Engineering of a Promoter Repressed by a Light-Regulated Transcription Factor in Escherichia coli.

Daniel Camsund,Alfonso Jaramillo,Peter Lindblad

doi:10.34133/2021/9857418

Abstract

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.

Highlights

Synthetic gene circuits in bacteria often rely on external inducers, and light offers the ability to regulate cellular processes with unsurpassed spatial and temporal resolution
We exploited the capability of the transcription factor EL222 to undergo light-stimulated dimerization and subsequently strongly bind its DNA target, to engineer a repressible optogenetic one-component system
As the capacity of EL222 to form a dimer and bind the promoter is controlled by light, we hypothesized that such a transcriptional system would be light-regulated

Summary

Introduction

Synthetic gene circuits in bacteria often rely on external inducers, and light offers the ability to regulate cellular processes with unsurpassed spatial and temporal resolution. Exogenous wild-type or modified photoreceptors are heterologously expressed and harnessed to control in vivo biological phenomena such as ion transport, protein interactions, or gene expression [1]. This versatility has led to great interest and diverse applications in, for instance, biomedical research or as characterization tools for synthetic and systems biology [2, 3]. Light-induced transcription factors have been engineered [8,9,10], which do not require membrane localization They rely on light-induced dimerization to bind and positively regulate their target promoters. Potential onecomponent optogenetic repression systems were either not implemented in vivo [11] or repressed gene expression in vivo with limited efficiency [12]

Methods

Results

Conclusion