Bacterial Cultivation

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E. coli

E. coli was grown in Lysogeny broth (LB) medium (1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl, pH 7± 0.2) and antibiotics for selection were added to the medium (0.01% (w/v) ampicillin; 0.01% (w/v) spectinomycin; 0.005% (w/v) kanamycin). Cultures were then incubated at 37°C under agitation at 120 rpm.

B. subtilis

B. subtilis strains were grown in LB medium (1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl, pH 7± 0.2), MOPS-based MCSE medium (modified chemically defined medium; 1x MOPS solution (0.837% (w/v) MOPS, 0.33% (w/v) (NH4)2SO4, 0.385 mM KH2PO4, 0.615 mM K2HPO4), 0.5% (w/v) tryptophane, 0.22% (w/v) ammonium ferric citrate, 1x III’ salts (0,023% (w/v) MnSO4 × 4 H2O, 1.23% (w/v) MgSO4 × 7 H2O), 40% (w/v) potassium glutamate, 30% (w/v) sodium succinate, 1.5% (w/v) fructose) or double yeast extract tryptone (2xYT) medium (2% (w/v) tryptone, 1% (w/v) yeast extract, 1% (w/v) sodium chloride, pH 7) and supplemented with respective antibiotic (0.0005% (w/v) chloramphenicol or 0.005% (w/v) spectinomycin).

Solibacillus silvestris

For propagation the S. silvestris strain CGN12 (Reeksting et al. 2020) was grown in LB medium. For sand consolidation or MICP assays CGN12 was grown in YA medium (Tris-HCl 50 mM, yeast extract 2 g/L, sodium acetate 100 mM, MnSO4 * 4x H2O 2.32 mg/L ; MgSO4 * 7x H2O 123 mg/L, pH 7.8 ± 0.2) and YAC medium (Tris-HCl 50 mM, yeast extract 2 g/L, calcium acetate 100 mM, MnSO4 * 4x H2O 2.32 mg/L ; MgSO4 * 7x H2O 123 mg/L, pH = 7.8 ± 0.2) if calcium was required. Incubation took place under aerobic conditions (shaking at 120 rpm) at 30 °C.

Xanthomonas campestris

X. campestris was grown in Lysogeny Broth (LB; tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH 7 ± 0.2) under aerobic conditions (shaking at 120 rpm) at 37 °C as preculture overnight. Production cultures were grown in xanthan production medium (citrate 0.5 g/L, K2HPO4 1.5 g/L, MgSO4 x 7 H2O 0.1 g/L, potassium glutamate 1.5 g/L, sucrose 25 g/L, yeast extract 5 g/L, pH 7 ± 0.2).

Agar plates were cast with the addition of 1.5% (w/v) agar-agar, towards the proper LB medium.

Codon Harmonisation of a pysp1 Gene

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Retrieval of DNA sequences

The pyriform silk gene pysp1 (MH37674) from A. ventricosus was used as the template for the synthetic pyriform silk gene. For the subsequent workflow, the complete sequence was downloaded and saved as a text file in the FASTA format. Furthermore, a consensus sequence of the 16 repeat domains was extracted from an alignment using Jalview (Waterhouse et al., 2009). This consensus sequence was used in all following steps.

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Structural analysis via AlphaFold

Structure predictions were generated using AlphaFoldV3 (Jumper et al., 2021) and subsequently analysed using UCSF ChimeraX (Pettersen et al., 2021). Multiple predictions with different synthetic pyriform silk constructs containing one, two, four, eight and 16 repeats between the N-terminal and C-terminal elements were carried out.

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Generation of codon usage tables

For the extraction of the B.subtilis codon usage table, first the annotated reference genome was downloaded from NCBI GenBank (B.subtilis: Genome assembly ASM904v1) and analysed using SnapGene Viewer (GSL Biotech LLC, Boston, MA, USA). All annotated CDS were selected, next the codon usage table was extracted utilizing the “Show Codon Frequencies” function.

A review of available A. ventricosus gene data yielded 28 entries across all NCBI databases. After filtering out redundant and synthetic constructs, 14 genes were selected for the generation of codon usage table. Their CDSs were extracted using the NCBI ORF Finder webtool and compiled into a single textfile dataset (total of 42561 bases). This dataset was then processed in SnapGene Viewer to extract codon usage data the same way described above.

