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Engineering Bacillus subtilis for the formation of a durable living biocomposite material

Engineered living materials (ELMs) are a fast-growing area of research that combine approaches in synthetic biology and material science. Here, we engineer B. subtilis to become a living component of a silica material composed of self-assembling protein scaffolds for functionalization and cross-linking of cells. B. subtilis is engineered to display SpyTags on polar flagella for cell attachment to SpyCatcher modified secreted scaffolds. We engineer endospore limited B. subtilis cells to become a structural component of the material with spores for long-term storage of genetic programming. Silica biomineralization peptides are screened and scaffolds designed for silica polymerization to fabricate biocomposite materials with enhanced mechanical properties. We show that the resulting ELM can be regenerated from a piece of cell containing silica material and that new functions can be incorporated by co-cultivation of engineered B. subtilis strains. We believe that this work will serve as a framework for the future design of resilient ELMs.


Introduction
The design of cells capable of producing self-organizing, biocomposite materials has the potential to enable the fabrication of new types of functional living materials with the ability of self-fabrication and self-repair. Engineered living materials (ELMs) are therefore a new and fast-growing area of research that combines approaches in synthetic biology and material sciences [1][2][3][4] . The fabrication of most ELMs, however, has so far largely relied on physical methods for incorporating a living component in an external material [4][5][6][7] . Engineering of "truly" living materials where the living component actively facilitates material fabrication and organization is much more challenging.
Despite the advances in ELM design, the diversity of different ELM types capable of autonomous self-fabrication and regeneration is still small. In addition, ELM fabrication is currently limited to common chassis organisms that lack the resilience and long-term viability capabilities to withstand conditions outside of the laboratory. To broaden the ELM landscape, we therefore sought to engineer a resilient ELM biocomposite that uses the spore-forming bacteria B. subtilis as its living component for the secretion of protein scaffolds for cell cross-linking and silica biomineralization.
B. subtilis is an industrially used GRAS (Generally Recognized as Safe) bacteria known to have excellent protein secretion capabilities. It forms spores that remain viable for a long time 29-31 , allowing it to persist in our ELM under unfavorable conditions until revival. This was recently demonstrated by bioprinting of Bacillus spores in an agarose matrix 6 . For the secretion of a selfassembling protein matrix, we chose a highly robust scaffolding system that we have previously characterized and engineered for the attachment of different cargo proteins 32-35 . Scaffolds selfassemble from hexameric units of the bacterial microcompartment shell protein EutM. EutM scaffold building blocks are highly amenable to engineering and tolerate N-and C-terminal fusions, including a C-terminal SpyCatcher domain for the covalent linkage of SpyTag-modified proteins to scaffolds 34,35 . We therefore rationalized that B. subtilis could be engineered to secrete EutM-SpyCatcher (EutM-SpyC) building blocks and attach itself via the SpyTag-SpyCatcher system to the formed scaffolds to become both a structural and the living component of the formed material. Further, by engineering the scaffolds to display a biomineralization peptide (EutM-BM), scaffold-silicification could then yield a durable biocomposite material for future development into functional coatings or plasters (illustrated in Fig. 1).
In this work, we first optimized secretion by B. subtilis of our EutM scaffold building blocks which are not normally secreted and in addition, rapidly self-assemble both in vivo and in vitro into large structures 32-38 . We then show that B. subtilis can be engineered to display a SpyTag on cluster, polar flagellar for cell attachment and cross-linking of EutM scaffolds that constitute the protein matrix of our ELM. To allow our production strain to remain a structural component of the formed material even in its dormant state, we created an endospore forming strain. Screening of known silica biomineralization peptides then allowed us to design scaffolds for silica polymerization to fabricate biocomposite materials with enhanced mechanical properties. Finally, we confirmed that our ELM can be regenerated from a piece of silica material and that new functions can be incorporated into the material simply by co-cultivation of engineered B. subtilis strains. We believe that this work will serve as a framework for the future design of resilient ELMs as functional, selfhealing materials such as responsive coatings and plasters.

Identification of growth conditions for ELM fabrication
Several factors need to be considered for the fabrication of a living silica biocomposite material.
Bacillus growth conditions must be identified that are permissible for silica biomineralization and preferably, do not create an overly discolored material. These conditions must also support scaffold building block expression and secretion during exponential to early stationary phase growth when cells are flagellated and have not yet initiated sporulation. Growth conditions for B.
subtilis 168 (referred to as WT) were therefore evaluated using two different media; a Luria Bertani (LB) and a clear, buffered Spizizen minimal medium (SMM). Bacillus cells were transformed with a plasmid (pCT-empty) derived from our previously developed cumate (p-isopropyl benzoate) inducible Bacillus expression vector (Table S1) 39 . Bacterial growth, media pH, flagella presence, and sporulation were then analyzed for 4 days in 24 h intervals at 20 and 30°C (Fig. S1, S2 and   S3). Although pH is not typically considered in culture conditions, silica polymerization is optimal between pH 6.0-8.0 and at pH levels above and below this range polymerization is inhibited 40 .
During growth, B. subtilis increasingly alkalifies the culture medium by switching from sugar to amino acid catabolism -releasing ammonia from tryptophan (SMM) or hydrolyzed amino acids from tryptone and yeast extract (LB) (along with the secretion of alkaline proteases) 41 . In buffered SMM at 20°C, the pH was maintained around 7.0 until 48 h of growth, while under all other conditions tested and during longer growth, media pH quickly increased above 7.0 (Fig. S1).
Peritrichous flagella were observed through 72 h of growth without significant sporulation (Fig.   S2). Buffered SMM and growth for 48 h at 20°C therefore appeared to be optimal for our ELM production by engineered B. subtilis.
We then repeated these growth experiments with B. subtilis WT transformed with a plasmid (pCT-EutMSpyC) for the secretion of the scaffolding protein EutM-SpyCatcher modified with the Nterminal secretion peptide SacB from B. subtilis (Fig. 2a). As before, pH increased above 7.5 regardless of medium at 30°C after 24 h in LB and after 48 h in SMM. (Fig. S1). Again, only cultures grown in SMM at 20°C maintained a stable pH around 7.0 for 48 h and even until 96 h after induction of protein expression. Notably, growth was significantly reduced at 20°C in SMM and LB during 48 h after induction compared to the empty vector control cultures. This was most notable in SMM at 20°C (Fig. 2b), suggesting that EutM-SpyCatcher protein overexpression and secretion may burden cells, slowing cell growth. Like the empty vector cultures above, EutM-SpyCatcher expressing cultures had peritrichous flagella through 72 h of growth, with the most flagella present until 48 h (Fig. S3a). However, spores were already observed after 48 h of growth at 20°C in LB media, indicating cells were stressed under this condition (Fig. S3b). Based on these results, we chose to perform all subsequent cultivations for ELM production in SMM at 20°C for 48 h as our standard condition.

