Lehrstuhl für Mikrobiologie, TU-München, Freising, Germany.
J Biotechnol. 2013 Sep 20;167(4):365-9. doi: 10.1016/j.jbiotec.2013.07.026. Epub 2013 Jul 31.
Conjugative shuttle vectors of the pKVM series, based on an IncP transfer origin and the pMAD vector with a temperature sensitive replication were constructed to establish a markerless gene deletion protocol for Bacilli without natural competence such as the exoenzyme producer Bacillus licheniformis. The pKVM plasmids can be conjugated to strains of B. licheniformis and B. subtilis. For chromosomal gene deletion, regions flanking the target gene are fused and cloned in a pKVM vector prior to conjugative transfer from Escherichia coli to B. licheniformis. Appropriate markers on the vector backbone allow for the identification of the integration at the target locus and thereafter the vector excision, both events taking place via homologous recombination. The functionality of the deletion system was demonstrated with B. licheniformis by a markerless 939 bp in-frame deletion of the yqfD gene and the deletion of a 31 kbp genomic segment carrying a PBSX-like prophage.
基于 IncP 转移原点和 pMAD 载体（具有温度敏感复制）的 pKVM 系列可移动穿梭载体被构建，以建立一种无标记基因缺失方案，用于缺乏自然转化能力的芽孢杆菌，如外毒素产生菌地衣芽孢杆菌。pKVM 质粒可以与地衣芽孢杆菌和枯草芽孢杆菌菌株共轭。对于染色体基因缺失，在目标基因侧翼的区域融合并克隆到 pKVM 载体中，然后从大肠杆菌共轭转移到地衣芽孢杆菌。载体骨架上的适当标记允许识别目标基因座的整合，然后进行载体切除，这两个事件都是通过同源重组发生的。通过对 yqfD 基因进行无标记的 939bp 框内缺失和删除携带 PBSX 样噬菌体的 31 kbp 基因组片段，证明了该缺失系统在地衣芽孢杆菌中的功能。