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Design and harmonisation of a synthetic pyriform silk gene

First, the wild-type gene sequence of pysp1 from A. ventricosus was split into three segments. Therefore, the sequences of the N-terminal and N-linker domain were summarized as N-terminal element (NTE) and the sequences of the C-terminal and C linker domain were summarized as C-terminal element (CTE). The previously acquired repeat consensus sequence was termed as repeat element (RPE). Next, the sequences of the segments were edited to be flanked by the fitting restriction endonuclease recognition sites via Benchling, which is explained in more detail in the results page.

The previously extracted codon usage tables of A. ventricosus and B.subtilis were then used for the codon harmonisation of all three segments. Therefore, the codon frequency of the wild-type gene sequence was extracted via SnapGene Viewer, the same way as the codon usage tables were extracted. The difference in codon usage of an organism and the codon frequency of a specific gene was coined “distance” from there on. Codons with a large distance were identified with the help of a python script. In brief, the script screened for codons, which are far less or greater used than the average codon usage of A. ventricosus. Furthermore, the script visualised regions in the gene sequence with high densities of codons with a large distance. Based on these indicators, codons were changed manually as follows. For each altered codon, a target frequency was calculated that matched the distance of the A. ventricosus codon usage and the wild-type pysp1 codon frequency, if compared with the B.subtilis codon usage. In case of a high density of a specific codon, replacements were carried out to maintain this density and vice versa. To validate the harmonisation, the codon frequency of the harmonised sequence was extracted and compared to the aimed values. Next, the sequence was translated into amino acid code via SnapGene Viewer and a sequence alignment with the wild-type sequence was performed by Clustal Omega (Madeira et al., 2024), to exclude the introduction of non-silent mutations or frameshifts. Then, the harmonised sequences were imported into Benchling (Biology software, 2024) and assembled into the complete gene via the assembly wizard function to identify logical mistakes in the design of the assembly. Finally, the harmonized sequences were submitted to the DNA synthesis service of Azenta Life Sciences (Griesheim, Germany).

DNA Cloning Methods

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DNA isolation

Vector and gDNA isolation

Vectors were isolated from 5 mL overnight LB cultures of E. coli NEB Turbo® cells carrying the respective vector and were supplemented with antibiotic. Vector extraction was conducted using the kit Monarch® Plasmid Miniprep Kit (NEB) according to the manufacturer’s instructions. Centrifugation steps were carried out at 17000x g.

gDNA extraction from Gram-negative bacteria was performed using the rapid protocol from Monarch® Spin gDNA Extraction Kit (NEB) according to the manufacturer’s instruction with small deviations: after addition of proteinase K, the sample was incubated at 56°C for 90 min without agitation and the incubation with RNase A was carried out for 10 min. gDNA elution was conducted using 80 µL elution buffer.

PCR product purification and gel extractions

For the purification of PCR products, the kit Monarch® PCR & DNA Cleanup Kit (NEB) was used. To extract DNA after verification in gel electrophoresis, gel extractions were carried out using the kit Monarch® DNA Gel Extraction Kit (NEB) according to the manufacturer’s instructions.

DNA quantification

Measurements of DNA concentrations of different samples were carried out using a Qubit Flex Fluorometer (ThermoFisher). To do so, 1 µL of sample was mixed with 199 µL Qubit 1x dsDNA High Sensitivity solution in Qubit Flex Pyrogen Free tubes. After vortexing the sample for 3 s and incubation at room temperature for 2 min, the measurement took place.

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Polymerase Chain Reaction

In order to amplify sequences of DNA, a polymerase chain reaction (PCR) was conducted with designed primers (Mullis et al., 1986).

Short DNA fragments

When short DNA fragments were amplified (≤2000 bp), the Q5® High-Fidelity DNA Polymerase (NEB) was used. The PCR was conducted according to the manufacturer's protocol with final concentrations of a 50 µL reaction mix being 0.02 U/µL Q5® polymerase, 1× Q5 reaction buffer, 0.3 µM of each forward and reverse primer, 200 µM dNTPs and 1-10 ng of vector DNA.