Establishing EutM scaffold building block secretion
The majority of industrially produced recombinant proteins by B. subtilis are extracellular enzymes (e.g. hydrolases) and achieving high-level secretion typically requires the identification of efficient secretion signal peptides to replace their native secretion signals [42][43][44] . In contrast, our scaffolding protein EutM is cytoplasmic that also rapidly self-assembles into scaffolds when overexpressed in E. coli [32][33][34][35][36][37][38] . We therefore screened different signal peptides and investigated secretion of EutM-SpyCatcher by Bacillus. In addition to SacB, we tested four other, commonly used secretion peptides (XynA, YngK, CelA and LipA) 45-49 by fusing them to the N-terminus of our scaffold protein ( Fig. S4a).
Each recombinant protein was expressed in B. subtilis WT in SMM, at 20°C for 48h as the optimal condition confirmed above. Protein secretion levels were then compared between the different cultures by SDS-PAGE analysis. We analyzed total protein in the culture, and after centrifugation of cultures, protein in the resulting culture supernatant and pellet. However, a very low amount of secreted protein was observed in the supernatant compared to visible bands in the pellet, and we suspected that secreted and scaffolded EutM proteins were pelleting together with the cells. We therefore solubilized scaffold proteins from pelleted cells with 4 M urea, which we have used previously to solubilize EutM scaffolds produced by E. coli 33,35 . At this concentration, urea does not lyse cells which we verified by comparing total protein released from completely lysed cells after urea treatment and protein solubilized from sediment (see Fig. S5). We compared by SDS-PAGE three fractions as shown in Fig. 2c: (A) culture supernatants (concentrated by trichloroacetic acid (TCA)), (B) urea supernatant after scaffold solubilization from pellets, and (C) total protein released from remaining pellet after urea solubilization and cell lysis. Only the SacB secretion signal peptide yielded significant yields of secreted protein in the culture supernatant and the solubilized fraction (Fig. 2d). Only small amounts of secreted EutM protein (only after 25x concentration) could be detected in culture supernatants with the other four signal peptides (Fig.   S4b). Compared to cultures expressing SacB-EutM-SpyCatcher, no scaffolds sedimented in the other cultures secreting low levels of EutM protein (Fig. S4b). From these results, we concluded that SacB is the best secretion signal peptide for EutM-SpyCatcher.

Engineering B. subtilis cells as integral material component
With scaffold building block secretion confirmed, we next set out to engineer our host strain to persist as a component within the matrix it fabricates by providing structure to the material and for material regeneration when favorable growth conditions are provided (Fig. 1). B. subtilis spores are extremely durable and can remain dormant indefinitely until proper environmental conditions return whereupon the spores will germinate 29,31 . During sporulation, endospores are formed in the mother cell which then undergoes lysis for spore release. To retain Bacillus cells as structural material components while also allowing spore formation for long-term viability of our material, we deleted the cell wall autolysin LytC (N-acetylmuramoyl-L-alanine amidase) in B.
subtilis 50 by in-frame deletion of lytC to generate a B. subtilis ΔlytC strain ( Fig. 3a and Fig. S6).
As our objective is to link B. subtilis cells to secreted EutM-SpyCatcher scaffold via SpyTag display on its flagella (Fig. 1), we also introduced a second in-frame deletion of flhG ( Fig. 3a and We reasoned that reducing flagellation density and directing flagella to the poles will enable more spaced-out cross-linking on scaffolds and less "clumping" of cells (which we later serendipitously discovered appears to be deleterious for B. subtilis, see below).
The resulting double deletion strain B. subtilis ΔlytC ΔflhG was then transformed with expression vectors (pCT-empty, pCT-EutMSpyC) for confirmation of expected cell phenotypes and scaffold building block secretion ( Fig. 3b-d). Comparison of flagella pattern between wild-type B. subtilis and the double deletion strain confirmed the presence of clustered polar flagella and cell wall enclosed endospores in the deletion strain (Fig. 3b). Clustered polar flagella were most prevalent at 48 h or less after induction, and the most flagella both for the wild-type and the double deletion strain were observed in cultures grown at 20°C (Fig. S2a, S3a, S6d). Unlike the wild-type strain, no free spores were released by the autolysin deficient ΔlytC strain in SMM (Fig. S2b, S3b, S6c).
Endospore formation by B. subtilis ΔlytC ΔflhG harboring pCT-empty and pCT-EutMSpyC was not observed until 72 h of growth under the optimal growth conditions (SMM, 20°C) identified above (Fig. 3b).
Following the confirmation of clustered flagella and no sporulation during growth in SMM at 20°C for 48 h after induction, we next evaluated growth and protein expression under these conditions ( Fig. 3c, d). Interestingly, the growth rate of the double deletion strain was steadier and higher than the wild-type strain when transformed with either the empty control plasmid or pCT-EutMSpyC (compare Fig. 2b and Fig. 3c). The pH of the culture was also maintained at around 7.0 for 48 h. Expression and secretion of scaffold building blocks by the engineered strain was unchanged from the wild-type strain (Fig. 3d, compare to Fig. 2d) despite reaching a slightly higher optical density after 48 h.
We noticed that the secreted EutM-SpyCatcher protein appears as a major higher (24.4 kDa) and a minor lower (21.1 kDa) molecular weight band on SDS-PAGE gels in all three analyzed fractions (culture supernatant, urea solubilized scaffolds and lysed cell pellet, Fig. 2d and 3d). We previously observed that EutM proteins do not migrate according to size and/or as double bands 33,36,38 . Here, we wondered whether the differences in apparent molecular weights might be due to uncleaved SacB signal peptide. Secretion of native proteins with uncleaved Sec signal sequences into culture supernatants has been documented for bacteria 54 . LC-MS analysis showed that indeed the signal peptide was not cleaved from either EutM protein band (Fig. S7).
This suggests that EutM likely folds rapidly upon emerging from the Sec translocase complex 42,43 , preventing access for the signal peptidase and pulling the folding protein into the extracellular space. EutM and its homologs are known to rapidly fold and self-assemble in vitro and in vivo to form higher order structures 32-38 . In contrast, the release and folding of the B. subtilis levansucrase, which is the source of the SacB secretion signal sequence, has been shown to be a slow process 55 .
To determine whether the remaining SacB signal peptide effects EutM-SpyCatcher assembly and to compare EutM-SpyCatcher protein assembly in SMM medium to previous observations in Tris buffer, we purified uncleaved and secreted protein from recombinant B. subtilis ΔlytC ΔflhG cultures and His-EutM-SpyCatcher without signal sequence from recombinant E. coli cultures.
After metal affinity chromatography and dialysis into SMM, scaffolds were observed by transmission electron microscopy (TEM). In comparison to the negative control of SMM which shows many nonhomogenously sized and poorly stained structures and a few heavily stained structures (Fig. S8a, e), scaffolds isolated from E. coli formed rolled-up tubes (Fig. S8b) which have been observed previously for EutM proteins expressed in E. coli 33 . Bacillus secreted protein assembled into fewer large, rolled up tubes and smaller, spherical structures (that stain as donutlike structures) as well as sheets and less-ordered coral-like scaffolds (Fig. S8c, d, f-h). These structures have also been previously observed for EutM homologs 33,38 . This greater variety of scaffold assemblies may be due to the SacB sequence, modifying surface charges and assembly.
These results confirm that B. subtilis secreted EutM-SpyCatcher forms scaffolds and that the SMM growth medium does not inhibit self-assembly.

