Most of the gene expression systems available for Gram‐negative bacteria are afflicted by relatively high levels of basal (i.e. leaky) expression of the target gene(s). This occurrence affects the system dynamics, ultimately reducing the output and productivity of engineered pathways and synthetic circuits. In order to circumvent this problem, we have designed a novel expression system based on the well‐known XylS/Pm transcriptional regulator/promoter pair from the soil bacterium Pseudomonas putida mt‐2, in which the key functional elements are physically decoupled. By integrating the xylS gene into the chromosome of the platform strain KT2440, while placing the Pm promoter into a set of standard plasmid vectors, the inducibility of the system (i.e. the output difference between the induced and uninduced state) improved up to 170‐fold. We further combined this modular system with an extra layer of post‐translational control by means of conditional proteolysis. In this setup, the target gene is tagged with a synthetic motif dictating protein degradation. When the system features were characterized using the monomeric superfolder GFP as a model protein, the basal levels of fluorescence were brought down to zero (i.e. below the limit of detection). In all, these novel expression systems constitute an alternative tool to altogether suppress leaky gene expression, and they can be easily adapted to other vector formats and plugged‐in into different Gram‐negative bacterial species at the user's will.

中文翻译：

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XylS/Pm 调节元件的物理解耦和条件蛋白水解能够精确控制恶臭假单胞菌中的基因表达。

大多数可用于革兰氏阴性菌的基因表达系统都受到靶基因相对高水平的基础（即渗漏）表达的影响。这种情况会影响系统动力学，最终降低工程路径和合成电路的输出和生产力。为了规避这个问题，我们设计了一种新的表达系统，该系统基于来自土壤细菌恶臭假单胞菌mt-2 的著名的 XylS/ Pm转录调节剂/启动子对，其中关键功能元件在物理上解耦。通过将xylS基因整合到平台菌株 KT2440 的染色体中，同时将Pm启动子导入一组标准质粒载体后，系统的可诱导性（即诱导状态和未诱导状态之间的输出差异）提高了 170 倍。我们通过条件蛋白水解进一步将该模块化系统与额外的翻译后控制层相结合。在此设置中，目标基因被标记为指示蛋白质降解的合成基序。当使用单体超折叠 GFP 作为模型蛋白表征系统特征时，荧光的基础水平降至零（即低于检测限）。总之，这些新的表达系统构成了一种完全抑制泄漏基因表达的替代工具，它们可以很容易地适应其他载体格式，并根据用户的意愿插入到不同的革兰氏阴性细菌中。