Thermocycling was performed with initial denaturation at 95°C for 30 s. Then 30 cycles of denaturation at 95°C for 10 s, primer annealing at appropriate temperatures for 30 s and elongation at 72°C for 20-30 s/kb. The annealing temperature depended on the primer pair used. Lastly, a final extension was conducted at 72°C for 30 s.

Longer or difficult DNA fragments

To obtain longer (>2000 bp) or difficult DNA fragments, repliQa® polymerase (Quantabio) protocol was used. Each 50 µL reaction mix consisted of 0.3 µM of each forward and reverse primer, 1x repliQa® HiFi ToughMix®, and 10-50 ng of template DNA. At first, the 2-step protocol was conducted. When the 2-step PCR cycling program failed, the 3-step program was conducted.

The 3-step thermocycling program was performed with initial denaturation at 98°C for 30 s. Then, 30 cycles of denaturation at 98°C for 10 s were conducted, followed by primer annealing for 5 s and elongation at 68°C for 1 s (fragment size ≤ 1 kb), 5 s/kb (fragment size 1-10 kb) or 10 s/kb (fragment size ≥ 10 kb), respectively. For primer annealing, the annealing temperature was calculated with the equation: Tanneal = (Tm-5)°C.

Tm corresponds to the melting temperature of the primer pair. If the annealing temperature was above 68°C only 2-step thermocycling was conducted.

Colony PCR

To verify the correct assembly and successful incorporation of a vector into the respective bacterial strains, colony PCR was performed. OneTaq® 2x Master Mix with Standard Buffer (NEB) was used according to the manufacturer’s instructions.

Initially, 10 µL deionised water was pipetted into PCR tubes. Individual colonies were picked using a sterile pipette tip, streaked onto fresh LB agar plates supplemented with the appropriate antibiotic, and lastly the same pipette tip was dipped for 10 s in the water provided to release bacterial material. 2 µL of this bacterial suspension functioned as the template for the PCR reaction. Each 12 µL reaction mix consisted of 1x OneTaq® Master Mix, 0.2 µM of both forward and reverse primer, and 2 µL of the prepared template.

The thermocycling program began with an initial denaturation at 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 40 s, primer annealing for 40 s (temperature depending on primer Tm) and elongation at 68°C for 30-60 s/kb. The thermocycling was completed with a final extension step at 68°C for 5 min.

When the target amplicon exceeded 4 kb, Q5® High-Fidelity DNA Polymerase (NEB) was used instead. The reaction mixes and thermocycling conditions were adapted as described for short DNA fragments.

QuikChange® PCR

Site-directed mutagenesis was performed using QuikChange® PCR method. For that purpose, repliQa® polymerase (Quantabio) was used. Each reaction contained 50 ng vector, 0.2 µM of each forward and reverse primer and 1x repliQa® HiFi ToughMix®. Thermocycling was conducted as described for PCRs with long or difficult fragments. However, the amplification was carried out with 15 cycles to reduce nonspecific constructs. Background contamination was assessed using a reaction without 1x repliQa® HiFi ToughMix® but equal amount of nuclease-free water as negative control under identical thermocycling conditions.

After amplification, 20 units of DpnI were added to the reaction mix to selectively digest parental methylated vector template. The sample was incubated at 37°C for 1 h and then at 80°C for 20 min. Subsequently, 10 µL of the digested mix were used to transform chemo-competent E. coli NEB Turbo® cells according the methods section about bacterial transformation.

Agarose gel electrophoresis

To validate PCR results or restrictions digests of vectors, agarose gel electrophoresis was performed. For target DNA fragments of 1-5 kb in size, 1% (w/v) agarose (Biozym Scientific GmbH, Hessisch Oldendorf, Germany) in 1x TAE buffer (40 mM Tris pH 8.5, 20 mM acetic acid, 1 mM EDTA) and 1:50000 of GelRed® Nucleic Acid Gel Stain (Biotium Inc., Fremont, CA, USA). 2% (w/v) agarose gels were used for target fragments of <1.5 kb in size. After solidification of the gel, samples were mixed with 6x Purple Loading Dye (NEB) and typically 5 µL of each sample loaded onto the gel. For a size estimation of separated DNA fragments, 5 µL of 1 kb Plus DNA Ladder (NEB) were loaded on the gel. The electrophoresis was executed at constant 130 V for 40 min. Gel documentation was done in an E-BOX CX5 TS (Vilber, Collégien, France).

