用于自然可转化的泰国伯克霍尔德氏菌和类鼻疽伯克霍尔德氏菌的敲除和拔出重组。
Knockout and pullout recombineering for naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei.
机构信息
Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii, USA.
出版信息
Nat Protoc. 2011 Jul 7;6(8):1085-104. doi: 10.1038/nprot.2011.346.
Phage λ-Red proteins are powerful tools for pulling and knocking out chromosomal fragments but have been limited to the γ-proteobacteria. Procedures are described here to easily knock out (KO) and pull out (PO) chromosomal DNA fragments from naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei. This system takes advantage of published compliant counterselectable and selectable markers (sacB, pheS, gat and the arabinose-utilization operon) and λ-Red mutant proteins. pheS-gat (KO) or oriT-ColE1ori-gat-ori1600-rep (PO) PCR fragments are generated with flanking 40- to 45-bp homologies to targeted regions incorporated on PCR primers. One-step recombination is achieved by incubation of the PCR product with cells expressing λ-Red proteins and subsequent selection on glyphosate-containing medium. This procedure takes ~10 d and is advantageous over previously published protocols: (i) smaller PCR products reduce primer numbers and amplification steps, (ii) PO fragments suitable for downstream manipulation in Escherichia coli are obtained and (iii) chromosomal KO increases flexibility for downstream processing.
噬菌体 λ-Red 蛋白是用于提取和敲除染色体片段的有力工具，但仅限于γ-变形菌。本文介绍了一种简便的方法，可从天然可转化的伯克霍尔德菌和鼻疽伯克霍尔德菌中敲除（KO）和提取（PO）染色体 DNA 片段。该系统利用了已发表的符合要求的可反向选择和可选择标记（sacB、pheS、gat 和阿拉伯糖利用操纵子）和 λ-Red 突变蛋白。pheS-gat（KO）或 oriT-ColE1ori-gat-ori1600-rep（PO）PCR 片段是通过在 PCR 引物上加入侧翼 40-45bp 的同源性来生成针对目标区域的片段。通过将 PCR 产物与表达 λ-Red 蛋白的细胞孵育，并随后在含有草甘膦的培养基上进行选择，可实现一步重组。该过程耗时约 10 天，优于以前发表的方案：（i）较小的 PCR 产物减少了引物数量和扩增步骤，（ii）获得了适合在大肠杆菌中进行下游操作的 PO 片段，（iii）染色体 KO 增加了下游处理的灵活性。
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