Department of Microbiology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, People's Republic of China.
J Microbiol Biotechnol. 2010 Jan;20(1):45-53.
We have developed an efficient and precise method for genome manipulations in Bacillus subtilis that allows rapid alteration of gene sequence or multiple gene sequences without altering the chromosome in any other way. In our approach, the Escherichia coli toxin gene mazF, which was used as a counter-selectable marker, was placed under the control of a xylose-inducible expression system and associated with an antibiotic-resistance gene to create a mazF-cassette'. A polymerase chain reaction (PCR)-generated fragment, consisting of two homology regions joined to the mazF-cassette, was integrated into the chromosome at the target locus by homologous recombination, using positive selection for antibiotic resistance. Then, the excision of the mazF-cassette from the chromosome by a single-crossover event between two short directly-repeated (DR) sequences, included in the design of the PCR products, was achieved by counter-selection of mazF. We used this method efficiently and precisely to deliver a point mutation, to inactivate a specific gene, to delete a large genomic region, and to generate the in-frame deletion with minimal polar effects in the same background.
我们开发了一种在枯草芽孢杆菌中进行基因组操作的高效、精确方法，该方法可以在不改变其他染色体的情况下，快速改变基因序列或多个基因序列。在我们的方法中，大肠杆菌毒素基因 mazF 被用作可选择的标记，被置于木糖诱导表达系统的控制之下，并与抗生素抗性基因相连，从而构建了 mazF 盒。通过聚合酶链反应（PCR）生成的片段，由两个与 mazF 盒相连的同源区组成，通过同源重组整合到目标基因座的染色体上，通过抗生素抗性进行正选择。然后，通过在设计的 PCR 产物中包含的两个短直接重复（DR）序列之间的单交换事件，从染色体上切除 mazF 盒，通过 mazF 的反选择实现。我们使用这种方法在同一背景下有效地、精确地实现了点突变、失活特定基因、删除大片段基因组区域以及最小极性效应的框内缺失。
