National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
Appl Environ Microbiol. 2013 Mar;79(5):1718-29. doi: 10.1128/AEM.03695-12. Epub 2013 Jan 11.
Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research.
大肠杆菌-分枝杆菌穿梭载体是分枝杆菌中基因表达和基因替换的重要工具。然而，由于缺乏扩展的多克隆位点（MCS）和方便地在克隆的开放阅读框（ORF）上添加表位标签（s），目前大多数可用的载体在使用上受到限制。在这里，我们报告了一系列新的载体，允许在 N 和 C 末端分别添加肽标签序列，从而实现蛋白质的组成型和可调节表达。这些载体的适用性通过组成型和诱导表达结核分枝杆菌 pknK 基因来证明，该基因编码蛋白激酶 K，一种丝氨酸-苏氨酸蛋白激酶。此外，还创建了一个带有扩展 MCS 的自杀质粒，用于创建基因替换，一个用于在常用的 L5 attB 位点进行染色体整合的质粒，以及一个缺氧反应性载体，用于在模拟潜伏状态的缺氧条件下表达基因（s）。此外，我们还创建了一个用于两个由两个独立启动子控制的蛋白质的共表达的载体，每个蛋白质都与不同的标签融合。本研究中开发的穿梭载体是分枝杆菌中基因功能分析的优秀工具，是分枝杆菌研究现有载体库的有价值的补充。
