Light has emerged as a control input for biological systems due to its precise spatiotemporal resolution. The limited toolset for light control in bacteria motivated us to develop a light-inducible transcription system that is independent from cellular regulation through the use of an orthogonal RNA polymerase. Here, we present our engineered blue light-responsive T7 RNA polymerases (Opto-T7RNAPs) that show properties such as low leakiness of gene expression in the dark state, high expression strength when induced with blue light, and an inducible range of more than 300-fold. Following optimization of the system to reduce expression variability, we created a variant that returns to the inactive dark state within minutes once the blue light is turned off. This allows for precise dynamic control of gene expression, which is a key aspect for most applications using optogenetic regulation. The regulators, which only require blue light from ordinary light-emitting diodes for induction, were developed and tested in the bacterium Escherichia coli, which is a crucial cell factory for biotechnology due to its fast and inexpensive cultivation and well understood physiology and genetics. Opto-T7RNAP, with minor alterations, should be extendable to other bacterial species as well as eukaryotes such as mammalian cells and yeast in which the T7 RNA polymerase and the light-inducible Vivid regulator have been shown to be functional. We anticipate that our approach will expand the applicability of using light as an inducer for gene expression independent from cellular regulation and allow for a more reliable dynamic control of synthetic and natural gene networks.

中文翻译：

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动态蓝光诱导T7 RNA聚合酶（Opto-T7RNAPs）的精确时空基因表达控制。

由于其精确的时空分辨率，光已成为生物系统的控制输入。用于细菌中光控制的有限工具集促使我们开发光诱导的转录系统，该系统独立于通过使用正交RNA聚合酶的细胞调节。在这里，我们介绍了我们的工程化蓝光响应性T7 RNA聚合酶（Opto-T7RNAPs），其具有以下特性：在黑暗状态下基因表达的低渗漏性，在蓝光诱导下的高表达强度以及可诱导范围超过300 -折叠。在优化系统以减少表达差异之后，我们创建了一个变种，一旦关闭蓝光，该变种会在几分钟之内返回非活动状态。这样可以精确动态控制基因表达，对于使用光遗传学调节的大多数应用而言，这是一个关键方面。在细菌中开发并测试了仅需要普通发光二极管发出蓝光进行感应的调节器。大肠杆菌，由于其快速，廉价的培养以及对生理学和遗传学的了解，是生物技术的重要细胞工厂。经过轻微改动的Opto-T7RNAP应该可以扩展到其他细菌种类以及真核生物，例如哺乳动物细胞和酵母，其中T7 RNA聚合酶和光诱导的生动调节子已经显示出功能。我们预计，我们的方法将扩大使用光作为独立于细胞调节的基因表达诱导剂的适用性，并允许对合成和天然基因网络进行更可靠的动态控制。