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Repeat assembly via Pyricloning

For the assembly of a repeat dimer, first two reactions containing 100 ng of the repeat monomer plasmid were prepared. One was supplemented with 10 U of AgeI-HF® (NEB) while the other one was supplemented with 2.5 U of NgoMIV (NEB). Additionally, a third reaction containing 100 ng of the repeat assembly vector was supplemented with 10 U of SapI-HF® (NEB). All reactions contained rCutSmart™ Buffer (NEB) at a final concentration of 1× and filled up with nuclease free water to a total volume of 25 µL. The reactions were incubated for 3 h at 37 °C. After the first hour 10 U of SapI were added to the reactions with AgeI and NgoMIV. Next, the reaction mix containing NgoMIV was purified using the Monarch® PCR & DNA Cleanup Kit (NEB). The other two reactions were heat inactivated at 65 °C for 20 minutes. Afterwards, half of the three reactions were mixed and supplemented with T4 DNA Ligase Reaction Buffer and T4 DNA ligase at a final concentration of 1× and 20 U/µL T4, respectively. Ligation was carried out at 25 °C for 1 hour, followed by transformation of competent NEB Turbo (E. coli) cells using half the reaction. White colonies were submitted to alkaline lysis and subsequently a control digest to validate the correct repeat assembly.

Modular Cloning

The Golden Gate-based Modular Cloning (MoClo) was employed to generate modular expression constructs. Using restriction type IIS endonucleases and DNA ligase, the method allows for rapid, directional and scarless assembly of multiple DNA fragments for the construction of genetic constructs (Weber et al., 2011). The cloning strategy in this study followed the assembly logic established by Weber et al..

MoClo level 0 reaction

To assemble level 0 coding sequences, 20 µL reactions were prepared containing 100 ng of the level 0 destination vector (pMMS0), alongside with 100 ng corresponding part entry vectors or PCR products (PCR product sfgfp; pPySp1_NTE_, pPySp1_RPE, pPySp1_mCTE, pRPAV-His6), were combined. The reaction mix was supplemented with 0.5 U/µL SapI-HF, 20 U/µL T4 DNA ligase and 1x T4 DNA Ligase Reaction Buffer.

MoClo level 1 reaction

Level 1 reactions were used to assemble multiple level 0 parts to full transcriptional units. Each 20 µL reaction consisted of 100 ng level 1 destination vector (pMMS1) and 100 ng of each corresponding level 0 part. The level 0 parts used were promotors (PxylA, PbceA, PliaI and PhpaII), synthetic RBSs (st4, st7, st11 and wk8), coding sequences (CDS; sfgfp, Spidroin constructs such as His6-tagged synthetic pysp1 spidroin monomer) and the terminator L3S1P47. Level 1 reaction were supplemented with 0.5 U/µL BsaI-HF, 20 U/µL T4 DNA ligase and 1x T4 DNA Ligase Reaction Buffer.

MoClo level M reaction

For chromosomal integration into the amyE locus in B. subtilis, level M constructs were assembled in which the assembled level 1 transcriptional units were flanked by homology regions for the amyE locus. Each 20 µL reaction contained 100 ng of level M destination vector (pMMSM1) combined with 100 ng of level 1 transcriptional unit, 100 ng homology regions (pJM1-1L_0052-amyE LF-catR and pJM1-3L_0060-amyE_RF), as well as 100 ng of level M end-linker pICH50892_MeL3. Level M reactions were supplemented with 0.5 U/µL SapI-HF, 20 U/µL T4 DNA ligase and 1x T4 DNA Ligase Reaction Buffer.

MoClo reaction conditions and transformation

Each MoClo reaction was incubated at 37°C for 5 h, followed by 10 min at 50°C and 10 min at 80°C. 10 µL of the reaction mix were used for the transformation of chemo-competent E. coli NEB Turbo® cells. Transformants were selected on LB agar plates supplemented with 0.01% (w/v) spectinomycin (level 0 and level M) or 0.01% (w/v) ampicillin (level 1), respectively. Red-white screening was performed.

