Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland.
Department of Life Sciences, Imperial College London, London, UK.
Sci Rep. 2018 Jun 22;8(1):9539. doi: 10.1038/s41598-018-27687-5.
Molecular research on mycobacteria relies on a multitude of tools for the genetic manipulation of these clinically important bacteria. However, a uniform set of vectors allowing for standardized cloning procedures is not available. Here, we developed a versatile series of mycobacterial vectors for gene deletion, complementation and protein production and purification. The vectors are compatible with fragment exchange (FX) cloning, a recently developed high-throughput cloning principle taking advantage of the type IIS restriction enzyme SapI and its capacity to generate sticky trinucleotide ends outside of its recognition sequence. FX cloning allows for the efficient cloning into an entry vector and the facile transfer of the sequenced insert into a variety of destination vectors. We generated a set of mycobacterial expression vectors spanning a wide range of expression strengths, tagging variants and selection markers to rapidly screen for the optimal expression construct in order to purify membrane proteins from the model organism Mycobacterium smegmatis. Further, we generated a series of suicide vectors containing two counterselection markers and used them to delete twenty genes encoding for potential drug efflux pumps in M. smegmatis. The vectors will further facilitate genetic and biochemical research on various mycobacterial species.
分子生物学研究依赖于大量的工具来对这些具有临床重要性的细菌进行遗传操作。然而，目前还没有一套通用的载体能够实现标准化的克隆操作。在这里，我们开发了一系列通用的分枝杆菌载体，可用于基因缺失、互补和蛋白质的生产和纯化。这些载体与片段交换（FX）克隆兼容，FX 克隆是一种最近开发的高通量克隆原理，利用了 II 型限制酶 SapI 及其在识别序列之外产生粘性三核苷酸末端的能力。FX 克隆允许高效地克隆到入口载体中，并将测序插入片段轻松转移到各种目的载体中。我们生成了一组分枝杆菌表达载体，涵盖了广泛的表达强度、标记变体和选择标记，以便快速筛选出最佳的表达构建体，从而从模式生物耻垢分枝杆菌中纯化膜蛋白。此外，我们还生成了一系列含有两个反向选择标记的自杀载体，并将其用于删除耻垢分枝杆菌中 20 个编码潜在药物外排泵的基因。这些载体将进一步促进各种分枝杆菌物种的遗传和生化研究。
