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 expression levels in response to 465 nm blue light for wild type AsLOV2 and mutated AsLOV2* fusions with and without truncation. (e) Expression of mCherry-LOVtag in response to variable light intensities. Error bars show standard deviation around the mean (n = 3 biological replicates).

Incorporating light responsiveness into diverse proteins with the LOVtag. (a) Control of mCherry repression using a LacI-LOVtag fusion. (b) mCherry expression in response to light exposure for strains with LacI-LOVtag compared to IPTG induction (**p < 0.001, two tailed unpaired t-test). (c) Schematic of SoxS-based CRISPRa activation with a LOVtag 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 LOVtag appended to AcrB of the AcrAB-TolC efflux pump. IM, inner membrane; OM, outer membrane. (f) Minimum inhibitory concentration (MIC) curves of cells cultured in chloramphenicol. Wild type cells (BW25113) are compared to a ΔacrB strain complemented with AcrB-LOVtag and exposed to light or kept in the dark. (g) OD600 of strains shown in (f) at 2.5 μg/ml chloramphenicol (***p < 0.0001, two tailed unpaired t-test). Error bars show standard deviation around the mean (n = 3 biological replicates).

Modulating LOVtag frequency response with photocycle mutations. (a) Photocycle of AsLOV2. Upon light absorption, the Jα helix unfolds for a period of time dictated by 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 LOVtag 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-LOVtag and variant mCherry-LOVtag (V416I) in response to different frequencies of blue light pulses. Fluorescent values are normalized to dark state expression. Error bars show standard deviation around the mean (n = 3 biological replicates).

Enhanced light response using transcriptional control together with the LOVtag. (a) The mCherry-LOVtag expressed from an EL222 responsive promoter that is constitutively active in the absence of EL222. Addition of EL222 results in a circuit that both represses and degrades in response to light. (b) Light and dark expression of mCherry in the ‘degradation only’ (circles) or ‘repression + degradation’ (squares) strains from (a) over time. 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 LOVtag to create optogenetic control. (b) Octanoic acid titer from strains that express EL222 only, CpFatB1*-LOVtag only, or CpFatB1*-LOVtag + EL222 control measured 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; n.s., not significant; two tailed unpaired t-test; n = 3 biological replicates).