标记置换重组来捕获PCR扩增的DNA片段。该重组事件同时将扩增的序列转移到辅助质粒中并恢复耐药基因的完整性，从而提供用于片段捕获的直接选择。尽管这种策略在B中足够有效，可以恢复。枯草芽孢杆菌的转化子高达10（3）个转化子/μ g的PCR产物，等效质粒系统类似于A.醋酸钙与B的最佳转化所需的半合成培养基中的两步生长相比，乙酸钙不动杆菌还提供了在普通复合肉汤（如LB）中基本上组成型转化能力的优点。枯草杆菌。
We describe a general method for random mutagenesis of cloned genes by error-prone PCR or DNA shuffling that eliminates the need for post-amplification subcloning following each cycle of mutagenesis, This method exploits the highly efficient and recombinogenic nature of DNA uptake during natural transformation in the Gram-positive bacterium Bacillus subtilis and the Gram-negative bacterium Acinetobacter calcoaceticus. Plasmid systems were designed that allow capture of PCR-amplified DNA fragments by marker-replacement recombination with a structurally similar helper plasmid resident in the transformation recipient, This recombination event simultaneously transfers the amplified sequences into the helper plasmid and restores the integrity of a drug resistance gene, thereby affording a direct selection for fragment capture. Although this strategy was sufficiently effective to permit recovery in B. subtilis of up to 10(3) transformants/mu g of PCR product, equivalent plasmid systems were similar to 100 times more efficient in A. calcoaceticus. Acinetobacter calcoaceticus also offers the advantage of essentially constitutive transformation competence in ordinary complex broth, such as LB, in contrast to two-step growth in semisynthetic media required for optimal transformation of B. subtilis.