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Gibson Assembly

Gibson Assembly was used as a cloning strategy to construct vectors and introduce specific sequence modifications. The method enabled the seamless joining of multiple DNA fragments and incorporation of intended changes at defined positions within the DNA sequence (Gibson et al., 2009).

Target DNA fragments were amplified via PCR using primers that contained the intended sequence modifications as well as overlapping regions complementary to adjacent DNA fragments or the vector backbone. Ideally, these overhangs were 20-40 bp in length and avoided secondary structure formation.

Each 20 µL reaction contained 100 ng of linearised vector backbone, 3-fold molar excess of the insert fragments and 15 µL homemade Gibson Master Mix. The reaction mix was incubated at 50°C for 30-60 min. Subsequently, 10 µL of the reaction mix were used for the transformation of chemically competent E. coli NEB Turbo® cells. To assess the background colony formation, a negative control reaction lacking the insert fragments was set up and performed under identical conditions. Adjusting the vector backbone to insert fragments ratio provided an effective approach for improving the efficiency of difficult Gibson assemblies.

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Alkaline lysis

To isolate vector DNA from bacteria using alkaline lysis, bacterial clones were picked from an agar plate and used to inoculate 5 mL LB overnight cultures containing an appropriate antibiotic. Cells were harvested in 2 mL microcentrifuge tubes by spinning down at 13000 rpm for 1 min and the supernatant was removed. The cells were then resuspended in 300 µL resuspension buffer (50 mM Tris/HCl [pH 8], 10 mM EDTA [pH 8], 0.01% (w/v) DNase-free RNase). To lyse the cells, 300 µL lysis buffer (0.2 M NaOH, 1% (w/v) SDS) was added and the tubes inverted for six times. Following lysis, 300 µL neutralisation buffer (3 M potassium acetate, 5% (v/v) formic acid) was added and the tubes inverted for further six times.

Samples were centrifuged at 13000 rpm for 10 min to pellet cellular debris. Subsequently, the supernatant was transferred to a new microcentrifuge tube and plasmid DNA precipitated by addition of 0.7 volumes of 100% isopropanol. Following inversion of the tubes for six times, the precipitate was spun down at 13000 rpm for 15 min and the resulting supernatant decanted. The pellet was washed with 70% (v/v) ethanol and final traces of ethanol removed by spinning again and removing the supernatant. After that, the pellet was dried at 37°C for 10-15 min. Plasmid DNA was dissolved in 50 µL nuclease-free water.

Analytical restriction digest

Analytical restriction reactions were conducted in a total volume of 10 µL containing 3 µL of sample from alkaline lysis or vector preparation from Plasmid Miniprep, 0.5 U/µL of respective restriction enzyme and 1x rCutSmart buffer. Samples were incubated at 37°C for 1 h and subsequently subjected to gel electrophoresis.

DNA sequencing

To verify the correct assembly or incorporation of a change on a vector, DNA sequencing was conducted. Sanger sequencing was executed by StarSEQ (Mainz, Germany; Sanger et al., 1977). In a total volume of 7 µL 10 pmol primer and 300-600 ng vector DNA were prepared. For larger constructs, whole plasmid sequencing was performed by GENEWIZ® (Leipzig, Germany). Samples were set up in a total volume of 20 µL containing 50 ng/µL vector DNA. The analysis of sequencing results was conducted in Benchling.

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Bacterial transformation

Transformation of E. coli

50 µL chemo-competent E. coli NEB Turbo® cells were mixed with 10 µL cloning reaction mix (Gibson reaction mix, MoClo reaction or restriction ligation, respectively) and thawed on ice for 30 min. A heat shock was conducted at 42°C for 90 s, followed by incubation on ice for 2 min and recovery of the cells with additional 950 µL LB medium at 37°C for 45 min. 200 µL of the cell suspension was streaked on LB agar plates containing appropriate antibiotics. Remaining cells were spun down at 13000 rpm for 1 min, the supernatant discarded, and the cell pellet resuspended with the remaining supernatant. The cell suspension was streaked on a LB agar plate with appropriate antibiotics. Agar plates were then incubated at 37°C over night.

