Enhancing the performance of Magnets photosensors

Domain: biology
Published: 2026-03-18
Editorial outcome: major_revision

Abstract
Photosensory protein domains, derived from nature, are foundational for optogenetic protein engineering. Tailoring their properties enables their full exploitation for optogenetic regulation in basic research and applied bioengineering applications. Here, we present a simple, yet powerful strategy based on random mutagenesis coupled to high-throughput screening that allowed altering the most fundamental properties of the widely used nMag/pMag photodimerization system: its light sensitivity and activation. Variants were characterized in vivo in bacteria by flow cytometry and during the entire growth curve by spectrofluorometry. We identify mutations that either increase or decrease the light sensitivity at sub-saturating light intensities, while also improving the light activation and dark-to-light fold change. Notably, light sensitivity and activation levels could be changed independently. In addition, we demonstrated that the shapes of the dose-response curves can be finely tuned. This broadens the applicability of the Magnets photosensors for optogenetic regulation strategies.

Introduction
In bioengineering, light has emerged as a versatile input to steer and control various biological processes.1,2 It is crucial for photosynthetic processes or protection against harmful effects of electromagnetic radiation, such as photooxidative cellular damage. Organisms have evolved a variety of photosensitive proteins called “photoreceptors” or “photosensors”, which allow them to react to a light stimulus. Depending on their function, these domains can register different wavelengths and intensities of light. Light is usually absorbed through an organic chromophore, which induces structural changes within the apoprotein1,2. These structural changes revert to the ground or “dark” state of the photoreceptor in a thermally driven reaction.

Optogenetics employs such photosensors or photosensory domains for the engineering of genetically encoded light-sensitive proteins1–3. Since the development of the first genetically engineered light-sensitive proteins4, light has been used in different optogenetic approaches to interfere with and control a variety of cellular functions3,5,6. The use of light as input is particularly attractive because it enables precise spatial and temporal applications,7,8 both with a higher resolution when compared to chemical induction. Furthermore, in non-photosensitive organisms such as Escherichia coli, light is an orthogonal input that does not require uptake and/or conversion as with small molecule inducers.9–11 Thus, the use of light allows for minimally invasive spatiotemporal control.

Photosensitive domains are the key components of optogenetics. In general, optogenetic proteins comprise a photosensory domain as well as an actuating module.3,5 For example, many cellular sensors such as kinases initiate signaling and cellular responses through oligomerization.12,13 Such proximity-based regulation is also the basis of various natural light-sensing modules in which the interaction, and therefore the distance of the interaction partners, is controlled through light in a mechanism that leads to either homodimerization or oligomerization of the same photoregulator or heterodimerization and oligomerization of two or more different photoregulators. Such proximity control can be used to assemble inactive subcomponents into an active protein, or for recruitment of an active protein to a specific location of action. Light-controlled dimerization domains have been used to implement optogenetic control usually through homodimerization of e.g. receptors14–20 or heterodimerization for split proteins 8,21–23 as well as subcellular localization4,24,25.

A widely used class of photosensors are light, oxygen, and voltage (LOV) domains as they have features that are often especially attractive for optogenetic protein (Opto-protein) engineering, such as a small domain size and tunable kinetic properties. LOV domains employ flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are present in most organisms, as chromophores. One of the most used photosensory protein heterodimerization pairs is the “Magnets”26, which are derived from the homodimerization protein VIVID (VVD). The LOV domain VVD, from the filamentous fungus Neurospora crassa27, has been used in numerous optogenetic designs (14,19,28–39). Blue light induces a conformational change in the LOV domain, which initiates the homodimerization of two VVD domains and dimer stabilization.27,40,41 Based on a truncated version of this photosensory domain27, the Magnets heterodimerization system was created through rational protein engineering.26 For this, positively and negatively charged amino acids were introduced into the VVD dimer interface to create complementary pMag and nMag domains.26,42 We and others used these Magnets domains for the reconstitution of split proteins8,29–32 due to their small size (150 amino acids) , favorable structure in which N- and C-termini of the two domains come in proximity in the dimeric state, and low dark-state activity as well as reversion rate tunability.8 In prior work, mutations in the Magnets domains and VVD were identified using rational structure-guided protein engineering. These approaches enabled changing their kinetic properties20,26,27,43,44 or optimizing functionality in mammalian cells45.

The Opto-T7RNAP is a light-inducible transcription system that incorporates the VVD-based Magnets photoregulators into the heterologous T7 RNA polymerase (Figure 1 A). It consists of two split fragments of the T7RNAP, which are fused to the light-inducible heterodimerizing Magnet domains. nMagHigh1 is fused to the C-terminus of the T7RNAP N-terminus and pMag is fused to the N-terminus of the C-terminal T7RNAP fragment. Light induces a conformational change in the Magnet domains which leads to the binding of the two complementary nMagHigh1 and pMag domains. The resulting spatial proximity of the T7RNAP fragments reconstitutes the function of the enzyme, leading to the transcription of genes from T7 promoters. The screening system consists of two plasmids, one containing the two Opto-T7RNAP genes under the control of arabinose-inducible promoters, and a reporter plasmid containing the red fluorescent protein mCherry expressed from the T7 promoter. The gene expression system shows high activation with blue light induction and simultaneously low residual expression in the dark.8 This ratio between the expression level in blue light and in the dark is called the light-induced fold change. A crucial aspect for the use of this system during a screen is that it offers a high dynamic range, which enables screening at sub-saturating induction levels (Figure 1 B, C). We characterized this transcription system via the expression of the red fluorescent protein mCherry (Figure 1 A). We investigated the effect of different ratios of the two Opto-T7RNAP split parts and observed that adjusting the relative ratios of the two components led to different levels of reporter gene expression. However, the dose-response curves, and thus the light sensitivity of the Opto-T7RNAPs, were not changed due to the altered regulator expression levels. (compare Figure 2 BC and Figure S11 “Light dose-response curve is not changed by different domain expression levels” of Baumschlager et al. 8). Thus, changes in light sensitivity cannot be achieved by adjusting the ratios of the Opto-T7RNAP parts.

Furthermore, we investigated how the expression levels of both Opto-T7RNAP parts influence the properties of the system. Since both components are expressed from the same arabinose-inducible promoter, this was achieved by varying the concentration of the arabinose inducer. Despite an increased Opto-T7RNAP protein expression, we only observed moderate (4-fold) changes in the resulting reporter expression comparing both dark and light-induced conditions separately (Figure 4 of the publication Baumschlager et al.8). In addition, we observed that only relatively high arabinose induction (more than 0.05% arabinose) leads to significant changes in the resulting output (same figure). These observations suggest that the inherent light-sensing parameters of this system cannot simply be addressed through changing the levels of the regulatory proteins or by altering the relative concentrations of the individual split components.

Further, when incorporating the previously identified photocycle mutations I85V (pMagFast1) and I74V, I85V (pMagFast2), we could only observe a change in the dose-response curve when pMagFast2 was incorporated. (Figure 4 of Baumschlager et al.8) For both variants, we observed large changes in the reporter expression level at saturating conditions (Figure 4, Figure S13 of Baumschlager et al.8). Further, only the variant containing pMagFast2 exhibited a change in the light dose-response curve (Figure 4 and Figure S11 of Baumschlager et al.8), and only the other variant pMagFast1 showed faster off-switching kinetics. Importantly, this suggests that different properties of photosensitive proteins, such as light sensitivity, lit-state lifetime, or light-activation can be tuned independently. These results prompted us to investigate these hypotheses further, which resulted in the need to develop a method that enables tuning of different optogenetic parameters independently.

Here, we envisioned that the direct phenotypic output of our transcription system in E. coli enables one to screen for the desired photoregulator properties using a high-throughput approach. These properties range from “light sensitivity” alone to “light activation” alone or a combination of both, which extends previous protein engineering efforts. Light sensitivity is used in this work to describe changes in light intensities that lead to half-maximal activation. In contrast, light activation describes changes in the resulting reporter protein expression level in this manuscript. Fold change or light-induced fold change is defined as the ratio between the reporter expression in light and the expression in dark.

Specifically, in this work we aimed to alter the most fundamental properties of this photosensory domain: its light sensitivity and its light activation. Highly light-sensitive photosensors, for example, bear the advantage that lower-intensity light can be applied to achieve similar outputs. This reduces potential phototoxic effects (e.g. mammalian cell lines46), aids in light delivery into denser cell cultures5,47, and minimizes heat development by the light application. The same can be achieved if the activity of the optogenetic regulator is increased (light activation), even if the shape of the dose-response curve is not changed. In other scenarios, lower light sensitivity might be preferred, e.g., if a regulator shall not be activated by ambient light, for expression of (potentially) toxic proteins, or for multiplexing when combined with other, more sensitive, light controllers.

To allow for screening of Magnets variants with increased or decreased light sensitivities and light activation, we used the Opto-T7RNAP*(568), which directly links the activity of the photosensitive domain to a detectable fluorescence output, thus creating a genotype-phenotype linkage that allows for screening of variants with altered photosensitivity. Through a single round of mutagenesis, we identified several mutations in both the nMagHigh1 as well as the pMag domain, some of which led to dramatically increased light sensitivities while maintaining or even improving the dark-to-light fold-change or changing the light activation (reporter expression levels). While the mutations identified here will expand the applicability of these photosensory domains and enable a wider range of applications, we anticipate that our approach can also be used to identify mutations that change other properties such as dark-state reversion rate or enhancement of fold change, which will further expand the applicability and toolset of VVD-based photoregulators in particular, and optogenetic approaches in general.

Results
Library and experimental designs.
We used error-prone PCR (epPCR) to diversify both photosensory domains (nMagHigh1 and pMag) of our Opto-T7RNAP*(563) individually (Figure 1E B). Both Magnets domains are based on a 36-residue N-terminal truncated VVD photoregulator27, and differ in several amino acids that were rationally introduced to enable heterodimerization.26 nMagHigh1 is C-terminally fused to the N-terminal part of the T7RNAP, while pMag is N-terminally fused to the C-terminal part of the T7RNAP (Figure 1 A). Although both photosensory domains are derived from VVD, we chose to create libraries for each individually (Figure 1 A-B), as they differ in amino acid positions I52 and M55 that confer complementary binding, as well as M135 and M165 for nMagHigh1.26 This leaves the possibility for different compensatory mutations for each domain, as well as accounts for their fusion to either the N- or C-terminal part of the T7RNAP.8 These libraries were then transformed separately into E. coli strain AB3608, which contained a plasmid for expression of mCherry under T7RNAP control (Figure 1 B). We used two different ribosome binding sites (RBS) strengths, first the published RBS from pAB508, as well a version denoted “pAB50-11k” in which we weakened the TIR using the RBS calculator 2.048 to 15% of the original RBS (Supplementary Figure 1). The use of these two different expression strengths in separate libraries should allow one to find higher light activation variants while limiting the effect of metabolic burden induced through mutations that cause higher mCherry expression levels which might obscure a screening.

We chose photosensitivity and photoactivation as the target properties for tuning for four main reasons: First, as previously mentioned, the ability to sense light effectively is the defining feature of a photoregulator, and engineering enzymes for the direct improvement of activities on their natural function is often unsuccessful and generally considered challenging49. Second, a potential drawback of optogenetic methods for the control of cells is light-induced cellular damage caused by high-intensity light and/or long illumination durations. While this effect might be less pronounced in bacteria like E. coli, it can be problematic in higher organisms such as mammalian cell lines. Third, depending on the light application setting, a high light intensity can produce heat, which in turn might interfere with experiments or applications. Fourth, a set of photoregulators with different sensitivities for the same wavelength will enable the multiplexing of expression levels of different proteins, as well as the activation of genetic circuitry through variation of the light intensity.

Library induction and screening strategy.
We applied a multi-step Fluorescence-Activated Cell Sorting (FACS) for the screening and selection of variant libraries with altered light sensitivities/activation (Figure 1 C). For this, we used different sequences of induction conditions: No light induction (further denoted “dark”), in which variants with mCherry expression comparable to the non-induced original Opto-T7RNAP*(563) were sorted for, or with light intensities of 0.96 and 3.85 W m-2 (further denoted as low or high light induction respectively), which correspond to intermediate or saturating light induction (Figure 1 D). The example in Figure 1 C shows the screening approach for variants with increased light sensitivity. For the first enrichment (Figure 1 C left) nMagHigh1 or pMag libraries were induced with non-saturating light for 4h and then sorted for high mCherry expression, while cells with low or no mCherry fluorescence were discarded. Most variants showed reduced-to-non-functional mCherry expression, like the negative control (Supplementary Figure 2, left), indicating that most mutations inactivated the function of the photosensor, as to be expected through random mutagenesis. This removed the bulk of the library containing mutations that inactivated or reduced the light sensitivity of the photosensory domain. The enriched libraries were re-grown in the dark and then sorted for low mCherry expression for the second enrichment (Figure 1 C middle). The second step served to select variants that still showed a low dark state and removed constitutively active variants. These enriched libraries were then again induced for 4h with low-intensity 465-nm light blue light and single cells spotted on Omnitrays with LB-agar to isolate individual variants (Figure 1 C right), which were subsequently regrown in M9 medium and screened. Individual mutations were identified by Sanger sequencing and recloned into the original plasmid. For final characterization, cultures were grown and measured both through flow cytometry and monitored periodically through spectrophotometry until the stationary growth phase (Figure 1 E) as described in the Methods section.

Identified nMagHigh1 and pMag variants.
We compared the mCherry expression of the identified variants to the expression of the original wild-type Opto-T7RNAP*(563)8 and a negative control, containing only the mCherry reporter plasmid and an empty backbone with the same antibiotic resistance as the plasmid harboring the Opto-T7RNAP. For this, we took two experimental approaches: First, through single-cell analysis using flow cytometry measurement at a single time point, and second, through spectrophotometry over time.

Single-cell characterization of variants through flow cytometry was performed after 5h expression in log growth phase. This data served to extract changes in the basal dark (denoted b), as well as maximal light-induced (denoted t) mCherry expression, which are important to assess changes in the Opto-T7RNAP light activation. For the changes in light sensitivities, we compared the light intensities that lead to half-maximal gene expression (denoted I50), which were obtained from fits of the light-induced dose-response of mCherry expression to a Hill-type equation, as described in the Material and Methods section. Second, we also characterized the variants on the population level through spectrophotometry over time. The data were analyzed both using hierarchical clustering as well as through fits of the mCherry expression data of a single time point to a Hill-type equation to obtain the light-induced dose-response curve parameters.

After just one round of mutagenesis, we selected 19 different variants displaying a variety of interesting properties (Figure 2 at 37ºC, Figure 3 A-B at 30ºC, and Figure 3 C-D at 40ºC; shown in more detail in Supplementary Figure 3 and Supplementary Figure 4). From these variants, 5 single and 3 double mutations were found in nMagHigh1, and 9 single and 2 double mutations were located in pMag (Supplementary Table 1). All calculated properties for b, t, and I50 as well as percentage changes compared to the WT are summarized in Supplementary Table 2-Supplementary Table 12 with the underlying data shown in Supplementary Figure 5-Supplementary Figure 20. Apart from these variants, we also isolated a double mutant in the pMag library (M135I, M165I), which was previously described as pMagHigh126, and showed a higher mCherry expression level compared to pMag in the screening, thereby confirming the ability of this setup and approach to identify variants with improved properties for a given experimental setup.