Linking B. subtilis cells to secreted EutM-SpyCatcher scaffolds by SpyTag display
We next set out to engineer the display of a SpyTag on flagella for cell attachment to secreted scaffolds. For characterization of flagella SpyTag display, a cysteine variant (T209C) of the flagellin Hag protein was first created for labeling of flagella filaments with a sulfhydryl-specific (maleimide) dye 56 . The modified flagellin gene was then expressed from its native promoter on a plasmid in wild-type B. subtilis and the ΔlytC ΔflhG double deletion strain. The assembled flagella filaments are therefore composed of both engineered and native flagellin monomers. The recombinant Hag T209C protein was then modified to include a SpyTag domain at nine different positions chosen inside the variable D2/D3-region of the flagellin protein ( Fig. 4a, b) 57 . Four Hag insertions (bp positions 399, 426, 459, 537) were deleterious for both B. subtilis strains, meaning that either no transformants could be obtained or transformants contained missense mutations or deletions in the modified hag gene of the recovered plasmids (Fig. 4c). Fluorescence microscopy ( Fig. 4d) of the remaining mutants revealed flagella located at the mid cell for B. subtilis WT and poles for B. subtilis ΔlytC ΔflhG which adopt different filament conformations (Fig. 4e, S9a). Wildtype flagella form a left-handed helix 58 , which was retained for insertions at positions 612 and 645 in Hag T209C . Curly, right-handed forms (with half the pitch and amplitude) were observed for flagella expressing SpyTags at positions 555 and 588. SpyTag insertion at position 588 also resulted in shortened flagella compared to unmodified flagella (Fig. 4e). One strain expressing Hag T209C with a SpyTag at position 492 had both normal and curly flagella (Fig. S9a).
Functional display of the SpyTag on B. subtilis flagella by the different insertion mutants was then evaluated by adding purified red fluorescent tdTomato-SpyCatcher protein (Fig. 4d) to recombinant B. subtilis WT and ΔlytC ΔflhG cultures that were then grown overnight for flagella expression. No attachment of tdTomato-SpyCatcher to flagella of strains with Hag T209C SpyTag insertions at positions 492, 555, and 612 was observed by fluorescence microscopy (Fig. 4f,   S9b). Some red fluorescent labeling was observed for SpyTag display at position 645 (Fig. S9b), but the best attachment of tdTomato-SpyCatcher was achieved on flagella that display a SpyTag at position 588 (Hag T209C ::SpyTag 588 ), despite the shorter than wild-type flagella formed by the recombinant strains (Fig. 4f).
We then confirmed that Hag T209C ::SpyTag 588 modified flagella on B. subtilis ΔlytC ΔflhG are capable of forming isopeptide bonds with EutM-SpyCatcher proteins secreted during cultivation.
For this, two B. subtilis ΔlytC ΔflhG strains -one expressing Hag T209C -SpyTag 588 and the other EutM-SpyCatcherwere co-cultured under the identified conditions for scaffold building block secretion but extended to 96 h of induction (SMM, 20°C, 96 h induction). Flagella were then isolated from cells and analyzed by SDS-PAGE, confirming the presence of a higher-molecular weight protein complex (55 kDa) that was not present in the control (Fig. 4g). Based on these results, we therefore chose Hag T209C ::SpyTag 588 expression for cross-linking B. subtilis ΔlytC ΔflhG cells to EutM-SpyCatcher scaffolds.