Transformation of B. subtilis

Initially, 500 µL of Paris medium (1× phosphate citrate (1.07% (w/v) K2HPO4, 0.6% (w/v) KH2PO4, 0.1% (w/v) trisodium citrate), 1% (w/v) glucose, 20 mM potassium glutamate, 0.011% (w/v) ferric ammonium citrate, 0.1% (w/v) casamino acids, 0.004% (w/v) tryptophane, 3 mM MgSO4) were inoculated with the B. subtilis W168 wildtype strain and grown over night at 37°C shaking at 120 rpm. 10 µL of overnight culture was used to inoculate 500 µL fresh Paris medium. After 3 h incubation at 37°C under agitation (120 rpm), 1-2 µg of vector DNA was added. The culture was then incubated for an additional 5 h at 37°C shaking at 120 rpm. When transformations were conducted using a vector transferring a chloramphenicol resistance, in the last hour of incubation, chloramphenicol was added to the culture to a final concentration of 0.0000125% (w/v). Afterwards, 180 µL of the culture were each plated onto three separate LB agar plates with suitable antibiotic.

To validate successful chromosomal integration into the amyE locus, transformants were streaked onto starch agar plates (1.5% (w/v) agar, 1% (w/v) starch, 0.8% (w/v) peptone, 0.5% (w/v) NaCl) and incubated over night at 37°C. The wild-type strain served as negative control. After incubation, 2 mL of Lugol solution (0.11% (w/v) iodine) were pipetted onto the starch agar plate and incubated for 30 s at room temperature. Colonies were then inspected for starch hydrolysis halos. The wild-type strain producing a functional α-amylase from the amyE locus formed clear zones, whereas successful integrants lacking amyE showed no halo formation (Sekiguchi, Takada & Okada, 1975).

Protein production and Analysis

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Protein production and culture harvest

Intracellular spidroin production

For protein overproduction, overnight cultures of B. subtilis harbouring spidroin monomer (SPM) constructs in 3 mL LB medium were used to inoculate 10 mL production cultures in LB medium or 2xYT medium, respectively, to an OD600 of 0.05. Reaching an OD600 of 0.4-0.6, protein production was induced using 0.0003% (w/v) or 0.002% (w/v) bacitracin. Subsequently, the production cultures were grown for 3 h at 37°C or over night at 18°C shaking at 120 rpm.

For the protein overproduction in E. coli BL21 strains, 5 mL LB overnight cultures were prepared containing the appropriate antibiotic. These overnight cultures were used to inoculate 5 mL production cultures in LB medium to an OD600 of 0.05. After growth to mid-exponential phase, protein production was induced by addition of 0.5 mM IPTG. Incubation took place at 37°C under agitation at 120 rpm.

3 h post-induction, the cells were harvested equivalently to an OD600 of 2.4 and 7 in a volume of 100 µL by centrifugation at 13000 rpm for 5 min. Right after harvest, the cell pellets were stored at - 20°C until further usage.

Secretory spidroin production

5 mL LB overnight cultures were used to inoculate 10 mL production cultures to an OD of 0.05. Induction with 0.3% (w/v) xylose or 20 µg/mL bacitracin was performed at an OD between 0.4-0.5. After 2-3 h culture volumes equal to an OD of 2.4 and 4.8 were transferred into fresh Eppendorf tubes and harvested by centrifuging at 13,000 x g and 4 °C for 5 min. The supernatants were transferred into fresh 15 mL flacon tubes and stored on ice, whereas the cell pellets were frozen away at -70 °C until further usage. The rest of the cultures were harvested by centrifuging at 4,000 x g and 4 °C for 20 min. Supernatants were then transferred and pooled in the respective falcons from the cell pellets. If supernatants were not directly used for TCA precipitation, they were stored in an ice bath at 4 °C overnight and directly used the next day.

Cell lysis

To obtain whole cell lysate for the analysis of the proteome in SDS-PAGE, the cell pellets from production cultures of B. subtilis harbouring synthetic pysp1 constructs were resuspended with 100 µL lysis buffer (20 mM Tris-HCl pH 7.2, 15% (w/v) sucrose) and 3 µL of 0.005% (w/v) lysozyme were added. Subsequently, the samples were gently vortexed and incubated at 37°C for 5 min. Next, the samples were mixed with 33 µL 4x SDS sample buffer (240 mM Tris-HCl pH 6.8, 8% (w/v) SDS, 40% (v/v) glycerol, 20 mM EDTA, 400 mM DTT, 0.02% (w/v) bromophenol blue) and lastly incubated at 95°C for 5 min. The lysed cells were then used for analysis in SDS-PAGE.

