An Optimized Transformation Protocol for Escherichia coli BW3KD with Supreme DNA Assembly Efficiency.
An Optimized Transformation Protocol for Escherichia coli BW3KD with Supreme DNA Assembly Efficiency.
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具有最高 DNA 组装效率的优化大肠杆菌 BW3KD 转化方案
DOI：
10.1128/spectrum.02497-22
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发表时间：
2022-12-21
影响因子：
3.7
通讯作者：
中科院分区：
文献类型：
--
作者：
DNA克隆需要两个步骤：重组DNA分子的组装以及将产物转化到宿主生物体中进行复制。这两个过程的高效率可以提高成功率。最近，我们开发了大肠杆菌BW3KD菌株，其转化效率比常用的克隆菌株更高。在此，我们进一步开发了一种简单的感受态细胞制备方法，名为TSS-HI（通过Hannahan和Inoue方法优化的转化储存溶液），该方法结合了三种常用方法的操作简单和转化效率高的优点。采用该方法制备BW3KD感受态细胞时，转化效率高达(7.21±1.85) × 109 CFU/μg DNA，超过了市售化学感受态细胞和自制电感受态细胞的水平。 BW3KD 细胞在溶源肉汤琼脂平板上 7 小时内形成菌落，比众所周知的快速生长的大肠杆菌克隆菌株 Mach1 更快。感受态细胞可有效地一步转化1至7个片段组装的DNA，并提高大质粒转化或克隆的效率。该方法制备的BW3KD细胞的克隆效率比常规方法制备的大肠杆菌XL1-Blue MRF'细胞的克隆效率提高了828倍。因此，感受态细胞适用于不同的克隆工作，并有助于满足生物学研究和生物技术中对 DNA 组装不断增长的需求。重要性 DNA 转化通常用于克隆；然而，高转化效率成为许多应用的限制因素，例如CRISPR和DNA文库的构建、多个片段的组装以及大质粒的转化。我们开发了一种新的感受态细胞制备方法，具有无与伦比的转化效率。采用该方法制备的大肠杆菌BW25113细胞BW3KD菌株，其转化效率高达(7.21±1.85) × 109 CFU/μg DNA，打破了化学制备感受态细胞的记录。由于该菌株生长速度快，常规克隆只需1天即可完成。感受态细胞对于大质粒的转化或克隆以及多个片段的组装非常有效。结果强调了新方案的有效性以及 BW3KD 菌株作为宿主的有用性。
DNA cloning requires two steps: the assembly of recombinant DNA molecules and the transformation of the product into a host organism for replication. High efficiencies in both processes can increase the success rate. Recently, we developed an Escherichia coli BW3KD strain with higher transformation efficiency than commonly used cloning strains. Here, we further developed a simple method named TSS-HI (transformation storage solution optimized by Hannahan and Inoue method) for competent cell preparation, which combined the advantages of three common methods for operational simplicity and high transformation efficiency. When competent BW3KD cells were prepared using this developed method, the transformation efficiency reached up to (7.21 ± 1.85) × 109 CFU/μg DNA, which exceeded the levels of commercial chemically competent cells and homemade electrocompetent cells. BW3KD cells formed colonies within 7 h on lysogeny broth agar plates, quicker than the well-known fast-growing E. coli cloning strain Mach1. The competent cells worked effectively for the transformation of assembled DNA of 1 to 7 fragments in one step and promoted efficiencies of transformation or cloning with large plasmids. The cloning efficiency of BW3KD cells prepared by this method increased up to 828-fold over that of E. coli XL1-Blue MRF′ cells prepared by a common method. Thus, competent cells are suitable for different cloning jobs and should help with the increased demand for DNA assembly in biological studies and biotechnology. IMPORTANCE DNA transformation is commonly used in cloning; however, high transformation efficiency becomes a limiting factor in many applications, such as the construction of CRISPR and DNA libraries, the assembly of multiple fragments, and the transformation of large plasmids. We developed a new competent cell preparation method with unmatched transformation efficiency. When the BW3KD strain, derived from Escherichia coli BW25113 cells, was prepared by this method, its transformation efficiency reached up to (7.21 ± 1.85) × 109 CFU/μg DNA, which broke the record for chemically prepared competent cells. Routine cloning could be completed in 1 day due to the high growth rate of this strain. The competent cells were shown to be highly efficient for transformation or cloning with large plasmids and for the assembly of multiple fragments. The results highlight the effectiveness of the new protocol and the usefulness of the BW3KD strain as the host.
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DOI：
10.1128/genomea.01038-14
发表时间：
2014-10-16
期刊：
Genome announcements
影响因子：
--
作者：
Grenier F;Matteau D;Baby V;Rodrigue S
通讯作者：
Rodrigue S
影响因子：
13.8
作者：
Hengen, PN
通讯作者：
Hengen, PN
影响因子：
--
作者：
Green, Michael R;Sambrook, Joseph
通讯作者：
Sambrook, Joseph
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
5.4
作者：
Altermark, Bjorn;Niiranen, Laila;Moe, Elin
通讯作者：
Moe, Elin