Reproducible robotic culture inoculation, followed by characterization of the variants on the population level through spectrophotometry over time, further allowed us to compare population growth curves. Both the negative and the wild-type controls did not show apparent changes in their growth curves due to the light illumination and the mCherry reporter expression. This was also the case for most nMagHigh1 and pMag variants, with the exceptions of pMag P42T T69M, pMag R52L, pMag N100L, and pMag P66S Y94N at 30ºC. These variants showed a clearly decreased hill slope in the growth curve with increasing light intensity (Hill slopes shifted from 0.3013 to 0.1941 for pMag P42T T69M, from 0.3127 to 0.1858 for pMag R52L, from 0.2736 to 0.2012 for pMag N100L, from 0.3123 to 0.2174 for pMag P66 S Y94N in the dark and with 5.77 Wm-2 light induction respectively; see Supplementary Table 13 and Supplementary Table 14) compared to both the wild type (from 0.302 to 0.2631 Hill slope in the dark and with 5.77 Wm-2 light induction respectively; see Supplementary Table 13) as well as the negative control (from 0.3096 to 0.329 Hill slope in the dark and with 5.77 Wm-2 light induction respectively; see Supplementary Table 13). A lower hill slope corresponds to a slower growth rate (compare growth curves at 30ºC in Supplementary Figure 21, 37 ºC in Supplementary Figure 22 and 40ºC in Supplementary Figure 23). As a result, given similar starting ODs, also the time for the culture density to reach half maximum (T50) was shifted to later time points correspondingly and causing an up to 2 h delay for the two highest expressing variants pMag P42T T69M and pMag R52L, while for both the negative control and the wild type, no delay in reaching the T50 point was detected when comparing dark to 5.77 Wm-2 light induction conditions (see Supplementary Table 13 and Supplementary Table 14). These growth differences reflect that these four variants show the highest expression levels of all variants and that expression at 30ºC was highest in the three incubation conditions (30ºC, 37ºC, and 40ºC). This behavior can be explained by the strong mCherry reporter overexpression, which led to metabolic burden effects that result in reduced growth in light-induced conditions.

Alongside manual comparison and the extraction of relevant enzyme parameters as mentioned above, we also performed a principal component (PC) analysis of the datasets. The first PC could explain over 95% of the variance in the flow cytometry data (Supplementary Figure 24 A-C). In the case of the spectrophotometry data acquired over time, the first PC could explain only 62% of the variance. We reasoned that if the data were not scaled before the PC analysis, this would prevent giving excessive weight to the fluorometric data obtained at low OD values, when the bacterial cultures are starting to grow and gene expression could not be detected yet. Indeed, using non-scaled data, the first PC could explain over 95% of the variance in the data (Supplementary Figure 24 D-F). In both sets of data, all the variants were nicely distributed along the PC1 (x-axis) according to their expression level (compare with Figure 2 A-B and Figure 3 A-B). Visual inspection of the PCA plots suggested that the variants can be clustered according to their expression levels. We performed hierarchical clustering (HC) of the complete flow cytometry dataset of the different light intensities and the complete dataset of the spectrometry data over time and at the different light intensities using the Ward clustering method with Euclidean distances50 (Figure 3 E-F). Four clusters were identified that contained variants with very high (red), high (orange), medium (green), and low (blue) expression profiles (Figure 3 E-F). The negative control (NC) not harboring the Magnets constructs was in the low expression cluster, accordingly (reporter expression time courses shown in Supplementary Figure 25). Visual inspection of the PC and HC cluster analyses showed that they were in agreement. Therefore, this classification of clusters was used for the analysis of the data that was gathered at different incubation temperatures during the characterization of the mutations. Despite the two datasets using different measurement methods (single-cell by flow cytometry and population by fluorometry) and time considerations (single timepoint and expression measured over time), only slight changes were observed when comparing the corresponding dendrograms at the screening temperature (37 ºC) (Supplementary Figure 26). The red and orange clusters contained variants with mutations in the pMag, apart from M48V N100L, while the green and blue clusters contained variants with mutations both in pMag and nMag.

nMagHigh1 variants with increased light sensitivity and improved light activation at 37ºC.
The nMagHigh1 variants L39P and V160M were located in the medium expression (green) cluster (Figure 3 E) and showed an increased light sensitivity, described with a decrease in the half-maximal light intensity (I50=80 and 83% respectively compared to WT, Figure 2 A, Supplementary Table 2). We also observed a higher light activation, presumably due to improved dimerization properties in these variants, visible through the increased expression at a saturating light intensity (t=151 and 148% compared to WT respectively, Figure 2 A), while also showing increased basal expression (b=150 and 148% compared to WT, Figure 2 A). Interestingly, a double mutant also containing a mutation at position 39 (L39R) with a second mutation T38A located in the low expression cluster (blue) showed an even further increased light sensitivity compared to the single mutant (I50=68% compared to the WT). Together with the lower light activation, visible through reduced basal- and light-induced reporter expression (b=84%, t=77%, Figure 2 A), this indicates that expression levels and light sensitivity can be tuned separately. In addition, we identified the double mutant M48V N100L, which shows a dramatically increased light sensitivity (I50=50% compared to the WT, light dose-response curves, and expression at different intensities over time of WT and M48V N100L, Figure 2 C- D). In addition, both basal- and light-induced expression are increased in this variant (b=296%, t=267% compared to the WT, Figure 2 A). Accordingly, this variant clustered in the high expression group (orange).

nMagHigh1 variants with decreased light sensitivity at 37ºC.
nMagHigh1 variants with decreased light sensitivity all clustered in the low expression group (blue). This includes mutations A143S and V147M (with an I50=143 and 162%, respectively compared to the WT, Figure 2 E, Supplementary Table 2) that displayed expression levels comparable or slightly reduced compared to the WT (b=91 and 89%, t=95, and 95% respectively, Figure 2 C). Also here, expression level and light sensitivity were tuned separately. In addition, double mutant I135V K137R showed an increase in the half-maximal light intensity (I50=158% compared to the WT), a reduced dark expression (b=88%), and a slight increase in the maximal light-induced expression (t=111%, n.s., Figure 2 A).

nMagHigh1 variant with shifted light activation at 37ºC.
Variant K137E showed a light sensitivity that is comparable to the WT (I50=98%, Figure 2 F). However, the reporter gene expression was upshifted for both the dark- as well as the light-induced expression (t=128%, b=133% compared to the WT, Figure 2 C, Supplementary Table 2). Interestingly, this variant also clustered in the low expression group.

pMag variants with increased light sensitivity and light activation at 37ºC.
All pMag variants, except for positions N100, were unique (Figure 2 B) compared to the ones previously described for nMagHigh1 (Figure 2 A). While N100S was found in pMag, a mutation at position N100 was also found in nMagHigh1 double mutant M48V N100L. pMag variants N100L, N100K, and N100S as single mutations were all identified during site saturation mutagenesis (see below). All variants R52L, T69M, T83M, N100K, N100L, N100S, K119N, M165T, S178Y, P42T T69M, and P66S Y94N showed an increased light sensitivity of up to 47% compared to the WT (I50=79, 50, 47, 71, 59, 89, 78, 50, 64, 52 and 83% respectively, Supplementary Table 4). The increase in light sensitivity was most pronounced in variants T69M, T83M, N100L, M165T, and P42T T69M, all belonging to the high or very high expression clusters (I50=50, 47, 59, 50, and 52% respectively, Figure 2 G-I, Supplementary Table 4). In addition, for all variants except for K119N (located in the low expression cluster), the expression levels were increased, which was most pronounced (higher than 300%) in variants T69M, N100L, and P42T T69M up to 540% (t=326, 540 and 517% respectively) and comparably less increased for the basal expression (b=257, 362 and 422% respectively). In contrast, variant K119N showed a light activation and thus expression levels comparable to the WT enzyme (b=121%, t=105%) while having an increased light sensitivity (I50=78%, Figure 2 J), again indicating that these properties can be tuned separately. Comparing both T69M variants, the single (high expression cluster, orange) and double mutant with P42T (very high expression cluster, red), light sensitivities are similar (I50=50 and 52%), while expression levels are upshifted in the double mutant (b=257 and 422%, t=326 and 527% respectively), indicating an even further improved dimer assembly through the additional P42T substitution.

Dynamic off-switching at 37ºC.
The observed changes in light sensitivity and expression level could potentially also be caused by changes in the dynamic off-kinetics, although this was not specifically screened for. To investigate these effects, a pseudo-time course was performed (Supplementary Figure 27). Cells were induced with constant saturating light intensity (3.85 W m-2) for 120 min before turning off the light. nMagHigh1 variants with increased (M48V N100L, I50 = 50%), similar (K137E, I50=98%) and decreased (I135V K137R, I50=158%) light sensitivity at 37ºC were chosen (Figure 2 D, F, Supplementary Table 2-Supplementary Table 3). In addition to their difference in light sensitivity, M48V N100L also shows overall the highest expression of all identified nMagHigh1 variants. For pMag, N100L was chosen along with the two variants containing either T69M, either alone or in combination with P42T. N100L and P42T T69M are the two highest expressing pMag variants at 37℃ (Figure 2 H-I, Supplementary Table 4-Supplementary Table 5). All variants, except for T69M, follow the WT dynamics closely, suggesting no major change in the off-kinetics. The variant with mutation T69M shows sustained activity after the light input is removed, which might be due to slower dark-state reversal. A mutation at the same position (T69W), which is located at the dimer interface, was previously described to cause a constitutively active homodimer.41 One hypothesis could be that T69M renders the magnet domain either constitutively active or slower reverting, which could make inactivation mainly or soleely dependent on the complementary Magnet domain, which could be mechanistically investigated in follow-up studies.

Site saturation mutagenesis at hotspot positions T69 and N100.
Random mutagenesis can be used to identify important amino acid residues and positions. We chose the two sites T69 and N100 in the very high expression clusters (red) for further investigation through site saturation mutagenesis (SSM) as they appeared multiple times during our screening both as single and as double mutants and in addition showed highly interesting properties. T69M as single mutant, as well as double mutant with P42T, was one of the variants with the highest low light sensitivity in the pMag photosensor. N100 was the only residue in which variants were selected in both the nMagHigh1 as well as the pMag libraries, and in both cases increased the light sensitivity of the photosensor. As both mutations were found in the pMag photosensor, we also used this domain to construct SSM libraries either using the “22-codon trick”51 or as a “small intelligent”52 library. From these libraries, 93 randomly selected colonies were picked per 96-well plate, as well as three WT controls in the same plate and incubated either in the dark or induced with low-intensity light as previously described.

SSM of position T69 was performed with a reduced “small intelligent”52 library with NDT, VMA and TGG for position T69, but lacking the ATG, as T69M was already identified. Due to the large number of variants to screen, we only chose two light intensities (subsaturating 0.95 W m-2 and saturating 3.78 W m-2 for WT Opto-T7RNAP) and dark controls for the screening. Although this does not allow for the fitting of the dose-response model and identification of parameters b, t, and I50, it still enables one to identify variants that show higher light sensitivity or activation than what was previously identified. As shown in the inset in Supplementary Figure 28 A, T69, shown as red spheres, lies in the interface of the photoactivated VVD dimer. As a result of the SSM, only T69W was identified to perform significantly better at low light induction compared to the WT, while T69L and T69F performed similarly to the WT. However, variant T69M found during the screening outperformed T69W for mCherry expression in low as well as high light induction conditions (Supplementary Figure 28 B). Interestingly, despite T69W was reported to form VVD dimers in the dark41, here we found no difference in mCherry expression between the WT and the T69W in the dark, noting that the mutation is only present in one of the heterodimerization domains. T69M showed a 2.4-fold higher light activation when comparing low light expression levels to the WT expression level (Supplementary Figure 28 C). However, also the activation of T69W was improved 1.7-fold. For both T69M and T69W, but also for the leucine, phenylalanine and tryptophan substitution, the fold change at low light induction was improved by 2.8-, 1.5-, 1.6- and 1.3-fold respectively (Supplementary Figure 28 D). In general, all mutations improving mCherry expression are amino acids with hydrophobic side chains and therefore might play a role in the dimer formation, while substitutions that decreased the function of the photoregulator were mostly hydrophilic or polar e.g. aspartic acid, arginine, or serine (Supplementary Figure 28 A-D). In summary, no variant outperforming T69M, obtained during the initial screening, could be identified by site saturation mutagenesis of this residue.

SSM of position N100 was performed using the “22-codon trick”51. The position is indicated by red spheres, located at the surface of the protein close to the Ncap (Supplementary Figure 28 E inset). During the screening, we already identified N100L in combination with M48V and N100S. N100L showed the highest expression level at low light induction (Supplementary Figure 28 E-F), which was higher than the levels obtained with the double mutant M48V N100L (Figure 2 A-B). The light activity of the single mutant was increased 9.9-fold for the expression at the low light intensity (Supplementary Figure 28 G), compared to 5.7-fold for the double mutation, suggesting that M48V might have a slightly detrimental effect which is overcompensated by N100L in the double mutant compared to the WT. N100L also showed an increase in the fold change at low light intensity of 3.6-fold. Further, methionine, lysine, and arginine, along with the previously identified serine showed increased light activation of 3.2-, 2.9-, 2.9-, and 2.5-fold respectively. The dark-to-light fold change was also improved in all of these variants at low light intensities with the highest improvement of 3.6-fold for N100L and slightly improved or similar to the WT at high light intensities (Supplementary Figure 28 H). The different properties of improving mutations ranging from polar via positively charged to hydrophobic side chains do not allow for clear mechanistic conclusions and require additional assessment in future studies. Also for position N100, no further variant with a higher light sensitivity and activation than N100L could be identified by site saturation mutagenesis of this residue, revealing the benefits of a random mutagenesis coupled to a high-throughput screening compared to a rational design strategy.

Light-induced gene expression at 30, 37, and 40℃.
We performed the same functional characterization as described before (reporter expression at different light intensities) also at lower (30ºC) and higher (40ºC) temperatures than the standard 37ºC cultivation conditions. This should aid in further investigating the performance of the Opto-T7RNAP and the discovered variants in different culture conditions as well as its effect on the mutations themselves. As described for the experiments at 37ºC, characterization was done by flow cytometry and over time measured through fluorescence spectrophotometry. In general, we observed an approximately 4.5-times higher maximal mCherry fluorescence (t=128,601 arb. units, Supplementary Table 6) in cells containing the original Opto-T7RNAP*(563) regulator at 30ºC compared to 37ºC (t=28,360 arb. units, Supplementary Table 2), which might be caused by a decrease in protein stability and/or in dimerization ability at the higher temperatures. In addition, the light intensity required for half-maximal activation decreased 3.9-fold (from I50=0.67 W m-2 at 37℃ to I50=0.17 W m-2 at 30ºC). Therefore, experiments were performed at a lower light intensity range for experiments performed at 30℃ (from 0 to 0.96 W m-2) compared to the experiments at 37℃ (from 0 to 5.77 W m-2). Accordingly, similar effects were observed for experiments performed at 40℃. At this temperature, the highest used light intensities (5.77 W m-2) did not lead to a saturating reporter expression (Supplementary Figure 9, Supplementary Figure 19). To avoid additional heating of the samples caused by the light induction, we did not further increase the light intensities. As increased light intensities would be necessary to reach saturation, which would be required to mathematically fit the dose-response curve, we directly compare the fluorescent signal of the reporter expression in the dark and in the light intensity that led to saturation at 37℃ (3.85 W m-2). In comparison to the reporter expression at 37℃, the higher temperature (40℃) led to a 114-fold reduction in reporter expression with 3.85 W m-2 light induction (28,854 arb. units and 252 arb. units respectively, compare Supplementary Table 2 and Supplementary Table 10), while dark expression was reduced around 6.4-fold (517 arb. units and 82 arb. units respectively). To summarize, this pre-characterization of the wild-type Opto-T7RNAP system showed that light-induced reporter expression and light sensitivity were highest at 30℃ and decreased as the temperature was increased to 37℃ and 40℃. As described above at 37ºC, analogous PC analysis showed that all the variants were nicely distributed along the PC1 (x-axis) according to their expression level also at 30 and 40ºC (Supplementary Figure 24 A, C, D, F). Analysis of the number of clusters using the silhouette algorithm53 and the data at all temperatures suggested an optimal number of clusters of 4-5 or 4, depending if the dataset was single-cell or population. Four clusters were selected and visual inspection of the HC analyses suggested that both types of data were in agreement.

