Curated Optogenetic Publication Database

Abstract: Optogenetics is a powerful approach that enables researchers to use light to dynamically manipulate cellular behavior. Since the first published use of optogenetics in synthetic biology, the field has expanded rapidly, yielding a vast array of tools and applications. Despite its immense potential for achieving high spatiotemporal precision, optogenetics has predominantly been employed as a substitute for conventional chemical inducers. In this short review, we discuss key features of microbial optogenetics and highlight applications for understanding biology, cocultures, bioproduction, biomaterials, and therapeutics, in which optogenetics is more fully utilized to realize goals not previously possible by other methods.

Abstract: Recent progress in protein engineering has established optogenetics as one of the leading external non-invasive stimulation strategies, with many optogenetic tools being designed for in vivo operation. Characterization and optimization of these tools require a high-throughput and versatile light delivery system targeting micro-titer culture volumes. Here, we present a universal light illumination platform – Diya, compatible with a wide range of cell culture plates and dishes. Diya hosts specially-designed features ensuring active thermal management, homogeneous illumination, and minimal light bleedthrough. It offers light induction programming via a user-friendly custom-designed GUI. Through extensive characterization experiments with multiple optogenetic tools in diverse model organisms (bacteria, yeast and human cell lines), we show that Diya maintains viable conditions for cell cultures undergoing light induction. Finally, we demonstrate an optogenetic strategy for in vivo biomolecular controller operation. With a custom-designed antithetic integral feedback circuit, we exhibit robust perfect adaptation and light-controlled set-point variation using Diya.

Abstract: 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.

Abstract: Dynamic control of gene expression can have far-reaching implications for biotechnological applications and biological discovery. Thanks to the advantages of light, optogenetics has emerged as an ideal technology for this task. Current state-of-the-art methods for optical expression control fail to combine precision with repeatability and cannot withstand changing operating culture conditions. Here, we present a novel fully automatic experimental platform for the robust and precise long-term optogenetic regulation of protein production in liquid Escherichia coli cultures. Using a computer-controlled light-responsive two-component system, we accurately track prescribed dynamic green fluorescent protein expression profiles through the application of feedback control, and show that the system adapts to global perturbations such as nutrient and temperature changes. We demonstrate the efficacy and potential utility of our approach by placing a key metabolic enzyme under optogenetic control, thus enabling dynamic regulation of the culture growth rate with potential applications in bacterial physiology studies and biotechnology.