Engineering of silica biomineralization
Our next objective was to modify EutM scaffolds such that they would biomineralize silica for the fabrication of a resilient living biocomposite material. We selected four known silica biomineralization peptides (R5, CotB1p, SB7, and Synthetic 59-63 for C-terminal fusion to His-EutM ( Fig. S10a). We then characterized silica precipitation and confirmed scaffold formation with the fusion proteins overexpressed and purified from E. coli. We intentionally left the His-Tag on our scaffold building blocks as histidine residues are enriched in proteins involved in natural silica biomineralization processes such as e.g. in the histidine-rich glassin protein present in the siliceous skeletons of sponges 64 .
Silica precipitation by the modified EutM scaffolds was then quantified using a spectroscopic molybdate blue assay 65 (Fig. S10a). The CotB1p (hereafter referred to as CotB) peptide fusion was most efficient at precipitating silica at 7.44 ± 0.36 mmol/g protein and was also the most cationic peptide, rich in arginine residues. As expected, His-EutM scaffolds also precipitated silica at 1.98 ± 0.01 mmol/g protein. The structures (scaffolds) formed by the fusion proteins and His-EutM were then visualized by SEM ( Fig. 5a and Fig. S10b, left column). His-EutM formed tubular nanostructures that appeared to be rolled-up tubes fused together at multiple locations. In contrast, His-EutM-R5 formed globular structures fused into chains, while His-EutM-CotB formed similar scaffolds but with a greater surface roughness. Fusion of the SB7 and Synthetic peptide to EutM resulted in the formation of dense scaffolds comprised of fused together globular and poorly-formed tubular structures (Fig. S10b).
Silica precipitation on scaffolds was investigated by incubating 1 mg/mL scaffolds for 2 h at room temperature with 100 mM silica. SEM imaging of scaffolds (Fig. 5a, center and right) showed an increase in scaffold surface roughness, surface porosity, and the presence of silica nanoparticles fused to the scaffolds which are characteristics of silicification ( Fig. 5a and Fig. S10b, center and right columns). Silica nanoparticle formation was most prominent on His-EutM and His-EutM-SB7 scaffolds, suggesting that biosilicification may have been nucleated in the solution phase by these proteins. In contrast, a significant increase in surface roughness seen for His-EuM-R5 and His-EutM-CotB scaffolds suggests surface catalyzed biomineralization by these structures.
Based on these results, we chose the CotB biomineralization peptide for fusion to scaffold building blocks for secretion by B. subtilis. An expression construct was made where the SpyCatcher domain in pCT-EutMSpyC was replaced with CotB to yield plasmid pCT-EutMCotB, replicating the E. coli expressed His-EutM-CotB with the additional SacB signal sequence (Fig. 5b). EutM-CotB expression and secretion by B. subtilis ΔlytC ΔflhG transformed with pCT-EutMCotB (SMM, 20°C, 48 h induction) was confirmed by SDS-PAGE (Fig. 5c, lane 3). As in the case of EutM-SpyCatcher, the SacB signal sequence appears to be uncleaved based on the size of the observed protein band (16.8 kDa vs 13.8 kDa for the uncleaved and cleaved proteins, respectively). Secretion of EutM-CotB however was much lower than EutM-SpyCatcher, probably due to the added positive charged by the CotB peptide fusion 66 . Yet, despite the low amount of secreted EutM-CotB, cultures of B. subtilis strains transformed with pCT-EutMCotB significantly increased silica gel formation at low silica concentrations compared to empty vector control cultures (Fig. S11). After 48 h induction of protein expression (SMM, 20°C), samples of cultures were incubated with different concentrations of silica (50-500 mM) and silica gel formation followed for 24 h at 20°C. With 100 mM silica added, the EutMCotB expressing cultures solidified into a gel, while the control cultures only formed a liquid soft gel (Fig. S11).
We then proceeded with combining the expression and secretion of our EutM-SpyCatcher building block for cell cross-linking with the EutM-CotB scaffold building block for enhanced biomineralization, yielding the expression vectors as shown in Fig. 5b. For SpyTag-display on flagella of B. subtilis ΔlytC ΔflhG, a plasmid for co-expression of EutM scaffold building blocks with the modified flagellin subunit Hag T209C ::SpyTag 588 was also constructed. Each expression module contains its own promoter as shown. EutM building block expression and secretion by B.
subtilis ΔlytC ΔflhG transformed with the different plasmids was then confirmed and compared, showing again lower secretion levels for EutM-CotB compared to co-expressed EutM-SpyCatcher in SMM at 20°C after 48 h of induction (Fig. 5c). We also attempted to visualize by fluorescence microscopy the flagella phenotypes of B. subtilis ΔlytC ΔflhG cultures transformed with the EutM co-expression constructs with and without Hag T209C ::SpyTag 588 . We observed large structures labeled with cysteine reactive dye that interfere with cell labeling (Fig. S12). In contrast, cells that expressed only the modified Hag T209C or Hag T209C ::SpyTag 588 contained clearly labeled flagella under the same growth conditions (Fig. S12). We therefore presume that the dye labeled structures represent secreted EutM scaffold where one or both cysteine residues of the EutM proteins are labeled. To test whether the silica materials made by the engineered Bacillus strains above have different mechanical properties due to scaffold secretion and cross-linking, we optimized conditions for the formation silica blocks suitable for the rheological analysis (Fig. 6b). Rheological measurements have also been performed for other types of living materials, such as for example a cellulosebased material of a bacterial and yeast co-cultures with programmable weakening of material microstructure 27 . For measurements of our materials, induced culture samples from Fig. 6a were incubated in a syringe with 200 mM silica at 20°C or 25°C for up to 5 h until solid gel blocks were obtained (25°C, 5 h) for measurements with a rheometer. Small, but significant increases in storage moduli (G') were measured for cultures co-expressing EutM scaffold building blocks (1.23-fold for pCT-EutMCotB-EutMSpyC and 1.37-fold for pCT-EutMCotB-EutMSpyC-Hag T209C ::SpyT 588 ) compared to the empty plasmid control (pCT-empty) (Fig. 6c, Table S6). The loss modulus (G") remained the same for all samples and was lower than G' as would be expected for materials with lower viscous dissipation. The increases in G' shows that the silica gel blocks from scaffold secreting cultures have a higher degree of intermolecular bonding giving them greater mechanical rigidity. The engineered Bacillus strain with the SpyTag displaying flagella yielded the most robust material due to cross-linking of cells and scaffolds.