Variant characterization at 30℃.
As observed for the WT, all variants showed an increased reporter expression at the lower expression temperature (Figure 3 A-B) compared to 37ºC. Interestingly, due to the overall higher expression at 30ºC (Figure 3 A-B), the NC was the only variant in the low expression cluster, while at 37 and 40ºC the low expression cluster contained 6-7 and 8-11 variants, respectively. From the single-cell expression data, all the members of the medium expression cluster, except V160M, L39P, N100S, and N100K moved to the low expression cluster at 37ºC and 40ºC. In most cases, the general trends observed for the different variants (Supplementary Table 6) are comparable to 37℃ (Supplementary Table 2). Interestingly, nMagHigh1 variant A143S, which showed a reduced light sensitivity at 37℃ (I50=143% compared to the WT, Supplementary Table 2), displayed a similar sensitivity as the WT at 30℃ (I50=98%, Supplementary Table 6). Additionally, pMag variant R52L showed a dramatically increased expression level at 30℃, which was similar to pMag double mutant P42T T69M (t=206 and 207% respectively compared to the WT, Supplementary Table 8) in addition to the increased light sensitivity (I50=63 and 56% respectively, Supplementary Table 8). In comparison, at 37℃, the expression of R52L was still increased compared to the WT (t=275%, Supplementary Table 4) but significantly lower than P42T T69M (t=517%) or N100L (t=540%). Accordingly, variant R52L moved from the very high expression (red) cluster at 30 ºC to the high (orange) at 37 ºC and medium expression (green) cluster at 40 ºC in both datasets, while N100L moved from the high at 30ºC to the very high expression cluster at 37ºC (Figure 3 E-F).

Asymmetric changes for dark and light-induced expression.
nMagHigh1 variants V147M and I135V K137R both led to a more pronounced decrease in the basal expression at 30℃ (b=47 and 41% respectively, Supplementary Table 6) compared to the expression at 37℃ (b=89 and 88%, Supplementary Table 2), whereas the relative maximal expression was similar in both conditions (t=97 and 106% at 30℃ and t=95 and 111% at 37℃). The contrary was observed for nMagHigh1 variant M48V N100L, where light-induced expression was similar and the basal expression increased compared to the WT at 30℃ (t=109%, b=271%, Supplementary Table 6), but increased for both conditions at 37℃ (t=267%, b=296%, Supplementary Table 2) whereas the dramatic decrease in light sensitivity was comparable for both temperature conditions (I50=50% at 37℃; I50=52% at 30℃). For this variant, the extremely low light intensity of 0.09 W m-2 led to half-maximal activation of gene expression. Similarly, pMag variants T69M, T83M, N100KL, N100L, and P42T T69M showed a lower increase in the maximal expression at 30℃ compared to 37℃ (t=132, 125, 88, 165, and 207% at 30℃; t=326, 270, 190, 540, and 517% at 37℃, respectively), while the change in the basal expression at both temperatures was similar or comparably less (b=260, 245, 184, 253, and 468% at 30℃; b=257, 232, 145, 362, and 422% at 37℃). For all pMag variants, an increased light sensitivity was observed at 30 as well as 37℃ (Supplementary Table 8, Supplementary Table 4).

Variant characterization at 40℃.
Similar to the WT, the expression of all variants was dramatically reduced at 40℃ compared to 37℃ (Figure 3 C-D, Supplementary Table 10-Supplementary Table 12). However, all variants with increased light sensitivity and increased light activation (reporter expression) at 37℃, also showed an increased reporter expression compared to the WT in all light conditions, which might suggest an improved thermostability. The largest increase in expression compared to the WT was observed with nMagHigh1 variant M48V N100L, and pMag variants T69M, N100L, P42T T69M which showed an increase in reporter expression of 412, 407, 782, and 781% respectively at the highest light intensity (Supplementary Table 10-Supplementary Table 12), but only variant N100L moved from the high at 37ºC to the very high expression group at 40ºC to cluster together with P42T T69M. Variant nMagHigh1 T38A L39R, which displayed increased light sensitivity at both 30 and 37℃ (I50=75% at 30℃; I50=68% at 37℃), showed decreased expression levels at all temperatures (compare Supplementary Table 2 and Supplementary Table 10).

Population data over time supports single-cell data measured at one single time point.
The results obtained with the clustered spectrophotometry data over time were highly similar to the single-cell data as described above and confirmed that the behavior of the variants is not restricted to one single time point at low cell density, but rather consistent up to the early stationary phase of growth. To further analyze the expression at higher cell density we fitted the fluorescent data to light dose-response curves at one specific time point at which the variants reached the highest fluorescence levels. While in the single-cell analysis, cells were in the exponential growth phase at low cell density, the time point with the highest fluorescence in the population data was reached at the end of the exponential phase - beginning of the stationary phase. Despite this difference in growth stages, results were comparable. For example, nMagHigh1 variants L39P, T38A L39R, and V160M showed increased light sensitivity through a decrease in the half-maximal light intensity at 37ºC (I50=70, 70, 52% respectively, compared to the WT; Supplementary Table 3). Also in these experiments, nMagHigh1 K137E showed a light sensitivity that is comparable to the WT (I50=102%). As observed in the single cell data, also here all pMag variants revealed an increased light sensitivity (I50=79, 60, 55, 88, 71, 88, 78, 63, 58, 55 and 83% compared to the WT for R52L, T69M, T83M, N100K, N100L, N100S, K119N, M165T, S178Y, P42T T69M and P66S Y94N respectively; Supplementary Table 5). The previously described reduced light sensitivity at 37℃ and similar sensitivity as the WT at 30℃ for nMagHigh1 variant A143S was also observed in the population dataset (I50=160% at 37℃, I50=117% at 30℃; Supplementary Table 3, Supplementary Table 7). Also, the asymmetric changes for dark and light-induced expression of nMagHigh1 variants V147M and I135V K137R, which led to a more pronounced decrease in the basal expression at 30℃ compared to the expression at 37℃ and more similar maximal expression in both conditions (see discussion above) could be observed in the population dataset (V147M: b=103%, t=86%, I50=167% at 37℃ and b=47%, t=99%, I50=143% at 30℃; I135V K137R: b=111%, t=82%, I50=178% at 37℃ and b=51%, t=98%, I50=124% at 30℃; Supplementary Table 3, Supplementary Table 7).

Altogether, the hierarchical clustering, as well as the comparison of parameters, show that the mutations led to similar behaviors both for expression measured at a discrete time point during the early logarithmic growth phase as well as for gene expression over time. This is an important characteristic for future applications of this optogenetic system.

Methods
Bacterial strains and media.
E. coli Top10 was used for all cloning. For characterization, we used E. coli strain AB3608. The strain contains the transcription factor AraC, whereas arabinose metabolizing genes araBAD are deleted, and the mutated permease lacYA177C for titratable arabinose regulation. Plasmids were transformed using a one-step preparation protocol of competent E. coli for the transformation of plasmids in testing strains59 or made electrocompetent for subsequent electroporation60. E. coli CloneCatcherTM DH5G Gold Electrocompetent was used for high-efficiency transformation of mutagenesis libraries following the manufacturer-provided protocol (Genlantis, Inc.).

An autoclaved LB-Miller medium was used for strain propagation. Sterile-filtered M9 medium (M9 Minimal Salts 5X, Sigma Aldrich) supplemented with 0.2% casamino acids, 0.4% glucose, 0.001% thiamine, 0.00006% ferric citrate, 0.1 mM calcium chloride, 1 mM magnesium sulfate was used for all gene expression experiments. Antibiotics (Sigma-Aldrich Chemie GmbH) were used as necessary for plasmid maintenance at concentrations of 100 μg/mL ampicillin, 17 μg/mL chloramphenicol, and 50 μg/mL kanamycin.

Plasmids and genetic parts.
Plasmid pAB150 containing the Opto-T7RNAP*(563) as well as the reporter plasmid pAB50 with mCherry under T7 promoter expression control are taken from our previous study.8 All primers used were purchased from Microsynth AG, Switzerland and are listed in Supplementary Table 15. The plasmid with lower TIR was constructed by using the RBS Calculator V.2.048 and inserted via PCR amplification of mCherry with primers oAB819 and oAB507 from template pAB50, digested with BamHI and KpnI and ligated into the BamHI and KpnI digested backbone. Primer and plasmid sequences are described in Supplementary Table 15.

Error-prone PCR of Magnets domains was performed through amplification of nMagHigh1 and pMag from pAB150 with primers pairs oAB734/oAB736 and oAB810/oAB744 respectively and error-prone Pfu DNA Polymerase containing D141A, E143A mutations61, a gift from Dr. Luzius Pestallozi (DBSSE, ETH Zurich). We used template concentrations of 2.5, 5, and 10 ng to reach variable mutation rates. As backbone we used pAB150 amplified with Phusion HF polymerase (Phusion flash HF PCR master mix, Thermo Scientific) and primer pairs oAB589/oAB446 and oAB448/oAB809 for nMagHigh1 and pMag insertion, respectively, and for Gibson assembly62. The mutation rate was estimated at 1.3-2.9 per kilobase pair through Sanger sequencing. All identified mutations were recloned into the original pAB150 plasmid and used for final characterization. For nMagHigh, the inserts were amplified using primers oAB409/oAB696, and the backbone was amplified from pAB150 with primers oAB589 /oAB446. Before gel purification, the PCR product was digested with DpnI followed by restriction digestion with AvrII and BglII, gel extraction, and ligation. For pMag, inserts were amplified with primer pair oAB49/oAB285, and the backbone was amplified with primers oAB448/oAB286. Before gel purification, the PCR product was digested with DpnI followed by restriction digestion with PacI and KpnI-HF, gel extraction, and ligation. Plasmid library sizes were determined based on colony counts of dilution series to 1.2 * 106 and 1.9 * 106 for pMag and nMagHigh respectively.

Single point mutations in all mOptoT7 plasmids were based on published plasmids (mOptoT7 Version 1, V1)58 and created using CloneAmp HiFi PCR Premix (Takara Bio). All constructs were transformed into Top10 E. coli competent cells and checked through sequencing (Microsynth).

Screening and isolation of variants.
We picked the colonies obtained from the sorting into 96-well plates containing M9 medium and grew them overnight to full cell density. We again inoculated main cultures of the individual variants at low cell concentrations so that the cultures are in log growth phase throughout the experiment for single-cell measurements, before inhibition of transcription and translation for maturation of mCherry8 as follows. Sample was added to an inhibition solution in equal volumes in 96-well U-bottom plates (Thermo Scientific Nunc) on ice, resulting in a final inhibitor concentration of 250 μg/mL rifampicin (Sigma-Aldrich Chemie GmbH) and 25 μg/mL tetracycline (Sigma-Aldrich Chemie GmbH). The inhibition solution contained 500 μg/mL rifampicin and 50 μg/mL tetracycline in phosphate buffered saline (Sigma-Aldrich Chemie GmbH, Dulbecco’s phosphate buffered saline) and was filtered using a 0.2 μm syringe filter (Sartorius). After incubated on ice for at least 30 min the samples were transferred to a 37 °C incubator for 90 min for mCherry maturation. Then, the cells were kept on ice until measurement through flow cytometry. From every plate, we then inoculated two 96-well plates and grew them for 5h either in the dark or in the light before inhibition and measurement through flow cytometry. Based on these first characterizations, we chose a reduced subset of variants with differing mCherry expression levels as well as fold changes higher than the Opto-T7RNAP*(563) “wild-type” (WT) control. These variants were then recharacterized and again a subset of these variants was sent for sequencing. We then re-cloned mutations of unique Magnets variants identified through sequencing into the original Opto-T7RNAP*(563) plasmid. We transformed the recloned variants into AB360 strains containing the pAB50 reporter plasmid, grew them in 96-well plates overnight to full cell density, and then stored the plates in 25% glycerol at -80 C. For final characterizations, we inoculated main cultures in 96-well plates at low cell concentrations through pin replication (96-pin replicator, Scinomix, MO, USA, SCI-4010-0S) so that the cultures are in log growth phase throughout the experiment before inhibition of transcription and translation for maturation of mCherry through flow cytometry (Figure 1 D, left; for WT).

Growth and light induction conditions.
All experiments were performed in an environmental shaker. The shaking incubator consisted of a Kuhner ES-X shaking module (Adolf Kühner AG, Basel, Switzerland) mounted inside an aluminum housing (Tecan, TMaennedorf, Switzerland) and temperature controlled using an “Icecube” (Life Imaging Services, Basel, Switzerland) at 37°C with shaking at 300 rpm and black, clear bottom 96-well plates (Cell Culture Microplates 96 Well µClear® CELLSTAR®, Greiner Bio-One GmbH, Product #: 655090), which was sealed with peelable foil (Sealing foil, clear peelable for PlateLoc, No. 16985-001, Agilent) to eliminate liquid evaporation and guarantee sterility, as well as a plastic lid (Greiner Bio-One GmbH, Product #: 656171). We used a previously published light induction setup.39 In this setup, 96 LEDs (SK6812, Dongguang Opsco Optoelectronics) are arranged on a printed circuit board (PCB) at a pitch of 9 mm in an 8 × 12 grid and daisy-chained using their DIN and DOUT ports with a 0.1-nF capacitor placed in parallel with the VDD port of each LED and ordered preassembled (www.pcbway.com). 4 × 96-LED PCBs were powered using a single Adafruit #658 5-V 10-A switching power supply (digikey.ch). The LEDs were controlled by an Arduino Uno microcontroller (Arduino) using the fastLED library (http://fastled.io/). The 96-LED array was mounted inside the shaking incubator using custom three-dimensional (3D)-printed holders (Ultimaker S5 using black Ultimaker co-polyester), and a custom-made anodized aluminum plate (10-mm thick, with 96 holes of 4-mm diameter). A 3D-printed adapter was placed between the aluminum plate and the microtiter plate which were held in place by metal rods (4-mm diameter, 20-mm length). For experiments, cultures were pin replicated into fresh M9 medium containing the respective inducer concentrations as described in the methods section for the screening and isolation of variants. This high dilution ensures that the cells are still in logarithmic growth phase after 5 h, at the end of the experiment8. 200 µl of inoculated culture was incubated per well of the 96-well plates. Cells were grown for 5h before transcription and translation were stopped with rifampicin and tetracycline.8

Flow cytometry measurement.
Cell fluorescence was characterized using a CytoFlex SE flow cytometer (Beckman Coulter) equipped with CytExpert 2.1.092 software. mCherry fluorescence was measured with a 561 nm laser and 610/20 nm band pass filter and the following gain settings: forward scatter 100, side scatter 100, mCherry gain 300. Thresholds of 2,500 FSC-H and 1,000 SSC-H were used for all samples. The flow cytometer was calibrated before each experiment with QC beads (CytoFLEX Daily QC Fluorospheres, Beckman Coulter) to ensure comparable fluorescence values across experiments from different days. For the primary high-throughput characterization of the sorted variants, measurements were aborted after 5,000 events or after 30 seconds. As threshold, 15,000 events were recorded in a two-dimensional forward and side scatter gate, which was drawn by eye and corresponded to the experimentally determined size of the testing strain at logarithmic growth and was kept constant for analysis of all experiments and used for calculations of the median and CV using the CytExpert software (Supplementary Figure 31). At least 50,000 events were taken that were distributed amongst 3-9 biologically independent samples with an average of 27,000-31,000 events/sample, depending on the temperature for bacterial experiments. For mammalian experiments, at least 20,000 events were recorded per sample. Cells were inhibited with rifampicin and tetracycline and mCherry was matured before measurement.8

