1Microbial Chemistry, Department of Chemistry-Ångström, Uppsala University, Uppsala, Sweden
2Molecular Systems Biology, Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden
3Warwick Integrative Synthetic Biology Centre (WISB) and School of Life Sciences, University of Warwick, Coventry, UK
4Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, Evry, France
5Institute for Integrative Systems Biology (I2SysBio), CSIC – Universitat de València, 46980 Paterna, Spain
| Received 27 Apr 2021 |
Accepted 23 Aug 2021 |
Published 28 Sep 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 PEL that in combination with the light-regulated dimer EL222 constitutes a one-component repression system. Inspired by previously engineered synthetic promoters and the Escherichia coli lacZYA promoter, we designed PEL with two EL222 operators positioned to hinder RNA polymerase binding when EL222 is bound. PEL 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 PEL-EL222 system herein presented could aid the design and implementation of analogous one-component optogenetic repression systems. Finally, we compare the PEL-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.