Biocomposite ELM fabrication and characterization
Although we have shown that our final ELM Bacillus strain (B. subtilis ΔlytC ΔflhG pCT-EutMCotB-EutMSpyC-Hag T209C ::SpyT 588 ) improved material strength, we wanted to determine where the secreted scaffolds are located in the material. Uninduced and induced cultures used for silica material formation in Fig. 6 were incubated with SpyTag-eGFP or eGFP (control) to label SpyCatcher domains on EutM scaffolds not linked to SpyTag displaying flagella of cells (Fig.7a).
No labeling was observed with uninduced or eGFP control samples. In contrast, regions outside and in between cells were labeled in the induced samples, suggesting that EutM proteins are secreted and assemble into scaffolds between the cells. The relatively high binding of SpyTag-eGFP indicates that there are many available binding sites for future incorporation of additional, secreted SpyTagged cargo proteins for the fabrication of functional ELMs.
The same cultures examined by GFP labeling were then transformed into a silica blocks as in Fig.   6b for TEM visualization of cells in the biocomposite material. After curing, solid gel plugs were cut into 2 mm 3 pieces and processed for thin sectioning. Cell were observed in clusters for both the induced and uninduced cultures in relatively low density which corresponds to a final OD600 of ~3.5 (induced) or ~6 (uninduced) of the cultures prior to silica material formation (Fig. 7b).
Unexpectedly, no scaffolds could be visualized outside or in between cells in the material from the induced cultures. Instead, only thin sections from the induced cultures contained large clear zones around cells. These zones correspond to the SpyTag-eGFP labeled areas (Fig. 7a) and indicates that a material is physically taking up the space that does not stain after silica biomineralization. The same observations were made when the cell density in silica blocks was increased to an OD600 of 10 and 20 by gentle centrifugation and resuspension of cells prior to biomineralization (Fig. S14 b and c). We therefore theorize that silica deposition on EutM scaffolds surrounding the cells prevents staining. Although no scaffolds were visualized in thin sections, silica gels made in the same manner above show protein bands of the expected sizes for both EutM-SpyCatcher and biomineralizing scaffolds on SDS-PAGE analysis (Fig. S14a) supporting our theory that silica biomineralization is interfering with staining scaffolds during thin sectioning.

Regeneration and functional augmentation of biocomposite ELM
Ultimately, a living composite material for application as e.g. functional coating or plaster should be self-regenerating and integrate desired functions. We therefore first tested whether our ELM could be regenerated from a small plug (~5 mm 3 ) taken from silica blocks generated from cultures of our final ELM strain. Blocks fabricated with 200 mM silica were cured for 1 day at 25°C and then small pieces were used to inoculate fresh cultures for expression and secretion of scaffold building blocks (Fig. 8a). After 48 h of growth with and without induction (control), secretion of EutM proteins at similar levels than the parent culture could be confirmed in the induced cultures ( Fig. 8b). Similarly, we could reproduce the formation of cross-linked silica material in 6-well plates with the induced cultures (Fig. 8c). These results show that the recombinant Bacillus cells remain viable in the silica material and retain their engineered functions. We repeated the same experiments with silica blocks that were cured for 2 weeks at 25°C. Although cultures could be regrown in selective media, meaning that the plasmid is still present, no scaffold building block secretion could be detected. Due to plasmid-based expression of our proteins, we theorized this issue might be caused by mutations in their coding sequences. Sequencing of plasmids isolated from individual colonies derived from the revived culture indeed confirmed that all of them contained multiple mutation in the EutM gene regions. The rapid mutability of high copy number plasmids in Bacillus is a well-known issue 67,68 and can be solved in the future by integrating the expression cassettes currently located on a plasmid into the genome of our engineered Bacillus strain. Nevertheless, the ability to regrow the engineered strain with its plasmid after 2 weeks in a silica gel speaks to the resilience of B. subtilis as ELM chassis organism.
As a proof-of-concept, we sought to demonstrate the incorporation of another property into our material. Rather than adding another genetic burden to our engineered Bacillus strain, we opted to co-cultivate our engineered strain with a second engineered Bacillus strain that would provide the new property. Such a co-cultivation strategy but with two different microorganisms has recently been successfully explored for the fabrication of a living material that incorporates cellulose 27 . For demonstration purposes, we chose the formation of colored material by expressing a purple chromoprotein 69 . An additional plasmid pCT-Purple-Hag T209C ::SpyT 588 was constructed for transformation into B. subtilis ΔlytC ΔflhG and co-cultivation with our final ELM strain. Co-cultures (mixed at a 1:1 ratio in the induction step) were grown as before (Fig. 6) and biomineralization and silica material formation compared to co-cultures with an empty plasmid control (Fig. 8d). Only co-cultures that also expressed the EutM scaffolds formed a purple a crosslinked silica material due to the attachment of the purple protein expressing Bacillus cells (which also display SpyTags on their flagella) to the scaffolds. Likewise, a purple-colored silica block could be fabricated under the same conditions used above with monocultures of our engineered Bacillus strain (Fig. 8e). These results demonstrate an easy and modular approach for the fabrication of living materials with tailored functions and properties ranging e.g. from color and antimicrobial properties 70 to more complex functions such as sensing and responding.

Molecular biology kits and reagents
All supplies were purchased from New England Biolabs (NEB) (Ipswhich, MA, USA). Q5® High-Fidelity DNA polymerase was used for PCR amplifications. The Q5® Site-Directed Mutagenesis kit was used for site directed mutagenesis and the NEBuilder® HiFi DNA Assembly Master Mix was used for Gibson assembly of DNA fragments, and restriction enzymes (BglII, BamHI, and PstI) and T4 DNA Ligase were used for general cloning. Primers were ordered from Integrated DNA Technologies (Coralville, IA, USA).