Spectrophotometric and fluorometric measurements.
Cells were grown overnight in a 96-well master plate containing 200 µl of M9 liquid media and the appropriate antibiotics in the dark. This master plate was pin-replicated into 96-well black, clear bottom 96-well plates (Cell Culture Microplates 96 Well µClear® CELLSTAR®, Greiner Bio-One GmbH, Product #: 655090), containing 200 µl of fresh M9 liquid media plus antibiotics and sealed with peelable foil (Sealing foil, clear peelable for PlateLoc, No. 16985-001, Agilent). The 96-well plates contained each strain in technical triplicates. Measurements in a Tecan Infinite 200Pro and Firmware v. 3.40 were multiplexed for absorbance at 600 nm (to measure bacterial growth) and fluorescence at 535 nm (ex) / 595 nm (em) to quantify mCherry expression. The reader settings were adjusted to 9 nm bandwidth, 25 flashes, and 200 ms settle time for absorbance mode and 25 nm bandwidth for excitation, 35 nm bandwidth for emission, gain 100, 25 flashes, integration time 2,000 µs, and 200 ms settle time. Plates were incubated in an environmental shaker as described above. Light induction was performed using the 96-LED array described above at intensities ranging from 0 (dark control) to 0.96 W m-2 at 30 ºC and from 0 (dark control) to 5.77 W m-2 at 37 ℃ and 40 ºC. Measurements were taken at 1 h time intervals with the aid of a robotic arm.39 At 30 and 37 ºC, we observed overgrowth of the bacterial cultures in some of the wells that were accompanied by higher fluorescence levels out of the range observed within the replicates and in comparison to other variants. These samples were excluded from the analysis using the criterion: absorbance at 600 nm > 0.805. This excluded an average of 31 and 15 samples per experiment, coming up to 5.5% and 2.6% of the total number of samples at 30ºC and 37ºC, respectively. Background subtraction was only done at 40 ºC because at lower temperatures the background levels (fluorescent levels of the negative control not expressing mCherry) did not affect the analysis of the data.

Fluorescence-Activated Cell Sorting (FACS).
10 mL M9 was inoculated with 50μL of glycerol stocks of the libraries and grown in dark tubes overnight at 37°C and 230 rpm. AB363 controls were prepared accordingly. From these overnight cultures, 2 μL was used for inoculation of 10 mL of M9 (1:5000 v:v), mixed through vortexing and distributed to single wells of a 96-well plate which were immediately incubated and exposed to the respective light conditions for 4 hours at 37°C. The respective libraries or controls were then again pooled and pelleted at 3220 x g (4000 rpm) for 15 min and 4°C. The supernatant was discarded, and the pellet was resuspended in ice-cold PBS. The samples were kept on ice in dark tubes until they were sorted. It was determined before that mCherry fluorescence values of cells in PBS at 4°C remain constant. Single-cell sorting was performed using a Sony SH800S Cell Sorter and a 70 μm chip. Fluorescence of mCherry was measured with a 561 nm laser and a 617/30 nm bandpass filter. The sorting chamber was maintained at 4°C and sample chamber pressure was never set higher than 4 arbitrary units on the machine. Gain settings were set individually at the respective control samples. Sheath fluid was used as running buffer, and gates for setting the threshold for sorting were individually set. Their stringency varied and was determined by comparison to the respective control sample. For enrichment, cells were sorted into 8 ml LB medium in black tubes (Black Tubes CELLSTAR® (15 mL), Greiner Bio-One GmbH), while for single-cell sorting, selected events were spotted onto LB agar omnitrays (NuncTM OmniTrayTM, Thermo Scientific) supplemented with chloramphenicol and ampicillin. Plates were grown at 37°C overnight. Colonies were picked in 96-well plates (Microplates 96 well polystyrene, Product #: 655161, Greiner Bio-One GmbH) filled with 200 uL LB medium using sterile toothpicks and incubated overnight at 37°C and 230 rpm and sealed with gas-permeable membrane (BREATHseal™, Greiner Bio-One GmbH). The next day, 100 µL was pipetted into a new 96-well plate and 50% glycerol (PanReac Applichem ) was added into both plates which were then sealed and frozen to -80°C until use.

Fitting of light dose-response curves.
For fitting of mCherry expression in response to light intensity using least squares regression, we used Prism 9 for MacOS (Version 9.3.1 (#350, GraphPad Software, LLC.).

𝑓 (𝑥) = 𝑏+(𝑡−𝑏) (𝑥𝑛 / (𝐼50 𝑛+𝑥𝑛)) (1)

Where in equation (1) 𝑓 (𝑥) describes the gene expression controlled by the respective Opto-T7RNAP variant as a function of light intensity, 𝑥 represents light intensity, 𝑏 corresponds to the basal promoter under not activated conditions, 𝑡 is the maximal promoter expression, 𝐼50 is the light intensity for half-maximal activation, and 𝑛 is the Hill coefficient for Opto-T7RNAP. As weighting method, no weighting was chosen, and each replicate Y value was considered as an individual point.

Values for 𝑏, 𝑡, 𝐼50, 𝑛, and their lower and upper 95% profile-likelihood confidence limits as well as R squared values, were calculated and confidence bands are shown in the respective plots.

Flow cytometry measurements were performed after 5h induction during the exponential growth phase. As the time point for the spectrophotometric measurement, we chose 8h for cells grown at 37ºC and 13h for cells grown at 30ºC. To compensate for the slight shift in OD600 of variant N100L 30ºC, and thus slightly later fluorescence peak, we took the 15h time point only for this variant.

Fitting of growth curves.
For fitting of 600 nm absorbance in response to incubation time using least squares regression, we used Prism 9 for MacOS (Version 9.3.1 (#350, GraphPad Software, LLC.).

𝑓 (𝑥) = 𝑏+(𝑡−𝑏) / (1+10(𝑙𝑜𝑔𝑇50−𝑥)∗𝑛) (2)

Where in equation (2) 𝑓 (𝑥) describes the absorbance at 600 nm due to cell growth as a function of time, 𝑏 corresponds to the background 600 nm absorbance, 𝑡 is the maximal 600 nm absorbance, 𝑇50 is the time for half-maximal culture density, and 𝑛 is the Hill slope.

Values for 𝑏, 𝑡, 𝑇50, 𝑛, and their lower and upper 95% profile-likelihood confidence limits as well as R squared values, were calculated.

Cell culture and Transfection.
HEK293T cells (ATCC, strain number CRL-3216) were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 10% FBS (Sigma-Aldrich), 1% penicillin/streptomycin, 1× GlutaMAX (Gibco) and 1 mM Sodium Pyruvate (Gibco). Cells were kept at 37 °C and 5% CO2. Transfections were carried out in suspension using 24 well plates (either black for light experiments, PerkinElmer, or transparent for dark control, ThermoFisher) at a density of 1.5x105 cells/well; DNA and Polyethylenimine (PEI) (Mw 40 000; Polysciences, Inc.) complexes were incubated for 25 minutes at room temperature before addition to the cells.

Light induction and Flow Cytometry analysis of HEK293T cells.
Cells were illuminated with constant light using 470nm LEDs (Super Bright LEDs Inc) in an optimized version of the Light Plate Apparatus (LPA).58 HEK293T cells were analyzed 24h after illumination using CytoFLEX S Flow Cytometer (Beckman Coulter).58 Measurement was done using 488 and 561 lasers with 530/11 nm and 610/20 nm OD1 bandpass filters, respectively. Cells were washed once with DPBS (Thermo Fisher) and incubated with 100 μL of Accutase solution (Sigma-Aldrich) to allow detachment prior to measurement. FCS/SSC parameters were used to select the main population of cells and singlets. For each sample, light-induced expression was normalized to mCitrine constitutive plasmid (dividing the mRuby3 reporter fluorescence values by the mCitrine fluorescence values) to account for transfection efficiency. Data was analyzed using Cytoflow Software.

Clustering analysis.
Principal component analysis and hierarchical clustering were performed in R using the following packages: cluster63, factoextra64, dendextend65, and ggplot266. The optimal number of clusters was determined using the silhouette algorithm53 implemented in the factoextra package of R.

The flow cytometry (single cell) data was scaled before PC analysis. We observed that scaling the data before PC analysis for the fluorometric (population) data gave too much weight to the measurements in the lag phase of growth when the mCherry activity was below the detection level of the plate reader due to the low number of cells. Therefore, the fluorometric data was not scaled before PC analysis.

Hierarchical clustering was performed using the Ward method with Euclidean distances.50 When we analyzed all the data at the three different temperatures together (in both cases: single cell-flow cytometry and population-fluorometric data), we employed the principal components scores to perform the hierarchical clustering rather than the raw data to have the same mean in the data at all temperatures and again to avoid giving special weight to the data at 30 ºC, where the mCherry showed the highest values.

