Department of Pathology and Infectious Diseases, The Royal Veterinary College, Centre for Emerging, Endemic and Exotic Disease, Hawkshead Lane, Hertfordshire, AL9 7TA, United Kingdom.
J Microbiol Methods. 2010 Oct;83(1):34-41. doi: 10.1016/j.mimet.2010.07.014. Epub 2010 Jul 27.
Targeted mutagenesis is one of the major tools for determining the function of a given gene and its involvement in bacterial pathogenesis. In mycobacteria, gene deletion is often accomplished by using allelic exchange techniques that commonly utilise a suicide delivery vector. We have adapted a widely-used suicide delivery vector (p1NIL) for cloning two flanking regions of a gene using ligation independent cloning (LIC). The pNILRB plasmid series produced allow a faster, more efficient and less laborious cloning procedure. In this paper we describe the making of pNILRB5, a modified version of p1NIL that contains two pairs of LIC sites flanking either a sacB or a lacZ gene. We demonstrate the success of this technique by generating 3 mycobacterial mutant strains. These vectors will contribute to more high-throughput methods of mutagenesis.
靶向诱变是确定特定基因功能及其在细菌发病机制中的作用的主要工具之一。在分枝杆菌中，基因缺失通常通过使用自杀载体的等位基因交换技术来完成。我们已经使用连接独立克隆（LIC）使一种广泛使用的自杀载体（p1NIL）适应于克隆基因的两个侧翼区域。产生的 pNILRB 质粒系列允许更快、更高效和更少费力的克隆程序。在本文中，我们描述了 pNILRB5 的构建，这是 p1NIL 的一个修饰版本，其中包含两对侧翼为 sacB 或 lacZ 基因的 LIC 位点。我们通过生成 3 株分枝杆菌突变株证明了该技术的成功。这些载体将有助于更多的高通量诱变方法。