Strains
Escherichia coli TOP10 (Invitrogen, Carlsbad, CA, USA) was used for cloning and plasmid propagation. E. coli T7 Express (C2566) (NEB) and used for protein expression and purification.
Bacillus subtilis 168 (ATCC 23857) was used as a host strain (referred to as WT strain). All plasmids and strains used in this study are listed in Table S1. For plasmid maintenance, LB was supplemented with 100 μg/mL ampicillin for E. coli strains while LB and SMM were supplemented with 10 μg/mL tetracycline or 20 μg/mL erythromycin for Bacillus strains.

Transformation, strain storage
Transformation of E. coli with plasmids followed standard molecular biology techniques.
Transformation of B. subtilis strains was done using a method previously described by Bennallack

Construction of plasmids and in-frame deletion in B. subtilis
All plasmids and strains used in this study are described in Table S1. Plasmid sequences were verified by Sanger sequencing (ACGT, Inc. Wheeling, IL, USA). Amino acid sequences and nucleotide sequences for all proteins and genes reported in this work are found in Table S2 and   Table S3. Primer pairs for Bacillus genomic manipulations and flagellin engineering are listed in Table S5.

Plasmids for protein expression in E. coli and B. subtilis
Plasmids were constructed using a combination of Gibson Assembly (HiFi® DNA assembly kit, NEB) for fragment assembly, site directed mutagenesis (Q5® mutagenesis kit, NEB) for shorter insertions, deletions and insertions, or restriction enzyme cloning and ligation for larger fragment assembly following manufacturers' instructions and as described previously 1,11 .
Briefly, for the C-terminal fusion of biomineralization peptides to His-EutM in pCT5-His-EutM 1,11 , primers were designed to insert the corresponding into the plasmid using the Q5® mutagenesis kit according to the manufacturers' instructions.
To construct the pCT-His-dtTomato-SpyCatcher plasmid, His-EutM was combined with the fluorescent protein in a previously constructed pCT5-His-EutM-SpyC plasmid 2 by HiFi Assembly.
To create the EutM Bacillus expression vectors, His-EutM-SpyCatcher was amplified from pCT5-His-EutM-SpyC and inserted into our in-house, cumate inducible Bacillus expression vector pCT5-bac2.0 11 by HiFi Assembly. For a control plasmid, pCT-empty was created by deletion of the sfgfp gene from pCT5-bac2.0 using the the Q5® mutagenesis kit according to the manufacturers' instructions. All signal peptides (Table S4) were fused to His-EutM-SpyCatcher by site directed mutagenesis (Q5® mutagenesis kit, NEB) with appropriate primers. The pCT-EutMCotB plasmid for Bacillus expression was made by replacing the SpyCatcher sequence in pCT-EutMSpyC with the CotB sequence by site directed mutagenesis. To create pCT-EutMCotB-EutMSpyC, the complete SacB-His-EutM-SpyCatcher expression cassette (including cumate inducible promoter) from pCT-EutMSpyC was amplified and subcloned into pCT-EutMCotB. The hag T209C ::SpyTag 588 expression cassette (including its native promoter) was added to pCT-EutMCotB-EutMSpyC by HiFi assembly with the hag expression module amplified from the corresponding pBBRm34 plasmid (see below). Finally, to construct pCT-Purple-Hag T209C ::SpyT 588 , the chromoprotein was obtained from pSB1C3-tsPurple 13 and assembled into pCT-EutMCotB-EutMSpyC-hag T209C ::SpyT 588 by replacing the EutM expression modules.

Plasmids for SpyTag display on B. subtilis flagella
The flagellin gene (hag gene) was amplified from B. subtilis genomic DNA using primer pair (NP-hag_fwd and NP-hag_rev) and cloned into the pRBBm34 backbone using HiFi assembly. The flagellin T209C mutation was introduced by Q5®-mutagenesis with primer pairs (NP-hag-T209C_fwd and NP-hag-T209C_rev). A SpyTag was inserted into nine different sites between D1b-N and D1-C domains using Q5®-mutagenesis with primer pairs listed in Table S5.

Plasmids for in-frame deletions of lytC and flhG in B. subtilis
To generate the ΔlytC marker-less deletion construct, the pHBintE vector carrying a temperature sensitive origin of replication, erythromycin and ampicillin resistance was used as the backbone.
The lytC upstream region was amplified from B. subtilis genomic DNA using primer pair dlytC_upstream_fwd and dlytC_upstream_rev while the lytC downstream region was amplified using primer pair dlytC_downstream_fwd and dlytC_downstream_rev (Table S5) and the deletion cassette was cloned into pHBintE vector using HiFi assembly. The plasmid for ΔflhG marker-less deletion was created in a similar manner: The flhG upstream region was amplified using primer pair dflhG_upstream_fwd and dflhG_upstream_rev while the flhG downstream region was amplified using primer pair dflhG _downstream_fwd and dflhG_downstream_rev ( Table S5). The flhG deletion cassette was cloned into pHBintE vector using HiFi assembly.

In-frame deletion of lytC and flhG
The lytC deletion plasmid (temperature sensitive pHBintE-dlytC) was transformed into B. subtilis  Table S5. Transformation of flhG deletion construct was done into B. subtilis ΔlytC host using the pHBintE-dflhG plasmid. In frame deletion of flhG followed by screening was performed with same method as above for lytC but the primer pair used for screening of colonies for flhG deletion mutants was dflhG_confirmation_fwd and dflhG_confirmation_rev.  The following workflow was used for sample preparation: 5 mL of culture broth was centrifuged at 3,220 x g, at 4°C for 10 mins to separate culture supernatant from pelleted cells and coprecipitated scaffolds.

