Veltman Douwe M, Keizer-Gunnink Ineke, Haastert Peter J M Van
Cell Biochemistry, University of Groningen, Department of Biology, Haren, The Netherlands.
Plasmid. 2009 Mar;61(2):119-25. doi: 10.1016/j.plasmid.2008.11.002. Epub 2008 Dec 13.
Inducible expression systems are essential for the expression of toxic proteins and are very convenient for proteins that induce strong side effects such as retardation of growth or development. Currently available systems for use in Dictyostelium either do not have a very tight control over expression levels or use a combination of an integrating and an extrachromosomal vector. We designed a new vector in which all components of the available 2-plasmid tetracycline-inducible system were combined onto a single extrachromosomal vector. Two types of inducible plasmids are presented, in which transcription is induced by adding or removing doxycycline, respectively. The location and orientation of the components was optimized in order to obtain a low background expression combined with high inducibility. The resulting vectors have a very low expression in the uninduced state (>1000-fold lower expression compared to that resulting from the act15 promoter), show a 10,000-fold induction of gene expression in a doxycycline concentration-dependent manner and are comparatively small (8.5 kb). With these new vectors, inducible gene expression is as easy as constitutive gene expression.
诱导型表达系统对于毒性蛋白的表达至关重要，对于那些会引发强烈副作用（如生长或发育迟缓）的蛋白质而言也非常便利。目前在盘基网柄菌中可用的系统，要么对表达水平的控制不够严格，要么使用了整合型和染色体外载体的组合。我们设计了一种新载体，将现有的双质粒四环素诱导型系统的所有组件整合到单个染色体外载体上。本文展示了两种类型的诱导型质粒，其中转录分别通过添加或去除强力霉素来诱导。对组件的位置和方向进行了优化，以获得低背景表达和高诱导性。所得载体在未诱导状态下表达极低（与act15启动子产生的表达相比，表达降低超过1000倍），在强力霉素浓度依赖性方式下显示出10000倍的基因表达诱导，并且相对较小（8.5 kb）。使用这些新载体，诱导型基因表达与组成型基因表达一样简便。