Protein structure visualization.
Protein structures were visualized using the PyMOL Molecular Graphics System, Version 2.5.2 Schrödinger, LLC.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this paper, Baumschlager et al applied random mutagenesis followed by high-throughput screening in both dark and light conditions on the nMagHigh1 and pMag in the context of opto-T7RNAP with the goal of improving their light sensitivity. They identified many hits with altered mCherry expression compared to the original Magnet photoreceptors, and proceeded to characterize these changes at three different temperature and over time, with a final brief look at T7-based gene expression in mammalian cells.\nIn general, I think the author’s idea on improving light sensitivity through mutagenesis and characterization to be interesting. Among the issues facing optogenetic tool application in mammalian contexts, light toxicity and inefficient tissue penetration could indeed be solved through improvement of light sensitivity. However, what this paper has in breadth it lacks in depth – we are left with very little idea of what exactly is changed in many of these mutants, nor do the authors leave us with a fully-characterized, improved tool to use in future experiments.\nMajor concerns:\n- Most broadly, the concept of “light sensitivity” here is poorly defined, and an amalgamation of many different possible biological processes. A higher mCherry expression level at a particular light intensity could be due to any of following: (1) increased expression levels of the split-T7 components, (2) slower reversion kinetics from the lit to dark state, (3) a tighter binding affinity between the Magnet components, leading to a higher concentration of reconstituted T7s at lower light intensities, or (4) an actual change in the absorbance of photons by individual Magnet photoreceptors. To fully understand the new mutants, it is important to have some idea whether they are simply expressed at higher levels in bacteria (something that may not translate to other contexts), or if they maintain the photoactivated state for longer (something that would substantially affect experimental design, such as the use of pulsatile inputs), or if they are simply a higher-affinity binding pair.\nThe manuscript would be greatly improved if the authors would fully characterize at least one improved Magnet variant (e.g. the one with the most improved light sensitivity) to understand exactly what is changing, and rule out the other mechanisms. Many of these experiments would be quite straightforward to do: the levels of T7-Magnet fusions (or a T7-Magnet-GFP variant, say) can be measured to test for changes in expression, and dark state reversion kinetics can be measured spectroscopically, by measuring Magnet association/dissociation after a change from light to darkness, or potentially by looking at mCherry decay kinetics after a switch to darkness.\nThe authors appear to argue that any such mechanistic insights are “out of the scope of our engineering-driven approach” (p. 18) but this is a pretty extreme position. Mutagenesis experiments have been carried out by biologists for a half century (error-prone PCR is itself at least 30 years old), yet here the authors appear to deem these approaches “engineering approaches” and absolve themselves of any subsequent mechanistic studies. This is in stark contrast to excellent screening & characterization work done on LOV domains over the past decade (e.g., Guntas et al, PNAS) who isolated mutants, measured changes in binding affinity, and characterized binding kinetics in mammalian cells. As it stands, any user who wishes to use an improved Opto-T7 (or other Magnet system) based on these results would have no idea which mutation to use for a particular application, nor how it works!\n- A related issue: the authors did many experiments in bacterial cells where light sensitivity is not typically a major issue, but did not measure the light sensitivity in the mammalian cell context where it is likely to be more important. In mammalian cells, only one light intensity condition was tested. An investigation of light sensitivity in this context would help the reader to know whether the light sensitivity property screened for in bacteria could be easily translated to other model systems.\nMinor concerns:\n- The title is misleading – “direct evolution” refers to iterative rounds of mutagenesis and selection. Here the authors don’t carry out any iterative process – just a single round of mutagenesis followed by screening.\n- It seems that the authors only focused on mutants either in nMag or in pMag. Have the authors ever tried combining the mutants with increased light sensitivity in nMag and pMag together and see if they could have synergistic effect?\n- It appears that opto-T7-induced expression is relatively weak in mammalian cells, in comparison to excellent Gal4-based gene expression systems (e.g. GAVPO from Wang et al Nature Methods). The authors might consider applying their improved Magnets in a mammalian context where they have already been used more effectively, such as split Cas9 (Nihongaki et al., 2015).", "reviewer_2": "The authors use directed evolution and a high-throughput screening strategy to identify new photosensitive Magnet domains with altered light-activity dose-response curves. In this approach the nMag and pMag components are optimized separately but some common amino acids (T69 and N100) that alter photosensitivity as well as the basal dark and the maximal light induced expression are identified. Optogenetic tools with significantly higher photosensitivity would certainly be of value in decreasing light toxicity and opening the possibility of multiplexing. Yet, the here obtained new Magnets only have slightly altered light sensitivity of ca. 50% and the overlap of the response windows with the original Magnets is still very high. Therefore, multiplexing does not seem feasible with the new tool and is also not shown. Some of the mutations result in improvements in Magnets in terms light response and thermal stability but these are shadowed by already reported enhanced magnets (eMag) (Benetti et al. eLife 2020;9:e63230. DOI: https://doi.org/10.7554/eLife.63230). Actually, some of the mutations found here in the high-throughput screen overlap with the eMag system. At present this work feels better suited to a more specialized journal and does not open completely new perspectives in optogenetics that would justify the publication in Nature Communications. Below are some suggestions for improvement of the manuscript.\n1) The Opto-T7RNAP tool requires both the pMag and nMagHigh1 component to be photoactivated. Therefore, the increased light sensitivity of one component might be limited by the less efficient activation of the second one. Beneficial mutations found in one component should be tested in the other one, considering that the here described library certainly doesn’t cover the entire sequence space and that the photochemistry in the pMag and nMagHigh1 are the same.\n2) In the current study, one component is optimized at a time and the other one is kept constant. While this certainly is sensible in the initial high throughput screen, the combination of the best performing pMag and nMagHigh1 variants should be tested to investigate if the benefits are additive or not.\n3) In the study of saturating mutagenesis at the hotspots T69 and N100 the terminology of the manuscript changes and fold mCherry under high intensity blue light are reported and not the previously used parameters b, t and I50. The term light sensitivity should be used only in one sense: either the light intensity required to induce half-maximal gene expression or the light intensity used to obtain a certain gene expression. It is misleading that these differences in mCherry expression are set equal to higher light sensitivity as they might simply arise from a higher overall expression under blue light. For the most promising mutations the full characterization including dark activity and full light sensitivity profiles should be measured.\n4) The thermal stability is an obvious limitation of the Magnets. This begs the question if the increased light sensitivity originates from a higher fraction of correctly folded proteins in the cells or is it an intrinsic property of the protein that can be explained through its photochemistry. Spectroscopic characterization with purified proteins and molecular modeling would help answer these questions.\n5) In many cases the increased maximal light induced expression comes at the cost of higher basal expression in the dark. A ratio of blue light to dark expression should be added as an additional parameter of merit.\n6) For the expression in mammalian cells the improvements remain minor and the increased fold expression under light illumination does not necessarily show increased light sensitivity of the protein itself but might just be due to more efficient dimerization between the nMag and pMag fragment or improved thermal stability. A full light sensitivity profile would be required to support this claim. A light intensity of 0.01 mW cm-2 is set as sub-saturating light induction but at what intensity does the system saturate and what is the range that the expression can be tuned over? These values could be rather different in E.coli and mammalian cells.\nMinor points:\n1) The library coverage of the high-throughput screen should be mentioned and discussed in the manuscript.\n2) There are some inconsistencies in the units of light intensity. On page 5 the high light intensity is given as 3.85 W/cm2, in the Figures 1-3 the intensity is in up to 6 W/m2 and in Figure 4 the intensities are in mW/cm2. These numbers differ orders of magnitude and should be verified."}, "author_response": "We have revised our manuscript according to the reviewers’ comments. Reviewer comments are in black, our responses have blue font. All changes in the manuscript and supplementary material have font color blue.\nReviewer #1 (Remarks to the Author):\nIn this paper, Baumschlager et al applied random mutagenesis followed by high-throughput screening in both dark and light conditions on the nMagHigh1 and pMag in the context of opto-T7RNAP with the goal of improving their light sensitivity. They identified many hits with altered mCherry expression compared to the original Magnet photoreceptors, and proceeded to characterize these changes at three different temperature and over time, with a final brief look at T7-based gene expression in mammalian cells.\nIn general, I think the author’s idea on improving light sensitivity through mutagenesis and characterization to be interesting. Among the issues facing optogenetic tool application in mammalian contexts, light toxicity and inefficient tissue penetration could indeed be solved through improvement of light sensitivity. However, what this paper has in breadth it lacks in depth – we are left with very little idea of what exactly is changed in many of these mutants, nor do the authors leave us with a fully-characterized, improved tool to use in future experiments.\nMajor concerns:\n- Most broadly, the concept of “light sensitivity” here is poorly defined, and an amalgamation of many different possible biological processes. A higher mCherry expression level at a particular light intensity could be due to any of following: (1) increased expression levels of the split-T7 components, (2) slower reversion kinetics from the lit to dark state, (3) a tighter binding affinity between the Magnet components, leading to a higher concentration of reconstituted T7s at lower light intensities, or (4) an actual change in the absorbance of photons by individual Magnet photoreceptors. To fully understand the new mutants, it is important to have some idea whether they are simply expressed at higher levels in bacteria (something that may not translate to other contexts), or if they maintain the photoactivated state for longer (something that would substantially affect experimental design, such as the use of pulsatile inputs), or if they are simply a higher-affinity binding pair.\nWe agree that “light sensitivity” is a generic term that we use to describe a change in the observed phenotype outcome (increased or decreased expression of the reporter protein) in response to different constant intensities of light. We used different experimental approaches for our in vivo characterization approach, to disentangle different effects that might lead to this observed phenotype (e.g., reversion kinetics, temperature stability).\nChanges in the manuscript:\nWhile we kept the overall term “light sensitivity” to refer to this general phenotype, we understand the point of the reviewer and now defined the term and described the different properties in the results and discussion section. We now also clearly separate the two directly observable properties of our characterization: changes in I50 values, to which we now refer to as “light sensitivity” and changes in basal and maximal expression to which we now refer to as increased or decreased “light activation”. We added an additional description to clarify this and changed the terminology throughout the manuscript. Regarding the expression levels of the Opto-T7RNAP parts, please see our response to the next comment. We agree that in our in vivo characterization, kinetics were missing. As the reviewer correctly mentions, this can substantially affect experimental design. We thus performed further experiments to complement our previous characterization. Please also refer to our response to the following comment, which, in part, resolves around the same question.\nThe manuscript would be greatly improved if the authors would fully characterize at least one improved Magnet variant (e.g. the one with the most improved light sensitivity) to understand exactly what is changing, and rule out the other mechanisms. Many of these experiments would be quite straightforward to do: the levels of T7-Magnet fusions (or a T7-Magnet-GFP variant, say) can be measured to test for changes in expression, and dark state reversion kinetics can be measured spectroscopically, by measuring Magnet association/dissociation after a change from light to darkness, or potentially by looking at mCherry decay kinetics after a switch to darkness.\nThe authors appear to argue that any such mechanistic Insights are “out of the scope of our engineering-driven approach” (p. 18) but this is a pretty extreme position. Mutagenesis experiments have been carried out by biologists for a half century (error-prone PCR is itself at least 30 years old), yet here the authors appear to deem these approaches “engineering approaches” and absolve themselves of any subsequent mechanistic studies. This is in stark contrast to excellent screening & characterization work done on LOV domains over the past decade (e.g., Guntas et al, PNAS) who isolated mutants, measured changes in binding affinity, and characterized binding kinetics in mammalian cells. As it stands, any user who wishes to use an improved Opto-T7 (or other Magnet system) based on these results would have no idea which mutation to use for a particular application, nor how it works!\nWe want to respond to the two paragraphs above in a single response, as we think that they contain arguments that are connected. We understand, and it is mentioned in the manuscript, that mechanistic comprehension would add an interesting angle to the manuscript. In our opinion, in the excellent article by Guntas el at PNAS (2015) authors performed a screening for mutants with enhanced dimerization properties, and then characterized these mutations from a biochemical perspective, therefore providing a very nice focus on the role of the PAS domain residues in dimerization. Applications of the dimerization enhanced variants are also shown in the article. However, the aim of this manuscript is to provide an engineering workflow for the fast tuning of optogenetic protein with the application in regulatable expression tools for synthetic biological applications. Therefore, we focused on developing a very different tool from the previous articles, and consequently our study required a different set of experiments. We thus performed an extensive set of characterization experiments, which cover a very wide range of application scenarios and important parameters all related to the regulation of gene expression. Our focus is on the in vivo behavior of the mutant variants to provide a complete portfolio of tools for different applications. For this, we combined both time-resolved expression data that covers all growth phases, with single-cell expression data to further investigate expression heterogeneity. These clearly support and demonstrate our goal to tune the properties of the proteins toward desired features. This follows a different objective than the wonderful work of mentioned publications and the mechanistic insights they generated. The main objective was to enable the tuning of these optogenetic tools and thus enable a broader use in applications and not mechanistic investigations of photochemistry, which we better leave to specialized groups. While such analysis is highly interesting on a theoretical level, we provided a large dataset that goes beyond most experimental sets in protein engineering. By characterizing 1) the behavior of the variants experimental over time at the population level, we provide crucial information for use in bioprocesses and bulk experiments. Such information cannot be extracted from mechanistic studies, but could be essential for practical applications. Users can infer from these data how the variants behave from the inoculation point to stationary phase, all at different conditions (e.g., light intensities or temperatures) and in addition have information about population heterogeneity. To obtain this characterization, we had to develop an automation setup that transfers the samples between the light induction device and the measurement device. 2) In addition to these spectrophotometry measurements, which would already be a very good characterization of the newly generated variants, we further investigated changes in heterogeneity on the single-cell level through flow cytometry, important not only for targeted induction but also to more precisely control populations of cells to desired outputs. 3) On top of this, we tested altered behavior under different temperatures, again with both methods.\nChanges in the manuscript:\n1) We now further expanded this extensive in vivo characterization and added a set of practical experiments for on-off kinetics and thus not only provide a wide set of variants that show different behavior but also created a comprehensive experimental dataset that covers all relevant aspects for the application of these variants in biotechnological and synthetic biology contexts.\n2) We also thank this reviewer for bringing up how a future user can decide which variant to use for a particular application. To further assist users in choosing the appropriate mutation for their specific applications, we have now included an overview table classifying the different variants according to their properties. We think that this makes applying the results of this work easier for other researcher and thus thank the reviewer for this suggestion.\nWe also want to clarify specifically why changes in internal protein concentrations cannot explain the altered behavior of the variants we identified. We agree that differences in the concentration of transcriptional regulators can lead to changes in the resulting expression level. Mutations might cause slight differences in the expression levels of a protein. However, we did not quantify potential slight changes in the translation of the regulator that are caused by the mutations in our particular case, because of the following reasons:\n1) Light sensitivity changes (in terms of I50) cannot be attributed to changes in expression levels of Opto-T7 parts:\nDuring the development of the Opto-T7RNAPs, we tested different expression levels of the two split parts. Even though the changes in the expression levels of the regulator parts led to dramatic changes in the reporter gene expression (compare Figure 2 BC of the publication Baumschlager et al. ACS Synth Biol 2017), the dose-response curves were not changed due to the altered regulator expression levels. This is highly comparable to potential changes caused by altered translation rates, especially because also there, the expression level of one of the split parts in relation to the other part would be altered. (Figure S11 “Light dose-response curve is not changed by different domain expression levels” of the publication Baumschlager et al. ACS Synth Biol 2017). We even tested this for different Magnet versions as shown in this figure (Opto-T7RNAP*(563-F1) and Opto-T7RNAP*(563-F2)) In case there would be changes in the expression level caused by a mutation, this would also only alter the expression level of one of the Opto-T7RNAP parts, as simulated in these experiments. Thus, changes in light sensitivity cannot solely be attributed to expression level changes of the Opto-T7RNAP parts.\n2) Large changes in Opto-T7RNAP expression are required to lead to moderate changes in the resulting Opto-T7RNAP transcription, which we previously determined using an arabinose-inducible promoter. This promoter is known for large differences in uninduced vs arabinose-induced transcription. From leaky expression of the promoter to full induction for expression of the Opto-T7RNAP parts, which means a dramatic change in the concentration of both Opto-T7RNAP elements (usually this promoter increases protein of interest concentrations by several hundred-fold), led to a 4-fold increase in the resulting reporter gene expression comparing both dark and light-induced conditions separately (Figure 4 of the publication Baumschlager et al. ACS Synth Biol 2017). In addition, we observed that only relatively high arabinose induction (more than 0.05% arabinose) leads to significant changes in the resulting output (same figure). Thus, we think it is very unlikely that such large differences of expression can be caused by a single or double amino acid substitution. In addition, in the variants presented in this manuscript, we observed fold changes of up to 10-fold compared to the wild-type. This leads us to conclude that other properties than expression level changes need to be the root cause of the observed phenotypes of the identified variants.