Extracellular secretion and intracellular accumulation of scaffold building blocks by recombinant
For SDS-PAGE analysis of soluble scaffolds in the culture supernatant (fraction i, or A in Fig. 1 For SDS-PAGE of total protein (fraction iv), pelleted cells and co-precipitated scaffolds from 5 mL of culture was resuspended into 500 μL of 50 mM Tris-HCl pH 7.5 in an eppendorf tube. The suspension was sonicated in a coldwater bath sonicator (Bransonic 3510R-MTH, CT, USA) followed by lysis with lysozyme as above. The resulting suspension was mixed with SDS-loading buffer.
All protein samples were denatured by boiling for 10 mins at 100°C and 8 μL of each sample was loaded onto a 15% SDS-PAGE gel. A PowerPAC 300 power supply (Bio-Rad) was used for SDS-PAGE analysis. Coomassie brilliant blue staining was then used for visualization of protein bands.
His-EutM and His-EutM-silica binding peptide fusion proteins were purified using the EutM lysis buffer, while EutM-SpyCatcher was purified using the EutM-SpyCatcher buffer described in 2 . His-tdTomato-SpyCatcher protein was expressed in E. coli and purified using the EutM-SpyCatcher For silica binding assays and SEM analysis of purified His-EutM and His-EutM silica binding fusion protein, purified proteins were dialyzed against deionized water.   16 .

Silica binding assays on EutM scaffolds
Silica precipitation was measured using the blue silicomolybdate method 17,18 . Silica was added to purified protein scaffolds, at a concentration of 1 mg/mL in deionized water, to a concentration of 100 mM and the suspension was mixed for 2 h at room temperature (RT). The precipitate was then collected by centrifugation (3 mins at 18,800 x g), and the resulting pellet was washed 3 times with deionized water. Precipitated silica in the scaffold pellet was depolymerized by adding 1 M NaOH for 10 mins. The samples were then diluted to 1.6 mL and 0.15 mL of solution A (containing 20 g/L ammonium molybdate tetrahydrate and 60 mL/L concentrated hydrochloric acid in deionized water) was added to the sample. After 10 mins, 0.75 mL of solution B (containing 20 g/L oxalic acid, 6.67 g/L 4-methylaminophenol sulphate, 4 g/L anhydrous sodium sulfite, and 100 mL/L concentrated sulphuric acid in deionized water) was added. The blue color was allowed to develop for 2 h at RT, and the absorbance was measured at 810 nm. All samples were run in triplicate.

Silica block formation and mechanical testing
Silica gel plugs were prepared in 10 mL syringes (ID = 15 mm) with the tops removed and then tops were covered instead with sterilized caps to maintain sterile conditions. Silica was added to

Flagella light microscopy
One mL of Bacillus culture was centrifuged at 2,000 x g for 5 mins and gently resuspended in 1 mL of PBS, pH 7.5. The sample was pelleted as above, and the pellet was resuspended to an OD600 of 10. Five µL of sample was loaded onto a polysine microscope slide, covered with a coverslip, and 10 µL Remel RYU flagella stain was applied to the edge of the coverslip.
Slides were examined using a Nikon Eclipse 90i microscope with a 100X 1.3 numerical aperture oil-immersion objective (University Imaging Center (UIC), University of Minnesota (UMN)).
Images were captured using a Nikon D2-Fi2 color camera running on Nikon Elements software.

Spore staining
The Schaeffer and Fulton Spore stain kit was used for spore staining with a modified protocol.
Briefly, 500 µL of Bacillus culture was placed into a glass test tube. One drop (≈150 µL) of malachite green was added to the tube and immersed into boiling water for 10 min. If samples were dehydrating before 10 min, deionized water was added dropwise.
Following boiling, 50 µL of sample was spread by pipette onto a plain, precleaned glass slide.
The slide was allowed to air dry and then heat fixed with flame. The slide was decolorized with distilled water, counterstained with safranin for ≈20 seconds and again decolorized.
Slides were examined using a Nikon Eclipse 90i microscope with a 100X 1.3 numerical aperture oil-immersion objective (UIC UMN). Images were captured using a Nikon D2-Fi2 color camera running on Nikon Elements software.

SEM imaging
EutM scaffolds (1 mg/mL purified proteins in water) were attached to silicon wafers by incubating the protein suspensions on a silicon wafer for 1 h. In samples with silica, the wafers with attached protein were incubated in 100 mM silica for 2 h. Samples were washed with water, fixed with Trump's Fixative for two min, and washed with water again. Samples were washed with increasing ethanol concentrations of 25%, 50%, 75%, and 100%, and then supercritically dried using a Tousimis Critical-Point Dryer (Model 780A). Samples were sputter-coated with 8 nm of iridium and viewed with a Hitachi SU8230 field emission gun scanning electron microscope (University of Minnesota Characterization Facility).

T209C flagella staining
Glycerol cultures were inoculated into 4 mL of selective LB medium for plasmid maintenance and grown overnight at 30°C and 220 rpm. One mL of culture was centrifuged at 2,000 x g for 10 mins.
The supernatant was discarded and pelleted cells were gently resuspended in 1 mL of PBS, pH

TEM of purified scaffolds from E. coli and Bacillus
Purified His-EutM-SpyCatcher protein scaffolds were diluted to 1 mg/mL. Ten µL was placed onto 200 mesh copper grids with 10 nm formvar and 1 nm carbon for 3 mins. The fluid was removed and 10 µL of Trump's fixative (4% formalin, 1 % glutaraldehyde, 0.1 M sodium cacodylate) was applied for 5 mins to fix the proteins. The fluid was removed and the grids were rinsed with 10 µL of deionized water three times. Negative staining was completed using 1% aqueous uranyl acetate which was applied and removed immediately to prevent over-staining. Grids were examined using a JEOL-JEM-1400Plus transmission electron microscope with a LaB6 tungsten filament at 60 kV (UIC UMN). Images were acquired using an Advanced Microscopy Techniques XR16 camera using AMT capture Engine software version 7.0.0.187. cultures were centrifuged at 3,220 x g for 10 mins and the supernatant was removed. Pellets were resuspended to OD600 10 or 20 using the media removed from the pellet. Silica gel plugs were prepared as described above for mechanical testing using 200 mM silica. Gels were pushed out of the syringes and 2 mm 3 gel portions were cut from the center of both induced and uninduced samples. The gel pieces were fixed at 4°C and 10 rpm overnight in 100 mM sodium cacodylate, 2% glutaraldehyde, and 2% paraformaldehyde. The gel pieces were then rinsed with 100 mM sodium cacodylate three times for 30 mins each at 4°C and 10 rpm. An overnight post-fixation using 4% osmium tetraoxide in 100 mM sodium cacodylate was completed at 4°C and 10 rpm.

