Cell-Free Optogenetic Gene Expression System

Abstract

Optogenetic tools provide a new and efficient way to dynamically program gene expression with unmatched spatiotemporal precision. To date, their vast potential remains untapped in the field of cell-free synthetic biology, largely due to the lack of simple and efficient light-switchable systems. Here, to bridge the gap between cell-free systems and optogenetics, we studied our previously engineered one component-based blue light-inducible Escherichia coli promoter in a cell-free environment through experimental characterization and mathematical modeling. We achieved >10-fold dynamic expression and demonstrated rapid and reversible activation of the target gene to generate oscillatory response. The deterministic model developed was able to recapitulate the system behavior and helped to provide quantitative insights to optimize dynamic response. This in vitro optogenetic approach could be a powerful new high-throughput screening technology for rapid prototyping of complex biological networks in both space and time without the need for chemical induction.

Controlling gene expression with light: a multidisciplinary endeavour

“…EL222 fused to an AD has been shown to rapidly induce gene expression in mammalian cells [ 127 ] and zebrafish embryos following irradiation [ 128 ], and has also been used in yeast to improve their chemical production capabilities [ 129 ]. Single component gene expression systems in bacteria [ 130 ] and cell-free expression systems [ 131 ] have used the transcription factor activity of wild type EL222.…”

Bioluminescent Synthetic Cells Communicate with Natural Cells and Self-Activate Light-Responsive Proteins

“…Control over protein expression was performed using the light-inducible bacterial transcription factor EL222, that dimerizes when exposed to blue light and binds to a specific region in the pBLind promoter to initiate transcription (Fig. 4a) 37,38 . To simplify the integration of EL222 into the synthetic cell inner solution, calibration of the required EL222 concentration was initially performed in CFPS reactions (before encapsulation in synthetic cells) using an external LED blue light source for activation.…”

“…Control over protein expression was performed using the light-inducible bacterial transcription factor EL222, that dimerizes when exposed to blue light and binds to a specific region in the pBLind promoter to initiate transcription (Fig. 4a) 37,38 .…”

Reconstituting Natural Cell Elements in Synthetic Cells

“…Synthetic cells are used as chassis for investigating biological processes, including reconstitution of signaling pathways [ 137,138 ] and construction of complex genetic cascades. [ 110 ] Synthetic cells can be chassis for constructing biosensors, [ 139–141 ] for metabolic engineering, [ 142–144 ] for production of viruses; [ 145 ] it was even demonstrated that synthetic cells can shrink solid tumor in live mice. [ 146 ]…”