Cell-free synthetic biology approaches enable engineering of biomolecular systems exhibiting complex, cell-like behaviors in the absence of living entities. Often essential to these systems are user-controllable mechanisms to regulate gene expression. Here we describe synthetic RNA thermometers that enable temperature-dependent translation in the PURExpress in vitro protein synthesis system. Previously described cellular thermometers lie wholly in the 5′ untranslated region and do not retain their intended function in PURExpress. By contrast, we designed hairpins between the Shine–Dalgarno sequence and complementary sequences within the gene of interest. The resulting thermometers enable high-yield, cell-free protein expression in an inducible temperature range compatible with in vitro translation systems (30–37 °C). Moreover, expression efficiency and switching behavior are tunable via small variations to the coding sequence. Our approach and resulting thermometers provide new tools for exploiting temperature as a rapid, external trigger for in vitro gene regulation.

中文翻译：

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用于PURExpress系统的RNA温度计

无细胞合成生物学方法使工程化的生物分子系统在缺乏生物实体的情况下表现出复杂的，类似细胞的行为。这些系统通常必不可少的是用户可控制的调节基因表达的机制。在这里，我们描述了能够在PURExpress体外蛋白质合成系统中实现温度依赖性翻译的合成RNA温度计。先前描述的细胞温度计完全位于5'非翻译区域，并且在PURExpress中未保留其预期功能。相比之下，我们在Shine-Dalgarno序列与目标基因内的互补序列之间设计了发夹。所得温度计可在与体外相容的可诱导温度范围内实现高产量，无细胞的蛋白质表达翻译系统（30–37°C）。而且，表达效率和切换行为可通过对编码序列的微小变化来调节。我们的方法和由此产生的温度计提供了利用温度作为体外基因调控的快速，外部触发的新工具。