Image Analysis
Images were cropped, scale bars added, and channels were colorized and merged using Fiji v.
1.53 20 . Fig. S9 image edits for part A include B. subtilis ΔlytC ΔflhG strain SpyTag 555 in which the red and green channels were multiplied by 1.25 before merging. Fig. S12 image edits for part A include the Hag T209C control in which the red channel was multiplied by 1.5 before merging with the green channel. In addition, for Hag T209C ::SpyTag 588 the red channel was multiplied by 1.25 before merging. Fig. S12 image edits in part B include the Hag T209C control in which the red channel was multiplied by 1.5 before merging. In addition, for Hag T209C ::SpyTag 588 the red channel was multiplied by 1.25 before merging.
Flagella RYU staining images were converted to grayscale and had the contrast and brightness increased by 10 using GNU Image Manipulation Program (GIMP) v. 2.8.18 (https://www.gimp.org/).   Figure S1) for staining and imaging by light microscopy. Cells were stained with RYU to observe flagella (a) or malachite green and safranin red to visualize spores (b). Flagella were observed through 72 h of growth, with the most flagella present at 48 h of growth or less under all conditions. Only one, unreleased spore was observed at 48 h of growth in SMM at 30°C, corresponding to reaching stationary phase. Spore formation and flagellation of B. subtilis WT transformed with pCT-EutMSpyC was analyzed under different growth conditions. At 24 h time intervals, samples were removed from SMM and LB cultures grown at 20°C or 30°C (shown in Fig. S1) for staining and imaging by light microscopy. Cells were stained with RYU to observe flagella (a) or malachite green and safranin red to visualize spores (b). Flagella were observed through 72 h of growth, with the most flagella present at 48 h of growth or less under all conditions. Sporulation was observed after 48 h in LB medium at 20°C, correlating with a corresponding decrease in OD600 due to cell lysis (see Fig. S1) while the few spores visible at 48 h in SMM at 30°C did not impact OD.   Deletions of genes encoding the autolysin LytC (a) and flagellar basal body protein FlhG (b) were made in B. subtilis 168 (WT strain) by consecutive rounds of homologous recombination with a temperature sensitive plasmid. In frame deletions were confirmed by PCR, yielding amplification products with the expected sizes. Endospore formation (c) and polar flagella clusters (d) were then confirmed for the double deletion strain transformed with different expression plasmids used in this work. B. subtilis ΔlytC ΔflhG strains were cultured in SMM at 20°C under induction condition. Spores were visualized with malachite green and safranin red at days after induction as indicated (when endospores formed). Red was replaced with magenta using FIJI software (c). Flagella of the same cultures RYU stained and imaged after 48 h of induction. Images were converted to grayscale using GIMP software (d).  a Fluorescence microscopy to evaluate flagella morphologies due to SpyTag insertion. Individual colonies from plates were inoculated in 4 mL LB medium (supplemented with tetracycline) and grown overnight at 30°C and 220 rpm. The hag gene is expressed from its native promoter. Flagella were stained with Alexa Fluor TM 488 C5 maleimide and cell membranes with SynaptoRed TM C2. Samples were examined using a Nikon Eclipse 90i microscope at 100X. dsRed (excitation 530-560 nm, emission 590-650 nm) and GFP (excitation 450-490 nm, emission 500-550 nm) channels were captured in black and white. Channels were false colored magenta for RFP and yellow for GFP and merged using FIJI.

SUPPORTING FIGURES
b Fluorescence microscopy to evaluate SpyTag function when inserted at different locations of the Hag protein. Cultures were grown as above but with the addition of 0.15 mg purified tdTomato-SpyCatcher protein to label functionally displayed SpyTags on flagella. Flagella were stained with Alexa Fluor TM 488 C5 maleimide and cells visualized with the nucleic acid stain Syto TM 16. Samples were imaged and channels false colored as above. a Four silica binding peptides were fused to His-EutM (top panel) and silica binding to the purified proteins (1 mg/mL protein incubated with 100 mM silica for 2 h) compared to His-EutM. Silica precipitation was quantified spectrophotometrically with the molybdate blue assay. Error bars represent standard deviations calculated for values from three sample replicates.
b The same protein scaffolds (1 mg/mL) incubated without (-silica; left column) and with (+ silica (100 mM); middle, and right) for 2 h were visualized by SEM. Deposition of silica increases surface roughness and porosity, and also forms silica nanoparticle aggregates on the surface. His-EutM-CotB shows the strongest silica precipitation. His-EutM also precipitates silica due to its His-tag and positive charge 3-6 .   b, c Silica blocks generated from concentrated cultures with cell densities adjusted to OD600 of 10 or 20 were viewed by TEM for comparison to silica gel blocks directly formed from cultures (Fig.  7). B. subtilis ΔlytC ΔflhG pCT-EutMCotB-EutMSpyC-Hag T209C ::SpyT 588 was first grown under inducing (induced) or non-inducing (uninduced) conditions for protein expression (SMM, 20°C, 48 h). Cultures were centrifuged to collect secreted scaffolds and cells and resuspended in collected SMM supernatants to OD 10 (b) or 20 (c). Silica gel blocks were then formed by adding 200 mM silica and 1 mL aliquots of the cultures were transferred to syringes as molds for curing at 25°C. After 5 h of curing at 25°C, solid gel plugs were cut into 2 mm 3 pieces and prepared for thin sectioning. Cells were observed in clusters for both the uninduced and induced cultures, however only the induced culture contained clear zones around cells as was observed with unconcentrated cultures (Fig. 7).