Camsund Daniel, Jaramillo Alfonso, Lindblad Peter
Microbial Chemistry, Department of Chemistry-Ångström, Uppsala University, Uppsala, Sweden.
Molecular Systems Biology, Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.
Biodes Res. 2021 Sep 28;2021:9857418. doi: 10.34133/2021/9857418. eCollection 2021.
Light-regulated gene expression systems allow controlling gene expression in space and time with high accuracy. Contrary to previous synthetic light sensors that incorporate two-component systems which require localization at the plasma membrane, soluble one-component repression systems provide several advantageous characteristics. Firstly, they are soluble and able to diffuse across the cytoplasm. Secondly, they are smaller and of lower complexity, enabling less taxing expression and optimization of fewer parts. Thirdly, repression through steric hindrance is a widespread regulation mechanism that does not require specific interaction with host factors, potentially enabling implementation in different organisms. Herein, we present the design of the synthetic promoter P that in combination with the light-regulated dimer EL222 constitutes a one-component repression system. Inspired by previously engineered synthetic promoters and the promoter, we designed P with two EL222 operators positioned to hinder RNA polymerase binding when EL222 is bound. P is repressed by EL222 under conditions of white light with a light-regulated repression ratio of five. Further, alternating conditions of darkness and light in cycles as short as one hour showed that repression is reversible. The design of the P-EL222 system herein presented could aid the design and implementation of analogous one-component optogenetic repression systems. Finally, we compare the P-EL222 system with similar systems and suggest general improvements that could optimize and extend the functionality of EL222-based as well as other one-component repression systems.
光调控基因表达系统能够在空间和时间上高精度地控制基因表达。与先前整合了需要定位在质膜上的双组分系统的合成光传感器不同，可溶性单组分抑制系统具有几个有利特性。首先，它们是可溶的，能够在细胞质中扩散。其次，它们更小且复杂性更低，使得表达负担更小且需要优化的部件更少。第三，通过空间位阻进行抑制是一种广泛存在的调控机制，不需要与宿主因子进行特异性相互作用，这有可能使其在不同生物体中得以应用。在此，我们展示了合成启动子P的设计，它与光调控二聚体EL222组合构成了一个单组分抑制系统。受先前工程化的合成启动子和[此处原文缺失相关启动子名称]启动子的启发，我们设计的P带有两个EL222操纵子，当EL222结合时，它们的位置会阻碍RNA聚合酶的结合。在白光条件下，P被EL222抑制，光调控抑制率为5。此外，在短至一小时的明暗交替循环条件下，结果表明抑制是可逆的。本文展示的P - EL222系统的设计有助于类似单组分光遗传学抑制系统的设计与应用。最后，我们将P - EL222系统与类似系统进行了比较，并提出了一些一般性的改进建议，这些建议可以优化和扩展基于EL222的以及其他单组分抑制系统的功能。