\nWe thus focused on protein stability, and in particular temperature stability, as the reviewer correctly mentions that mutations might lead to increased stability of the protein. Indeed, what we found during this characterization is that some variants, such as N100L, show improved temperature stability.\nChanges in the manuscript:\nWe agree with the reviewer that association/dissociation can also play an important role in the observed light sensitivity. We thus conducted further experiments to characterize the mCherry decay kinetics as suggested by the reviewer. We thank the reviewer for motivating us to these additional experiments as they are valuable contributions for implementing these mutations for different purposes.\n- A related issue: the authors did many experiments in bacterial cells where light sensitivity is not typically a major issue, but did not measure the light sensitivity in the mammalian cell context where it is likely to be more important. In mammalian cells, only one light intensity condition was tested. An investigation of light sensitivity in this context would help the reader to know whether the light sensitivity property screened for in bacteria could be easily translated to other model systems.\nIt is true that the focus was to develop and test the variants for use in bacteria. However, we show that – in principle – these mutations could also be highly interesting in mammalian cells.\nChanges in the manuscript:\nTo make this suggestion even stronger, we followed the advice of the reviewer and added additional experiments in mammalian cells. We performed experiments in the dark, at a sub-saturating light induction condition (0.1 W m-2) and close to saturating regulator activity (0.8 W m-2). Through the additional characterization conditions, we could further boost the activity of the mOptoT7 to about 50-fold compared to the WT, whereas the WT control increased by 14-fold. We thank the reviewer for suggesting these further experiments as they significantly improved our manuscript.\nMinor concerns:\n- The title is misleading – “direct evolution” refers to iterative rounds of mutagenesis and selection. Here the authors don’t carry out any iterative process – just a single round of mutagenesis followed by screening.\nChanges in the manuscript:\nWe changed the title to ‘’Enhancing the performance of Magnets photosensors”.\n- It seems that the authors only focused on mutants either in nMag or in pMag. Have the authors ever tried combining the mutants with increased light sensitivity in nMag and pMag together and see if they could have synergistic effect?\nWe observed synergistic effects within the individual components. Some of the mutations were characterized in tandem with a second mutation. Of course, also combinations of different mutations in the different dimerization domains might lead to further improvements and further possibilities for tuning.\nChanges in the manuscript:\nWe expanded on this in the discussion section to provide an overview of our findings and maybe also inspire further research as combinatorial assays enable extremely large sets of investigations.\n- It appears that opto-T7-induced expression is relatively weak in mammalian cells, in comparison to excellent Gal4-based gene expression systems (e.g. GAVPO from Wang et al Nature Methods). The authors might consider applying their improved Magnets in a mammalian context where they have already been used more effectively, such as split Cas9 (Nihongaki et al., 2015).\nWe agree with this reviewer and acknowledge that this reviewer appreciates that there are numerous potential applications for the mutations discovered in this work. The mOptoT7 is just one of these. We also believe that these suggestions would be a fantastic follow-up after this work gets published, and we are looking forward to seeing our results applied to these systems. For now, it is out of the scope of this study.\nChanges in the manuscript:\nWith new experiments added to the manuscript, we tackle exactly one point of the reviewer, which is the weak expression of mOptoT7 in mammalian cells. As previously mentioned, we could boost the activity of the mOptoT7 to about 50-fold compared to the WT, successfully addressing this issue.\nWe thank the reviewer for their insightful comments and suggestions which significantly improved the manuscript.\nReviewer #2 (Remarks to the Author):\nThe authors use directed evolution and a high-throughput screening strategy to identify new photosensitive Magnet domains with altered light-activity dose-response curves. In this approach the nMag and pMag components are optimized separately but some common amino acids (T69 and N100) that alter photosensitivity as well as the basal dark and the maximal light induced expression are identified. Optogenetic tools with significantly higher photosensitivity would certainly be of value in decreasing light toxicity and opening the possibility of multiplexing. Yet, the here obtained new Magnets only have slightly altered light sensitivity of ca. 50% and the overlap of the response windows with the original Magnets is still very high. Therefore, multiplexing does not seem feasible with the new tool and is also not shown. Some of the mutations result in improvements in Magnets in terms light response and thermal stability but these are shadowed by already reported enhanced magnets (eMag) (Benetti et al. eLife 2020;9:e63230. DOI: https://doi.org/10.7554/eLife.63230). Actually, some of the mutations found here in the high-throughput screen overlap with the eMag system. At present this work feels better suited to a more specialized journal and does not open completely new perspectives in optogenetics that would justify the publication in Nature Communications. Below are some suggestions for improvement of the manuscript.\nWe are delighted about the shared opinion of the reviewer that higher photosensitivity of optogenetic tools is of high value for biological research. We disagree on the assessment of novelty, as we believe that the fact that some of the mutations were already published validates our study. Benetti et al eLife (2020) employed a rationale design approach and four out of the eight residues that they identified were also found in our screening. However, by using random mutagenesis and a selection screening we identified different mutations in those residues. Furthermore, our saturation mutagenesis of two residues allowed us to identify the best variants in those residues, something that is very difficult to achieve by pure rational design. Our work also demonstrates the feasibility and power of directed evolution to enhance the properties of current syn bio tools. The light sensitivity of the wild-type Opto-T7RNA is up to two-fold compared to the variants found in this study. Furthermore, these variants showed up to 5-fold increase in the maximum activity and more importantly, up to 5-fold increase in the fold change at high temperature. The study by Benetti et al eLife (2020) focused on the application of the Magnets for dimerization of proteins and the consequent regulation of protein recruitment and inter-organellar contacts. This work n offers an alternative new approach for photosensor tuning with a completely different focus: the regulation of gene expression.\n1) The Opto-T7RNAP tool requires both the pMag and nMagHigh1 component to be photoactivated. Therefore, the increased light sensitivity of one component might be limited by the less efficient activation of the second one. Beneficial mutations found in one component should be tested in the other one, considering that the here described library certainly doesn’t cover the entire sequence space and that the photochemistry in the pMag and nMagHigh1 are the same.\nIndeed, it can be expected that some mutations will be easily transferable between the two domains. This is the case for mutation N100L, which we characterized in both domains separately.\nChanges in the manuscript:\nWe expanded on the transferability of the mutations between the domains to make future users of the method and the mutations aware of the possibility of transferability between the domains and combinatorial assays and applications. We also created an overview table (Supplementary Table 13) to give the reader a better overview of the findings and potential expansions. Please also see the corresponding response to the comment of reviewer 1.\n2) In the current study, one component is optimized at a time and the other one is kept constant. While this certainly is sensible in the initial high throughput screen, the combination of the best performing pMag and nMagHigh1 variants should be tested to investigate if the benefits are additive or not.\nWe did observe synergistic effects within the individual components. Some of the mutations were characterized in tandem with a second mutation. Since the different variants have different specific properties, it is not clear what the best set of mutations would be as the choice will be application-dependent, and combining all mutations would not be feasible and out of scope for this study, but would be a nice follow-up.\nChanges in the manuscript:\nWe describe the double mutants as examples of these, where we could entangle these effects further in the text. We further elaborated on these points to make the reader aware of these valid points. Please also see our response to reviewer 1.\n3) In the study of saturating mutagenesis at the hotspots T69 and N100 the terminology of the manuscript changes and fold mCherry under high intensity blue light are reported and not the previously used parameters b, t and I50. The term light sensitivity should be used only in one sense: either the light intensity required to induce half-maximal gene expression or the light intensity used to obtain a certain gene expression. It is misleading that these differences in mCherry expression are set equal to higher light sensitivity as they might simply arise from a higher overall expression under blue light. For the most promising mutations the full characterization including dark activity and full light sensitivity profiles should be measured.\nIt is true that we did not use parameters b, t, and I50 for comparison. This is due to the lower number of light intensities (only two, low and high), which does not allow for fitting our dose-response model, from which these parameters are derived from. Due to the large number of variants for each library, it is not feasible to perform a full dose curve for each variant. Also, this was never the goal. Our goal was to investigate the two main hotspots we identified and see if variants with even higher light sensitivity or light activation can be identified, which might not have been accessible via single nucleotide exchanges. For this, a saturation light induction (to identify increased light activation) and a sub-saturating light intensity (to variants with a higher light sensitivity) were sufficient.\nAlthough we did not identify variants with improved properties compared to our previously identified ones, this was a good confirmation that we are not missing other even more beneficial variants. As suggested by the reviewer, the -by far- most promising variants were characterized with full light sensitivity profiles (T69M, N100L).\nChanges in the manuscript:\nWe clarified the above-mentioned points in the text of the corresponding paragraph. We thank the reviewer for making us aware that this was not clearly communicated before.\n4) The thermal stability is an obvious limitation of the Magnets. This begs the question if the increased light sensitivity originates from a higher fraction of correctly folded proteins in the cells or is it an intrinsic property of the protein that can be explained through its photochemistry. Spectroscopic characterization with purified proteins and molecular modeling would help answer these questions.\nWe agree that different effects can lead to the same phenotype, so we characterized the ones that are most relevant for experimental design (light intensity, temperature stability) and we further strengthened these characterizations with new experiments of temporal dynamics. We agree that in vitro characterization of the protein variants would be very interesting to further elucidate the exact mechanisms of the observed phenotypes, and this is a very nice suggestion for a follow-up of this work. The idea for the follow-up study could be the complete biochemical characterization of some selected variants to decipher the molecular consequences of the mutations. In this work, we focused on changes toward a desired phenotype, and characterized the behavior of the variants under different conditions to provide a complete portfolio of variants for different applications.\nChanges in the manuscript:\nWe have addressed the question on the molecular mechanism experimentally by 1) providing additional in vivo characterization of important variants (especially in response to stability and the resulting dynamics as described in the new section “Dynamic off-switching at 37ºC”), and 2) a detailed explanation on the effect of Opto-T7RNAP concentration changes in our response to reviewer 1, comment 2.\n5) In many cases the increased maximal light induced expression comes at the cost of higher basal expression in the dark. A ratio of blue light to dark expression should be added as an additional parameter of merit.\nChanges in the manuscript:\nAs suggested, we added both the fold change as well as the comparison to the wild type to all the data tables in the supplementary information. Thank you for the comment.\n6) For the expression in mammalian cells the improvements remain minor and the increased fold expression under light illumination does not necessarily show increased light sensitivity of the protein itself but might just be due to more efficient dimerization between the nMag and pMag fragment or improved thermal stability. A full light sensitivity profile would be required to support this claim. A light intensity of 0.01 mW cm-2 is set as sub-saturating light induction but at what intensity does the system saturate and what is the range that the expression can be tuned over? These values could be rather different in E.coli and mammalian cells.\nIndeed, as the reviewer correctly mentions, these values are different in E.coli and mammalian cells. We chose 0.01 mW cm-2 for showing changes in light sensitivity, as this value was determined to be sub-saturating (see Figure 1d of publication Dionisi et al., ACS Synthetic Biology, 2022 and Figure 4 c of the same publication).\nChanges in the manuscript:\nWe have added this missing explanation in the manuscript. We have further added additional experiments in mammalian cells to expand the characterization. These experiments now show dark, sub-saturating and close-to-saturating light induction of the regulator. At the additional conditions, we could further boost the activity of the mOptoT7 to about 50-fold compared to the WT, whereas the WT control increased by 14-fold. We thank the reviewer for guiding us to these additional experiments.\nMinor points:\n1) The library coverage of the high-throughput screen should be mentioned and discussed in the manuscript.\nChanges in the manuscript:\nWe added this information to the methods section “Plasmids and genetic parts”.\n2) There are some inconsistencies in the units of light intensity. On page 5 the high light intensity is given as 3.85 W/cm2, in the Figures 1-3 the intensity is in up to 6 W/m2 and in Figure 4 the intensities are in mW/cm2. These numbers differ orders of magnitude and should be verified.\nThank you for spotting this!\nChanges in the manuscript:\nWe have corrected this and harmonized the units.\nWe express our sincere gratitude to the reviewer for their insightful review and invaluable suggestions that have contributed to our manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I thank the authors for their response to my prior review and have a few additional comments:\n(1) First and foremost, I still not convinced that this manuscript is a strong candidate for Nature Communications. The authors argue that their paper is principally focused on tuning a synthetic biology gene expression system, not understanding mechanism, which is fine. However, it narrows the scope of the paper considerably - we might learn how OptoT7 in bacteria change, but we do not gain much insight in how these changes will translate to any other application, since we are left with little mechanistic insight. We also are not left with a demonstration that the higher light sensitivity obtained from the screen is useful, say for example by regulating metabolism in high-OD culture. I agree with the other reviewer's first review that this manuscript would be better suited for a more specialized journal.\n(2) In their rebuttal the authors reference some supplementary data from their prior paper (Baumschlager 2017). In my view, that paper has a wealth of additional results that support the premise of the current study, and it would be *very* helpful if they were explained in the introduction of this study. The 2017 paper investigates two classic VVD mutants, I85V and I74V/I85V, which have well known effects on photocycling time, but observe that one of these mutants exhibits a massive change in light sensitivity (Fig 4 and S11), whereas the other does not. This shows that sensitivity and photocycle time can be tuned independently - they are not one-to-one - and that wild-type VVD is far from optimal in sensitivity. I agree with the authors' rebuttal that these results argue against sensitivity arising simply from variation in protein expression level. They also argue against the sensitivity being solely a function of lit-state lifetime.\nThe manuscript would be strengthened if the authors would describe something like this logic in their current manuscript, because it is absolutely not clear simply by reading this paper whether light sensitivity itself is an engineerable parameter, since so many other variables could in principle influence a dose response curve.\nThis prior work also suggests that the authors have already identified a very sensitive variant in I74V/I85V. Why was this variant not included in the current analyses, at least as a positive control? Would it be competitive with the best (most sensitive) variants discovered here?\n(3) The authors' table detailing the sensitivity changes (Supp Table 13) is very useful. It is one of the key pieces of information for users who might be interested in implementing these mutations for further study. I would strongly prefer that this be included in the main text as a final figure panel or table, not on page 38 of the supplementary information where it is likely to escape notice by most readers.\n(4) I have a major concern about the quantification and analysis of the mammalian cell data in Figure 3. It is quite misleading to only compare fold-changes between mutants in the light to the wild-type variant in the dark. This is because many variants already show dramatically increased gene expression in the dark! Fold-changes should be reported for each variant between its dark and light conditions. Separately, the authors can describe changes in \"leakiness\" in the dark, for example showing that M48V N100L is already ~10-fold higher than wild-type in the dark. It is not at all clear that the changes between dark and low-intensity light are significant for many variants (e.g. N100L or M48V/N100L). Is the fold-change between dark and 0.1 W/m^2 statistically different for the mutants compared to the wild-type in mammalian cells? Without such a comparsion it is not clear that the changes in sensitivity extend to the mammalian context at all.", "reviewer_2": "We have revised our manuscript according to the reviewers’ comments. Reviewer comments are in black, our\nresponses have blue font. All changes in the manuscript and supplementary material have font color blue.\nReviewer #1 (Remarks to the Author):\nIn this paper, Baumschlager et al applied random mutagenesis followed by high-throughput screening in both dark\nand light conditions on the nMagHigh1 and pMag in the context of opto-T7RNAP with the goal of improving their\nlight sensitivity. They identified many hits with altered mCherry expression compared to the original Magnet\nphotoreceptors, and proceeded to characterize these changes at three different temperature and over time, with a\nfinal brief look at T7-based gene expression in mammalian cells.\nIn general, I think the author’s idea on improving light sensitivity through mutagenesis and characterization to be\ninteresting. Among the issues facing optogenetic tool application in mammalian contexts, light toxicity and\ninefficient tissue penetration could indeed be solved through improvement of light sensitivity. However, what this\npaper has in breadth it lacks in depth – we are left with very little idea of what exactly is changed in many of these\nmutants, nor do the authors leave us with a fully-characterized, improved tool to use in future experiments.\nMajor concerns:\n- Most broadly, the concept of “light sensitivity” here is poorly defined, and an amalgamation of many different\npossible biological processes. A higher mCherry expression level at a particular light intensity could be due to any\nof following: (1) increased expression levels of the split-T7 components, (2) slower reversion kinetics from the lit to\ndark state, (3) a tighter binding affinity between the Magnet components, leading to a higher concentration of\nreconstituted T7s at lower light intensities, or (4) an actual change in the absorbance of photons by individual\nMagnet photoreceptors. To fully understand the new mutants, it is important to have some idea whether they are\nsimply expressed at higher levels in bacteria (something that may not translate to other contexts), or if they maintain\nthe photoactivated state for longer (something that would substantially affect experimental design, such as the use\nof pulsatile inputs), or if they are simply a higher-affinity binding pair.