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Digitalizing heterologous gene expression in Gram-negative bacteria with a portable ON/OFF module.
Molecular Systems Biology ( IF 9.9 ) Pub Date : 2019-12-01 , DOI: 10.15252/msb.20188777
Belén Calles 1 , Ángel Goñi-Moreno 1 , Víctor de Lorenzo 1
Affiliation  

While prokaryotic promoters controlled by signal-responding regulators typically display a range of input/output ratios when exposed to cognate inducers, virtually no naturally occurring cases are known to have an OFF state of zero transcription-as ideally needed for synthetic circuits. To overcome this problem, we have modelled and implemented a simple digitalizer module that completely suppresses the basal level of otherwise strong promoters in such a way that expression in the absence of induction is entirely impeded. The circuit involves the interplay of a translation-inhibitory sRNA with the translational coupling of the gene of interest to a repressor such as LacI. The digitalizer module was validated with the strong inducible promoters Pm (induced by XylS in the presence of benzoate) and PalkB (induced by AlkS/dicyclopropyl ketone) and shown to perform effectively in both Escherichia coli and the soil bacterium Pseudomonas putida. The distinct expression architecture allowed cloning and conditional expression of, e.g. colicin E3, one molecule of which per cell suffices to kill the host bacterium. Revertants that escaped ColE3 killing were not found in hosts devoid of insertion sequences, suggesting that mobile elements are a major source of circuit inactivation in vivo.

中文翻译:

使用便携式开/关模块对革兰氏阴性细菌中的异源基因表达进行数字化。

虽然由信号响应调节器控制的原核启动子在暴露于同源诱导物时通常表现出一系列输入/输出比率,但实际上已知没有自然发生的情况具有零转录的关闭状态——这是合成电路理想需要的。为了克服这个问题,我们建模并实现了一个简单的数字化模块,该模块完全抑制其他强启动子的基础水平,从而完全阻碍在没有诱导的情况下的表达。该电路涉及翻译抑制性 sRNA 与目标基因与阻遏物(如 LacI)的翻译偶联之间的相互作用。该数字化仪模块通过强诱导型启动子 Pm(在苯甲酸盐存在下由 XylS 诱导)和 PalkB(由 AlkS/二环丙基酮诱导)进行了验证,并显示在大肠杆菌和土壤细菌恶臭假单胞菌中均有效发挥作用。独特的表达结构允许克隆和条件表达,例如大肠杆菌素E3,每个细胞的一个分子足以杀死宿主细菌。在缺乏插入序列的宿主中没有发现逃脱 ColE3 杀伤的回复体,这表明移动元件是体内电路失活的主要来源。
更新日期:2019-12-19
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