Light inducible protein degradation inE. coliwith the LOVdeg tag

Abstract

Molecular tools for optogenetic control allow for spatial and temporal regulation of cell behavior. In particular, light controlled protein degradation is a valuable mechanism of regulation because it can be highly modular, used in tandem with other control mechanisms, and maintain functionality throughout growth phases. Here, we engineered LOVtag, a protein tag that can be appended to a protein of interest for inducible degradation in Escherichia coli using blue light. We demonstrate the modularity of LOVtag by using it to tag a range of proteins, including the LacI repressor, CRISPRa activator, and the AcrB efflux pump. Additionally, we demonstrate the utility of pairing the LOVtag with existing optogenetic tools to enhance performance by developing a combined EL222 and LOVtag system. Finally, we use the LOVtag in a metabolic engineering application to demonstrate post-translational control of metabolism. Together, our results highlight the modularity and functionality of the LOVtag system, and introduce a powerful new tool for bacterial optogenetics.

Light-directed evolution of dynamic, multi-state, and computational protein functionalities

“…LOV domains make up the core of most non-ion-channel optogenetic systems [39][40][41][42][43][44][45][46][47][48][49][50][51][52][53] , presumably because they are small, reliable, and do not need exogenous chromophores. Interest in LOV domains is reflected in multiple diversification campaigns 34,[54][55][56] .…”

Dynamic Multiplexed Control and Modeling of Optogenetic Systems Using the High-Throughput Optogenetic Platform, Lustro

“…Such optogenetic technologies empower researchers to orchestrate cellular processes with exquisite spatiotemporal precision. Optogenetic technologies have found diverse applications, ranging from modulating gene expression [4][5][6][7] , dissecting intricate signaling pathways [8][9][10] , manipulating protein localization [11][12][13][14][15] , or inducing targeted protein degradation 16,17 . If independent and simultaneous control of distinct optogenetic systems is possible, engineering multiple optogenetic systems into the same cell or community of cells allows for a higher degree of control over complex biological functions.…”

Dynamic Multiplexed Control and Modeling of Optogenetic Systems Using the High-Throughput Optogenetic Platform, Lustro

“…Optogenetics leverages genetically encoded light-sensitive proteins fused to biological effectors to precisely control cellular behavior in response to light. − Such optogenetic technologies empower researchers to orchestrate cellular processes with exquisite spatiotemporal precision. Optogenetic technologies have found diverse applications, ranging from modulating gene expression, − dissecting intricate signaling pathways, − manipulating protein localization, − or inducing targeted protein degradation. , If independent and simultaneous control of distinct optogenetic systems is possible, engineering multiple optogenetic systems into the same cell or community of cells allows for a higher degree of control over complex biological functions. This independent control can be achieved via multiplexing, where multiple control signals are sent over a shared medium, in this case light. , One approach is orthogonal multiplexing, where optogenetic systems responsive to different wavelengths of light are used.…”