\nWe agree that “light sensitivity” is a generic term that we use to describe a change in the observed phenotype\noutcome (increased or decreased expression of the reporter protein) in response to different constant intensities\nof light. We used different experimental approaches for our in vivo characterization approach, to disentangle\ndifferent effects that might lead to this observed phenotype (e.g., reversion kinetics, temperature stability).\nChanges in the manuscript:\nWhile we kept the overall term “light sensitivity” to refer to this general phenotype, we understand the point of the\nreviewer and now defined the term and described the different properties in the results and discussion section. We\nnow also clearly separate the two directly observable properties of our characterization: changes in I50 values, to\nwhich we now refer to as “light sensitivity” and changes in basal and maximal expression to which we now refer to\nas increased or decreased “light activation”. We added an additional description to clarify this and changed the\nterminology throughout the manuscript. Regarding the expression levels of the Opto-T7RNAP parts, please see\nour response to the next comment. We agree that in our in vivo characterization, kinetics were missing. As the\nreviewer correctly mentions, this can substantially affect experimental design. We thus performed further\nexperiments to complement our previous characterization. Please also refer to our response to the following\ncomment, which, in part, resolves around the same question.\nThe manuscript would be greatly improved if the authors would fully characterize at least one improved Magnet\nvariant (e.g. the one with the most improved light sensitivity) to understand exactly what is changing, and rule out\nthe other mechanisms. Many of these experiments would be quite straightforward to do: the levels of T7-Magnet\nfusions (or a T7-Magnet-GFP variant, say) can be measured to test for changes in expression, and dark state\nreversion kinetics can be measured spectroscopically, by measuring Magnet association/dissociation after a\nchange from light to darkness, or potentially by looking at mCherry decay kinetics after a switch to darkness.\nThe authors appear to argue that any such mechanistic Insights are “out of the scope of our engineering-driven\napproach” (p. 18) but this is a pretty extreme position. Mutagenesis experiments have been carried out by biologists\nfor a half century (error-prone PCR is itself at least 30 years old), yet here the authors appear to deem these\napproaches “engineering approaches” and absolve themselves of any subsequent mechanistic studies. This is in\nstark contrast to excellent screening & characterization work done on LOV domains over the past decade (e.g.,\nGuntas et al, PNAS) who isolated mutants, measured changes in binding affinity, and characterized binding kinetics\nin mammalian cells. As it stands, any user who wishes to use an improved Opto-T7 (or other Magnet system)\nbased on these results would have no idea which mutation to use for a particular application, nor how it works!\nWe want to respond to the two paragraphs above in a single response, as we think that they contain arguments\nthat are connected. We understand, and it is mentioned in the manuscript, that mechanistic comprehension would\nadd an interesting angle to the manuscript. In our opinion, in the excellent article by Guntas el at PNAS (2015)\nauthors performed a screening for mutants with enhanced dimerization properties, and then characterized these\nmutations from a biochemical perspective, therefore providing a very nice focus on the role of the PAS domain\nresidues in dimerization. Applications of the dimerization enhanced variants are also shown in the article. However,\nthe aim of this manuscript is to provide an engineering workflow for the fast tuning of optogenetic protein with the\napplication in regulatable expression tools for synthetic biological applications. Therefore, we focused on\ndeveloping a very different tool from the previous articles, and consequently our study required a different set of\nexperiments. We thus performed an extensive set of characterization experiments, which cover a very wide range\nof application scenarios and important parameters all related to the regulation of gene expression. Our focus is on\nthe in vivo behavior of the mutant variants to provide a complete portfolio of tools for different applications. For this,\nwe combined both time-resolved expression data that covers all growth phases, with single-cell expression data to\nfurther investigate expression heterogeneity. These clearly support and demonstrate our goal to tune the properties\nof the proteins toward desired features. This follows a different objective than the wonderful work of mentioned\npublications and the mechanistic insights they generated. The main objective was to enable the tuning of these\noptogenetic tools and thus enable a broader use in applications and not mechanistic investigations of\nphotochemistry, which we better leave to specialized groups. While such analysis is highly interesting on a\ntheoretical level, we provided a large dataset that goes beyond most experimental sets in protein engineering. By\ncharacterizing 1) the behavior of the variants experimental over time at the population level, we provide crucial\ninformation for use in bioprocesses and bulk experiments. Such information cannot be extracted from mechanistic\nstudies, but could be essential for practical applications. Users can infer from these data how the variants behave\nfrom the inoculation point to stationary phase, all at different conditions (e.g., light intensities or temperatures) and\nin addition have information about population heterogeneity. To obtain this characterization, we had to develop an\nautomation setup that transfers the samples between the light induction device and the measurement device. 2) In\naddition to these spectrophotometry measurements, which would already be a very good characterization of the\nnewly generated variants, we further investigated changes in heterogeneity on the single-cell level through flow\ncytometry, important not only for targeted induction but also to more precisely control populations of cells to desired\noutputs. 3) On top of this, we tested altered behavior under different temperatures, again with both methods.\nChanges in the manuscript:\n1) We now further expanded this extensive in vivo characterization and added a set of practical experiments\nfor on-off kinetics and thus not only provide a wide set of variants that show different behavior but also\ncreated a comprehensive experimental dataset that covers all relevant aspects for the application of these\nvariants in biotechnological and synthetic biology contexts.\n2) We also thank this reviewer for bringing up how a future user can decide which variant to use for a\nparticular application. To further assist users in choosing the appropriate mutation for their specific\napplications, we have now included an overview table classifying the different variants according to their\nproperties. We think that this makes applying the results of this work easier for other researcher and thus\nthank the reviewer for this suggestion.\nWe also want to clarify specifically why changes in internal protein concentrations cannot explain the altered\nbehavior of the variants we identified. We agree that differences in the concentration of transcriptional regulators\ncan lead to changes in the resulting expression level. Mutations might cause slight differences in the expression\nlevels of a protein. However, we did not quantify potential slight changes in the translation of the regulator that are\ncaused by the mutations in our particular case, because of the following reasons:\n1) Light sensitivity changes (in terms of I50) cannot be attributed to changes in expression levels of Opto-T7\nparts:\nDuring the development of the Opto-T7RNAPs, we tested different expression levels of the two split parts.\nEven though the changes in the expression levels of the regulator parts led to dramatic changes in the\nreporter gene expression (compare Figure 2 BC of the publication Baumschlager et al. ACS Synth Biol\n2017), the dose-response curves were not changed due to the altered regulator expression levels. This\nis highly comparable to potential changes caused by altered translation rates, especially because also\nthere, the expression level of one of the split parts in relation to the other part would be altered. (Figure\nS11 “Light dose-response curve is not changed by different domain expression levels” of the publication\nBaumschlager et al. ACS Synth Biol 2017). We even tested this for different Magnet versions as shown\nin this figure (Opto-T7RNAP*(563-F1) and Opto-T7RNAP*(563-F2)) In case there would be changes in\nthe expression level caused by a mutation, this would also only alter the expression level of one of the\nOpto-T7RNAP parts, as simulated in these experiments. Thus, changes in light sensitivity cannot solely\nbe attributed to expression level changes of the Opto-T7RNAP parts.\n2) Large changes in Opto-T7RNAP expression are required to lead to moderate changes in the resulting\nOpto-T7RNAP transcription, which we previously determined using an arabinose-inducible promoter. This\npromoter is known for large differences in uninduced vs arabinose-induced transcription. From leaky\nexpression of the promoter to full induction for expression of the Opto-T7RNAP parts, which means a\ndramatic change in the concentration of both Opto-T7RNAP elements (usually this promoter increases\nprotein of interest concentrations by several hundred-fold), led to a 4-fold increase in the resulting reporter\ngene expression comparing both dark and light-induced conditions separately (Figure 4 of the publication\nBaumschlager et al. ACS Synth Biol 2017). In addition, we observed that only relatively high arabinose\ninduction (more than 0.05% arabinose) leads to significant changes in the resulting output (same figure).\nThus, we think it is very unlikely that such large differences of expression can be caused by a single or\ndouble amino acid substitution. In addition, in the variants presented in this manuscript, we observed fold\nchanges of up to 10-fold compared to the wild-type. This leads us to conclude that other properties than\nexpression level changes need to be the root cause of the observed phenotypes of the identified variants.\nWe thus focused on protein stability, and in particular temperature stability, as the reviewer correctly\nmentions that mutations might lead to increased stability of the protein. Indeed, what we found during this\ncharacterization is that some variants, such as N100L, show improved temperature stability.\nChanges in the manuscript:\nWe agree with the reviewer that association/dissociation can also play an important role in the observed light\nsensitivity. We thus conducted further experiments to characterize the mCherry decay kinetics as suggested by\nthe reviewer. We thank the reviewer for motivating us to these additional experiments as they are valuable\ncontributions for implementing these mutations for different purposes.\n- A related issue: the authors did many experiments in bacterial cells where light sensitivity is not typically a major\nissue, but did not measure the light sensitivity in the mammalian cell context where it is likely to be more important.\nIn mammalian cells, only one light intensity condition was tested. An investigation of light sensitivity in this context\nwould help the reader to know whether the light sensitivity property screened for in bacteria could be easily\ntranslated to other model systems.\nIt is true that the focus was to develop and test the variants for use in bacteria. However, we show that – in principle\n– these mutations could also be highly interesting in mammalian cells.\nChanges in the manuscript:\nTo make this suggestion even stronger, we followed the advice of the reviewer and added additional experiments\nin mammalian cells. We performed experiments in the dark, at a sub-saturating light induction condition (0.1 W m-\n2) and close to saturating regulator activity (0.8 W m-2). Through the additional characterization conditions, we could\nfurther boost the activity of the mOptoT7 to about 50-fold compared to the WT, whereas the WT control increased\nby 14-fold. We thank the reviewer for suggesting these further experiments as they significantly improved our\nmanuscript.\nMinor concerns:\n- The title is misleading – “direct evolution” refers to iterative rounds of mutagenesis and selection. Here the authors\ndon’t carry out any iterative process – just a single round of mutagenesis followed by screening.\nChanges in the manuscript:\nWe changed the title to ‘’Enhancing the performance of Magnets photosensors”.\n- It seems that the authors only focused on mutants either in nMag or in pMag. Have the authors ever tried\ncombining the mutants with increased light sensitivity in nMag and pMag together and see if they could have\nsynergistic effect?\nWe observed synergistic effects within the individual components. Some of the mutations were characterized in\ntandem with a second mutation. Of course, also combinations of different mutations in the different dimerization\ndomains might lead to further improvements and further possibilities for tuning.\nChanges in the manuscript:\nWe expanded on this in the discussion section to provide an overview of our findings and maybe also inspire further\nresearch as combinatorial assays enable extremely large sets of investigations.\n- It appears that opto-T7-induced expression is relatively weak in mammalian cells, in comparison to excellent\nGal4-based gene expression systems (e.g. GAVPO from Wang et al Nature Methods). The authors might consider\napplying their improved Magnets in a mammalian context where they have already been used more effectively,\nsuch as split Cas9 (Nihongaki et al., 2015).\nWe agree with this reviewer and acknowledge that this reviewer appreciates that there are numerous potential\napplications for the mutations discovered in this work. The mOptoT7 is just one of these. We also believe that these\nsuggestions would be a fantastic follow-up after this work gets published, and we are looking forward to seeing our\nresults applied to these systems. For now, it is out of the scope of this study.\nChanges in the manuscript:\nWith new experiments added to the manuscript, we tackle exactly one point of the reviewer, which is the weak\nexpression of mOptoT7 in mammalian cells. As previously mentioned, we could boost the activity of the mOptoT7\nto about 50-fold compared to the WT, successfully addressing this issue.\nWe thank the reviewer for their insightful comments and suggestions which significantly improved the manuscript.\nReviewer #2 (Remarks to the Author):\nThe authors use directed evolution and a high-throughput screening strategy to identify new photosensitive Magnet\ndomains with altered light-activity dose-response curves. In this approach the nMag and pMag components are\noptimized separately but some common amino acids (T69 and N100) that alter photosensitivity as well as the basal\ndark and the maximal light induced expression are identified. Optogenetic tools with significantly higher\nphotosensitivity would certainly be of value in decreasing light toxicity and opening the possibility of multiplexing.\nYet, the here obtained new Magnets only have slightly altered light sensitivity of ca. 50% and the overlap of the\nresponse windows with the original Magnets is still very high. Therefore, multiplexing does not seem feasible with\nthe new tool and is also not shown. Some of the mutations result in improvements in Magnets in terms light\nresponse and thermal stability but these are shadowed by already reported enhanced magnets (eMag) (Benetti et\nal. eLife 2020;9:e63230. DOI: https://doi.org/10.7554/eLife.63230). Actually, some of the mutations found here in\nthe high-throughput screen overlap with the eMag system. At present this work feels better suited to a more\nspecialized journal and does not open completely new perspectives in optogenetics that would justify the\npublication in Nature Communications. Below are some suggestions for improvement of the manuscript.\nWe are delighted about the shared opinion of the reviewer that higher photosensitivity of optogenetic tools is of\nhigh value for biological research. We disagree on the assessment of novelty, as we believe that the fact that some\nof the mutations were already published validates our study. Benetti et al eLife (2020) employed a rationale design\napproach and four out of the eight residues that they identified were also found in our screening. However, by using\nrandom mutagenesis and a selection screening we identified different mutations in those residues. Furthermore,\nour saturation mutagenesis of two residues allowed us to identify the best variants in those residues, something\nthat is very difficult to achieve by pure rational design. Our work also demonstrates the feasibility and power of\ndirected evolution to enhance the properties of current syn bio tools. The light sensitivity of the wild-type Opto-\nT7RNA is up to two-fold compared to the variants found in this study. Furthermore, these variants showed up to 5-\nfold increase in the maximum activity and more importantly, up to 5-fold increase in the fold change at high\ntemperature. The study by Benetti et al eLife (2020) focused on the application of the Magnets for dimerization of\nproteins and the consequent regulation of protein recruitment and inter-organellar contacts. This work n offers an\nalternative new approach for photosensor tuning with a completely different focus: the regulation of gene\nexpression.\n1) The Opto-T7RNAP tool requires both the pMag and nMagHigh1 component to be photoactivated. Therefore,\nthe increased light sensitivity of one component might be limited by the less efficient activation of the second one.\nBeneficial mutations found in one component should be tested in the other one, considering that the here described\nlibrary certainly doesn’t cover the entire sequence space and that the photochemistry in the pMag and nMagHigh1\nare the same.\nIndeed, it can be expected that some mutations will be easily transferable between the two domains. This is the\ncase for mutation N100L, which we characterized in both domains separately.\nChanges in the manuscript:\nWe expanded on the transferability of the mutations between the domains to make future users of the method and\nthe mutations aware of the possibility of transferability between the domains and combinatorial assays and\napplications. We also created an overview table (Supplementary Table 13) to give the reader a better overview of\nthe findings and potential expansions. Please also see the corresponding response to the comment of reviewer 1.\n2) In the current study, one component is optimized at a time and the other one is kept constant. While this certainly\nis sensible in the initial high throughput screen, the combination of the best performing pMag and nMagHigh1\nvariants should be tested to investigate if the benefits are additive or not.\nWe did observe synergistic effects within the individual components. Some of the mutations were characterized in\ntandem with a second mutation. Since the different variants have different specific properties, it is not clear what\nthe best set of mutations would be as the choice will be application-dependent, and combining all mutations would\nnot be feasible and out of scope for this study, but would be a nice follow-up.\nChanges in the manuscript:\nWe describe the double mutants as examples of these, where we could entangle these effects further in the text.\nWe further elaborated on these points to make the reader aware of these valid points. Please also see our response\nto reviewer 1.\n3) In the study of saturating mutagenesis at the hotspots T69 and N100 the terminology of the manuscript changes\nand fold mCherry under high intensity blue light are reported and not the previously used parameters b, t and I50.\nThe term light sensitivity should be used only in one sense: either the light intensity required to induce half-maximal\ngene expression or the light intensity used to obtain a certain gene expression. It is misleading that these\ndifferences in mCherry expression are set equal to higher light sensitivity as they might simply arise from a higher\noverall expression under blue light. For the most promising mutations the full characterization including dark activity\nand full light sensitivity profiles should be measured.\nIt is true that we did not use parameters b, t, and I50 for comparison. This is due to the lower number of light\nintensities (only two, low and high), which does not allow for fitting our dose-response model, from which these\nparameters are derived from. Due to the large number of variants for each library, it is not feasible to perform a full\ndose curve for each variant. Also, this was never the goal. Our goal was to investigate the two main hotspots we\nidentified and see if variants with even higher light sensitivity or light activation can be identified, which might not\nhave been accessible via single nucleotide exchanges. For this, a saturation light induction (to identify increased\nlight activation) and a sub-saturating light intensity (to variants with a higher light sensitivity) were sufficient.\nAlthough we did not identify variants with improved properties compared to our previously identified ones, this was\na good confirmation that we are not missing other even more beneficial variants. As suggested by the reviewer,\nthe -by far- most promising variants were characterized with full light sensitivity profiles (T69M, N100L).\nChanges in the manuscript:\nWe clarified the above-mentioned points in the text of the corresponding paragraph. We thank the reviewer for\nmaking us aware that this was not clearly communicated before.