Yang Yuqing, Yu Qiaoli, Wang Min, Zhao Rui, Liu Huaiwei, Xun Luying, Xia Yongzhen
State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China.
Institute of Marine Science and Technology, Shandong University, Qingdao, China.
Front Microbiol. 2022 Mar 24;13:838698. doi: 10.3389/fmicb.2022.838698. eCollection 2022.
strains are usually used for cloning to prevent insert instability RecA-dependent recombination. Here, we report that BW25113 ( ) competent cells prepared by using a previously reported transformation and storage solution (TSS) had 100-fold or higher transformation efficiency than the commonly used cloning strains, including XL1-Blue MRF'. The cloning success rates with BW25113 were 440 to 1,267-fold higher than those with XL1-Blue MRF' when several inserts were assembled into four vectors by using a simple DNA assembly method. The difference was in part due to RecA, as the deletion in BW25113 reduced the transformation efficiency by 16 folds and cloning success rate by about 10 folds. However, the transformation efficiency and the cloning success rate of the deletion mutant of BW25113 are still 12- and >48-fold higher than those of XL1-Blue MRF', which is a commonly used cloning strain. The cloned inserts with different lengths of homologous sequences were assembled into four vectors and transformed into BW25113, and they were stably maintained in BW25113. Thus, we recommend using BW25113 for efficient cloning and DNA assembly.
菌株通常用于克隆以防止插入片段不稳定（RecA 依赖性重组）。在此，我们报告称，使用先前报道的转化和储存溶液（TSS）制备的 BW25113（ ）感受态细胞的转化效率比常用的克隆菌株（包括 XL1-Blue MRF'）高 100 倍或更高。当通过简单的 DNA 组装方法将几个插入片段组装到四个载体中时，BW25113 的克隆成功率比 XL1-Blue MRF' 高 440 至 1267 倍。这种差异部分归因于 RecA，因为 BW25113 中的 缺失使转化效率降低了 16 倍，克隆成功率降低了约 10 倍。然而，BW25113 的 缺失突变体的转化效率和克隆成功率仍然比常用的克隆菌株 XL1-Blue MRF' 高 12 倍和 >48 倍。将具有不同长度同源序列的克隆插入片段组装到四个载体中并转化到 BW25113 中，它们在 BW25113 中稳定维持。因此，我们建议使用 BW25113 进行高效克隆和 DNA 组装。
