Design of AsLOV2-based degradation tag. (a) Primary sequence of AsLOV2(546) C-terminal sequence. A three amino acid truncation exposes E-A-A. (b) Structure of AsLOV2 (aa404-546, PDB: 2V1A). Amino acids 541-543 (E-A-A) are red and 544-546 (K-E-L) are gray at the C-terminal of the Jα helix. (c) Construct used to characterize optogenetic control using AsLOV2 variants. Each variant is translationally fused to mCherry expressed from an IPTG inducible promoter. Variants include wild type AsLOV2 (light blue) and a dark state stabilized version, AsLOV2* (dark blue), with and without the three amino acid truncation. (d) mCherry protein levels in response to 465 nm blue light for wild type AsLOV2, and mutated AsLOV2* fusions with and without truncation. AsLOV2*(543) is the variant we denote the ‘LOVdeg’ tag. (***p < 0.0001; **p < 0.001; *p<0.01; n.s., not significant; two tailed unpaired t-test; n = 3 biological replicates). (e) mCherry-LOVdeg in response to variable light intensities. (f) mCherry fluorescence levels and optical density of mCherry-LOVdeg with 4 hours of 465 nm blue light exposure applied at different points in the growth cycle. Light exposure programs are plotted above each subplot and are staggered 2 hours apart (starting at 2, 4, 6, or 8 hours), all lasting 4 hours. Expression levels are normalized to the dark state control (Fig. S8). Error bars show standard deviation around the mean.

Incorporating light responsiveness into diverse proteins with the LOVdeg tag. (a) Control of mCherry repression using a LacI-LOVdeg fusion. (b) mCherry expression in response to light exposure for strains with LacI-LOVdeg compared to IPTG induction (**p < 0.001, two tailed unpaired t-test). (c) Schematic of SoxS-based CRISPRa activation with a LOVdeg tag appended to the MCP-SoxS activator. (d) CRISPRa control of mRFP1 expression in response to light (***p < 0.0001, two tailed unpaired t-test). (e) Schematic of the LOVdeg tag appended to AcrB of the AcrAB-TolC efflux pump. IM, inner membrane; OM, outer membrane. (f) Chloramphenicol sensitivity tests. Wild type cells (BW25113) are compared to a ΔacrB (BW25113 ΔacrB) strain, ΔacrB complemented with AcrB-LOVdeg (ΔacrB + AcrB-LOVdeg) exposed to light or kept in the dark, and ΔacrB strain complemented with an IPTG-inducible AcrB (ΔacrB + AcrB). No IPTG was added to ΔacrB + AcrA or ΔacrB + AcrB-LOVdeg. (g) OD600 of strains shown in (f) at 2.5 μg/ml chloramphenicol (**p < 0.001; *p < 0.05; ns, not significant; two tailed unpaired t-test). Error bars show standard deviation around the mean (n = 3 biological replicates).

Modulating LOVdeg frequency response with photocycle mutations. (a) Photocycle of AsLOV2. Upon light absorption, the Jα helix unfolds for a period of time dictated by the stability of the light state conformation. If not degraded, the Jα helix refolds, blocking degradation. (b) The light program used to test frequency responses of LOVdeg photocycle variants in (c). A constant pulse of 5 sec is followed by a variable dark time that allows for Jα helix refolding. (c) Expression of mCherry-LOVdeg and variant mCherry-LOVdeg (V416I) in response to different light exposure frequencies. Fluorescence values are normalized to dark state expression. Error bars show standard deviation around the mean (n = 3 biological replicates).

Enhanced light response using EL222 transcriptional control together with LOVdeg. (a) mCherry-LOVdeg expressed from an EL222 responsive promoter that is constitutively active in the absence of EL222. Addition of EL222 represses mCherry production. These two forms of regulation are combined when mCherry-LOVdeg is expressed from an EL222 responsive promoter, resulting in a circuit that both degrades and represses in response to light. (b) Light and dark expression of mCherry in the ‘degradation only’ (closed circles), ‘repression only’ (open circles), or ‘repression + degradation’ (squares) strains. Error bars show standard deviation around the mean (n = 3 biological replicates).

Optogenetic control of octanoic acid production. (a) Schematic of fatty acid synthesis in E. coli. CpFatB1* catalyzes elongating C8-ACP molecules from this pathway to produce free octanoic acid. CpFatB1* is tagged with a LOVdeg tag to create optogenetic control. (b) Octanoic acid titer from strains that express CpFatB1* only, CpFatB1*-LOVdeg only, EL222 regulated CpFatB1* only, or CpFatB1*-LOVdeg + EL222. Octanoic acid is quantified by GC-MS. Strains were kept either in the dark or with continuous blue light exposure for the duration of the production period. Error bars show standard deviation around the mean (****p < 0.0001; ***p < 0.0001; ns, not significant; two tailed unpaired t-test; n = 3 biological replicates).