\n4) The thermal stability is an obvious limitation of the Magnets. This begs the question if the increased light\nsensitivity originates from a higher fraction of correctly folded proteins in the cells or is it an intrinsic property of the\nprotein that can be explained through its photochemistry. Spectroscopic characterization with purified proteins and\nmolecular modeling would help answer these questions.\nWe agree that different effects can lead to the same phenotype, so we characterized the ones that are most relevant\nfor experimental design (light intensity, temperature stability) and we further strengthened these characterizations\nwith new experiments of temporal dynamics. We agree that in vitro characterization of the protein variants would\nbe very interesting to further elucidate the exact mechanisms of the observed phenotypes, and this is a very nice\nsuggestion for a follow-up of this work. The idea for the follow-up study could be the complete biochemical\ncharacterization of some selected variants to decipher the molecular consequences of the mutations. In this work,\nwe focused on changes toward a desired phenotype, and characterized the behavior of the variants under different\nconditions to provide a complete portfolio of variants for different applications.\nChanges in the manuscript:\nWe have addressed the question on the molecular mechanism experimentally by 1) providing additional in vivo\ncharacterization of important variants (especially in response to stability and the resulting dynamics as described\nin the new section “Dynamic off-switching at 37ºC”), and 2) a detailed explanation on the effect of Opto-T7RNAP\nconcentration changes in our response to reviewer 1, comment 2.\n5) In many cases the increased maximal light induced expression comes at the cost of higher basal expression in\nthe dark. A ratio of blue light to dark expression should be added as an additional parameter of merit.\nChanges in the manuscript:\nAs suggested, we added both the fold change as well as the comparison to the wild type to all the data tables in\nthe supplementary information. Thank you for the comment.\n6) For the expression in mammalian cells the improvements remain minor and the increased fold expression under\nlight illumination does not necessarily show increased light sensitivity of the protein itself but might just be due to\nmore efficient dimerization between the nMag and pMag fragment or improved thermal stability. A full light\nsensitivity profile would be required to support this claim. A light intensity of 0.01 mW cm-2 is set as sub-saturating\nlight induction but at what intensity does the system saturate and what is the range that the expression can be\ntuned over? These values could be rather different in E.coli and mammalian cells.\nIndeed, as the reviewer correctly mentions, these values are different in E.coli and mammalian cells. We chose\n0.01 mW cm-2 for showing changes in light sensitivity, as this value was determined to be sub-saturating (see\nFigure 1d of publication Dionisi et al., ACS Synthetic Biology, 2022 and Figure 4 c of the same publication).\nChanges in the manuscript:\nWe have added this missing explanation in the manuscript. We have further added additional experiments in\nmammalian cells to expand the characterization. These experiments now show dark, sub-saturating and close-to-\nsaturating light induction of the regulator. At the additional conditions, we could further boost the activity of the\nmOptoT7 to about 50-fold compared to the WT, whereas the WT control increased by 14-fold. We thank the\nreviewer for guiding us to these additional experiments.\nMinor points:\n1) The library coverage of the high-throughput screen should be mentioned and discussed in the manuscript.\nChanges in the manuscript:\nWe added this information to the methods section “Plasmids and genetic parts”.\n2) There are some inconsistencies in the units of light intensity. On page 5 the high light intensity is given as 3.85\nW/cm2, in the Figures 1-3 the intensity is in up to 6 W/m2 and in Figure 4 the intensities are in mW/cm2. These\nnumbers differ orders of magnitude and should be verified.\nThank you for spotting this!\nChanges in the manuscript:\nWe have corrected this and harmonized the units.\nWe express our sincere gratitude to the reviewer for their insightful review and invaluable suggestions that have\ncontributed to our manuscript.\nPoint-by-point response to the reviewers ’comments.\nWe have addressed all points raised by reviewer #1. Reviewers’ comments are in blue font and\nthe response to the comments is shown in black font. All changes in the main manuscript and\nsupplementary material are shown in green font.\nReviewer #1 (Remarks to the Author):\nI thank the authors for their response to my prior review and have a few additional comments:\n(1) First and foremost, I still not convinced that this manuscript is a strong candidate for Nature\nCommunications. The authors argue that their paper is principally focused on tuning a\nsynthetic biology gene expression system, not understanding mechanism, which is fine.\nHowever, it narrows the scope of the paper considerably - we might learn how OptoT7 in\nbacteria change, but we do not gain much insight in how these changes will translate to any\nother application, since we are left with little mechanistic insight. We also are not left with a\ndemonstration that the higher light sensitivity obtained from the screen is useful, say for\nexample by regulating metabolism in high-OD culture. I agree with the other reviewer's first\nreview that this manuscript would be better suited for a more specialized journal.\nWe want to emphasize that this manuscript not only provides a novel set of optogenetic tools\ncovering a wide range of light sensitivities and expression levels, but also reports a framework\nfor any research or application that requires tuning an optogenetic tool or expression system. It is\na general methodology that is not limited to a specific application. By thoroughly characterizing\nthe effect on protein expression, we demonstrate the applicability for control of gene expression.\nOne of the main points that this work shows is that the most important features of an expression\nsystem, i.e., the sensitivity and the expression levels can be tuned to fit specific desired\nrequirements. Our pre-defined goal was to achieve a higher gene expression using the Opto-\nT7RNAP at lower light intensities, which was achieved as shown by the data. The data presented\nin this manuscript have been confirmed using two different technologies (flow cytometry and\nfluorescent plate reading), both of which are in perfect agreement, and cross-validating the\nfindings.\nTo address the comment about the behavior of the variants at different ODs, we added the full\ntime courses of optical density, reaching from lag to stationary growth phase, together with the\nreporter expression data and further analysis, providing full information on the expression levels\nat any stage (and OD) during the entire growth curve, including stationary phase.\nWe added text and further analysis for all spectrophotometry data for all variants at all the light\nintensities and the three incubation temperatures (30, 37 and 40 ºC) to the manuscript and\nprovided new data in Supplementary Table 13, Supplementary Table 14, Supplementary Figure\n21, Supplementary Figure 22, and Supplementary Figure 23. We thank the reviewer for this\nsuggestion, as this information might indeed be useful for readers.\n(2) In their rebuttal the authors reference some supplementary data from their prior paper\n(Baumschlager 2017). In my view, that paper has a wealth of additional results that support the\npremise of the current study, and it would be *very* helpful if they were explained in the\nintroduction of this study. The 2017 paper investigates two classic VVD mutants, I85V and\nI74V/I85V, which have well known effects on photocycling time, but observe that one of these\nmutants exhibits a massive change in light sensitivity (Fig 4 and S11), whereas the other does\nnot. This shows that sensitivity and photocycle time can be tuned independently - they are not\none-to-one - and that wild-type VVD is far from optimal in sensitivity. I agree with the authors'\nrebuttal that these results argue against sensitivity arising simply from variation in protein\nexpression level. They also argue against the sensitivity being solely a function of lit-state\nlifetime.\nThe manuscript would be strengthened if the authors would describe something like this logic in\ntheir current manuscript, because it is absolutely not clear simply by reading this paper whether\nlight sensitivity itself is an engineerable parameter, since so many other variables could in\nprinciple influence a dose response curve.\nThis prior work also suggests that the authors have already identified a very sensitive variant in\nI74V/I85V. Why was this variant not included in the current analyses, at least as a positive\ncontrol? Would it be competitive with the best (most sensitive) variants discovered here?\nFollowing the reviewer’s suggestion, we added all relevant information to the introduction of the\nmanuscript (pages 3-4). We thank the reviewer for this suggestion, as this additional text will\nimprove the understanding of readers.\nFor clarification, we did not use I74/I85V as the variant would fail as a positive control in the\ncurrent study because the increased sensitivity comes at the cost of a dramatically lower\nexpression level. For this, compare the fluorescence level of strain AB363 that contains Opto-\nT7RNAP*(563) with nMagHigh1 and pMag, and strain AB364 which contains Opto-\nT7RNAP*(563-F2) that harbors the mutations I74V/I85V in Figure S20 (Baumschlager et al.,\n2017). The mCherry fluorescence values at 329 µW/cm2, the maximal light intensity in this plot,\nwere 35,459 a.u. for Opto-T7RNAP*(563) and 20,041 a.u. for Opto-T7RNAP*(563-F2). This\nmeans that this variant leads to a reduced reporter gene expression of 57% compared to the wild\ntype. Even comparing the respective maximal expression of the two variants in all light\nconditions (20 µW/cm2 for AB364 and 329 µW/cm2 for AB363), the expression level in the\nI74V/I85V variant is reduced to 76% compared to the wild type. Since we did not want to\ncompromise the activity of the Opto-T7RNAP for a higher light sensitivity, which supposedly\nwould be easier to achieve, we focused on variants that show equal or higher expression levels\ncompared to the wild-type. Thus, the variants identified in this study show equal or higher\nexpression levels and increased light sensitivity.\n(3) The authors' table detailing the sensitivity changes (Supp Table 13) is very useful. It is one of\nthe key pieces of information for users who might be interested in implementing these mutations\nfor further study. I would strongly prefer that this be included in the main text as a final figure\npanel or table, not on page 38 of the supplementary information where it is likely to escape\nnotice by most readers.\nThe reviewer raises an important point. We have replaced the previous Figure 4 (the mammalian\nexperiments) with this description, and moved the figure describing the mammalian experiment\ninto the supplement according to the description in the next point.\n(4) I have a major concern about the quantification and analysis of the mammalian cell data in\nFigure 3. It is quite misleading to only compare fold-changes between mutants in the light to the\nwild-type variant in the dark. This is because many variants already show dramatically increased\ngene expression in the dark! Fold-changes should be reported for each variant between its dark\nand light conditions. Separately, the authors can describe changes in \"leakiness\" in the dark,\nfor example showing that M48V N100L is already ~10-fold higher than wild-type in the dark. It is\nnot at all clear that the changes between dark and low-intensity light are significant for many\nvariants (e.g. N100L or M48V/N100L). Is the fold-change between dark and 0.1 W/m^2\nstatistically different for the mutants compared to the wild-type in mammalian cells? Without\nsuch a comparsion it is not clear that the changes in sensitivity extend to the mammalian context\nat all.\nThe reason for using fold-changes compared to the wild-type variant was to highlight the\nincrease in overall expression level and not dark-to-light fold-changes of individual variants.\nHowever, fold changes in optogenetic experiments typically refer to dark-to-light-fold changes,\nas the reviewer correctly mentioned. We have chosen to show the absolute data (as fluorescence\nnormalized to mRuby3 expression) in a newly created supplementary figure, which replaces\nprevious Figure 4. Following the suggestion of the reviewer, we added the variant overview\n(previous Supp Table 13) as a new Figure 4 to bring further focus on the core aspects of this\nwork, as the mammalian experiment is a mere extension of the findings to other organisms. This\nnew supplementary figure clearly shows the difference in expression levels due to the different\nvariants, which is the main value of these variants for mammalian systems. In addition, we\nmention in the text, that we observed increased expression levels, while fold-changes were not\naltered, as the reviewer pointed out.\nWe want to thank the reviewer again for their suggestions as we think that they helped us\nimprove the manuscript."}, "author_response": "Point-by-point response to the reviewers ’comments.\nWe have addressed all points raised by reviewer #1. Reviewers’ comments are in blue font and the response to the comments is shown in black font. All changes in the main manuscript and supplementary material are shown in green font.\nReviewer #1 (Remarks to the Author):\nI thank the authors for their response to my prior review and have a few additional comments:\n(1) First and foremost, I still not convinced that this manuscript is a strong candidate for Nature Communications. The authors argue that their paper is principally focused on tuning a synthetic biology gene expression system, not understanding mechanism, which is fine. However, it narrows the scope of the paper considerably - we might learn how OptoT7 in bacteria change, but we do not gain much insight in how these changes will translate to any other application, since we are left with little mechanistic insight. We also are not left with a demonstration that the higher light sensitivity obtained from the screen is useful, say for example by regulating metabolism in high-OD culture. I agree with the other reviewer's first review that this manuscript would be better suited for a more specialized journal.\nWe want to emphasize that this manuscript not only provides a novel set of optogenetic tools covering a wide range of light sensitivities and expression levels, but also reports a framework for any research or application that requires tuning an optogenetic tool or expression system. It is a general methodology that is not limited to a specific application. By thoroughly characterizing the effect on protein expression, we demonstrate the applicability for control of gene expression. One of the main points that this work shows is that the most important features of an expression system, i.e., the sensitivity and the expression levels can be tuned to fit specific desired requirements. Our pre-defined goal was to achieve a higher gene expression using the Opto-T7RNAP at lower light intensities, which was achieved as shown by the data. The data presented in this manuscript have been confirmed using two different technologies (flow cytometry and fluorescent plate reading), both of which are in perfect agreement, and cross-validating the findings.\nTo address the comment about the behavior of the variants at different ODs, we added the full time courses of optical density, reaching from lag to stationary growth phase, together with the reporter expression data and further analysis, providing full information on the expression levels at any stage (and OD) during the entire growth curve, including stationary phase.\nWe added text and further analysis for all spectrophotometry data for all variants at all the light intensities and the three incubation temperatures (30, 37 and 40 ºC) to the manuscript and provided new data in Supplementary Table 13, Supplementary Table 14, Supplementary Figure 21, Supplementary Figure 22, and Supplementary Figure 23. We thank the reviewer for this suggestion, as this information might indeed be useful for readers.\n(2) In their rebuttal the authors reference some supplementary data from their prior paper (Baumschlager 2017). In my view, that paper has a wealth of additional results that support the premise of the current study, and it would be *very* helpful if they were explained in the introduction of this study. The 2017 paper investigates two classic VVD mutants, I85V and I74V/I85V, which have well known effects on photocycling time, but observe that one of these mutants exhibits a massive change in light sensitivity (Fig 4 and S11), whereas the other does not. This shows that sensitivity and photocycle time can be tuned independently - they are not one-to-one - and that wild-type VVD is far from optimal in sensitivity. I agree with the authors' rebuttal that these results argue against sensitivity arising simply from variation in protein expression level. They also argue against the sensitivity being solely a function of lit-state lifetime.\nThe manuscript would be strengthened if the authors would describe something like this logic in their current manuscript, because it is absolutely not clear simply by reading this paper whether light sensitivity itself is an engineerable parameter, since so many other variables could in principle influence a dose response curve.\nThis prior work also suggests that the authors have already identified a very sensitive variant in I74V/I85V. Why was this variant not included in the current analyses, at least as a positive control? Would it be competitive with the best (most sensitive) variants discovered here?\nFollowing the reviewer’s suggestion, we added all relevant information to the introduction of the manuscript (pages 3-4). We thank the reviewer for this suggestion, as this additional text will improve the understanding of readers.\nFor clarification, we did not use I74/I85V as the variant would fail as a positive control in the current study because the increased sensitivity comes at the cost of a dramatically lower expression level. For this, compare the fluorescence level of strain AB363 that contains Opto-T7RNAP*(563) with nMagHigh1 and pMag, and strain AB364 which contains Opto-T7RNAP*(563-F2) that harbors the mutations I74V/I85V in Figure S20 (Baumschlager et al., 2017). The mCherry fluorescence values at 329 µW/cm2, the maximal light intensity in this plot, were 35,459 a.u. for Opto-T7RNAP*(563) and 20,041 a.u. for Opto-T7RNAP*(563-F2). This means that this variant leads to a reduced reporter gene expression of 57% compared to the wild type. Even comparing the respective maximal expression of the two variants in all light conditions (20 µW/cm2 for AB364 and 329 µW/cm2 for AB363), the expression level in the I74V/I85V variant is reduced to 76% compared to the wild type. Since we did not want to compromise the activity of the Opto-T7RNAP for a higher light sensitivity, which supposedly would be easier to achieve, we focused on variants that show equal or higher expression levels compared to the wild-type. Thus, the variants identified in this study show equal or higher expression levels and increased light sensitivity.\n(3) The authors' table detailing the sensitivity changes (Supp Table 13) is very useful. It is one of the key pieces of information for users who might be interested in implementing these mutations for further study. I would strongly prefer that this be included in the main text as a final figure panel or table, not on page 38 of the supplementary information where it is likely to escape notice by most readers.\nThe reviewer raises an important point. We have replaced the previous Figure 4 (the mammalian experiments) with this description, and moved the figure describing the mammalian experiment into the supplement according to the description in the next point.\n(4) I have a major concern about the quantification and analysis of the mammalian cell data in Figure 3. It is quite misleading to only compare fold-changes between mutants in the light to the wild-type variant in the dark. This is because many variants already show dramatically increased gene expression in the dark! Fold-changes should be reported for each variant between its dark and light conditions. Separately, the authors can describe changes in \"leakiness\" in the dark, for example showing that M48V N100L is already ~10-fold higher than wild-type in the dark. It is not at all clear that the changes between dark and low-intensity light are significant for many variants (e.g. N100L or M48V/N100L). Is the fold-change between dark and 0.1 W/m^2 statistically different for the mutants compared to the wild-type in mammalian cells? Without such a comparsion it is not clear that the changes in sensitivity extend to the mammalian context at all.\nThe reason for using fold-changes compared to the wild-type variant was to highlight the increase in overall expression level and not dark-to-light fold-changes of individual variants. However, fold changes in optogenetic experiments typically refer to dark-to-light-fold changes, as the reviewer correctly mentioned. We have chosen to show the absolute data (as fluorescence normalized to mRuby3 expression) in a newly created supplementary figure, which replaces previous Figure 4. Following the suggestion of the reviewer, we added the variant overview (previous Supp Table 13) as a new Figure 4 to bring further focus on the core aspects of this work, as the mammalian experiment is a mere extension of the findings to other organisms. This new supplementary figure clearly shows the difference in expression levels due to the different variants, which is the main value of these variants for mammalian systems. In addition, we mention in the text, that we observed increased expression levels, while fold-changes were not altered, as the reviewer pointed out.\nWe want to thank the reviewer again for their suggestions as we think that they helped us improve the manuscript."}]

Decision letter