Trichloroacetic acid precipitation

To obtain the secreted protein fraction, 10 mL of harvested culture supernatants from the production cultures were mixed with 2.5 mL of 100% (w/v) trichloroacetic acid (TCA, Sigma-Aldrich, St. Louis, USA) and incubated at 4 °C for 10 min to precipitate the proteins. Afterwards, the solutions were split into 2 mL aliquots and spun down at 17,000 x g for 5 min. The supernatants were discarded and the protein pellets resuspended and combined in 200 µL acetone. Another centrifugation at 17,000 x g for 5 min followed. Again, the supernatant was discarded and the protein pellets washed in 200 µL acetone. After centrifuging again at 17,000 x g for 5 min, the supernatants were discarded and the protein pellets dried at 37 °C for 1 h. Finally, the protein pellets were resuspended in 40 µL Monarch® DNA Elution Buffer, mixed with 13.3 µL 4× SDS sample buffer and incubated at 75 °C for 5 min. In case the samples turned yellow after adding the SDS sample buffer, 1-2 µL of 1 M Tris-HCl (pH 8) was added to increase the pH until the samples turned blue again.

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SDS-PAGE and Coomassie staining

Preliminarily to the analysis in SDS-PAGE, 12% resolving gels with topping stacking gel were poured. The composition of those gels is stated in the table below. When not used immediately for analysis, the poured gels were covered in deionised water-soaked papers and stored at 4°C until usage.

Tab. 1: Composition of 12% SDS gels.

Solution/ reagent	Resolving gel	Stacking gel
ddH2O	1.6 mL	1.25 mL
Resolving gel buffer (1.5 M Tris-HCl pH 8.8; 0.4% (w/v) SDS)	1.3 mL	-
Stacking gel buffer (0.5 M Tris-HCl pH 6.8; 0.4% (w/v) SDS)	-	0.5 mL
30% (w/v) acrylamide/ bisacrylamide	2 mL	0.25 mL
TEMED*	2.5 µL	6 µL
10% (w/v) APS**	50 µL	12 µL

*N,N,N′,N′-Tetramethylethane-1,2-diamine (TEMED); **Ammonium persulfate (APS)

Prepared samples of the cell lysis or protein precipitation were centrifuged at 13000 rpm for 5 min and 16 µL of supernatant were loaded on separate wells on a SDS gel. For size comparison, 5 µL of Color Prestained Protein Standard, Broad Range (10-250 kDa, NEB) were loaded as protein size marker. Electrophoresis was carried out in 1x running buffer (25 mM Tris-HCl, 192 mM Glycine, 3.5 mM SDS) at constant 180 V for 70 min. Following electrophoresis, the stacking gel was discarded as well as remaining running front at the end of the resolving gel. The resolving gel was washed three times in 100 mL deionised water for 5 min. Afterwards, the gel was kept for 20 min in fixation solution (10% (v/v) acetate, 25% (v/v) isopropanol) and thereafter stained for 1 h in 40 mL Coomassie blue staining solution (10% (v/v) acetate, 0.02% (w/v) Brilliant Blue R250). Lastly, the gel was destained in deionised water over night and for further 2 h in 10% (v/v) acetate. Images of the gel were captured.

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Western Blot

Preliminarily, samples were prepared and run in an SDS-PAGE. A polyvinylidene fluoride (PVDF; Merck, Darmstadt, Germany) membrane was activated in pure methanol for 1 min. For electroblotting, two Whatman paper soaked in 1× running buffer (25 mM Tris-HCl, 192 mM glycine, 3.5 mM SDS, 20% (v/v) methanol) were stacked together with the activated PVDF membrane, the SDS gel and two further Whatman paper soaked in 1× running buffer in the order mentioned. To avoid air bubbles, the blotting sandwich was pressed together by rolling on it with a 50 mL falcon. Subsequently, the transfer was conducted at constant 75 mA for 70 min. In order to prevent antibody from binding unspecific, the membrane was blocked with 5% (w/v) milk in 1× TBS-T (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% (v/v) Tween-20) for 1 h at 4°C shaking. Thereafter, 20 mL of 2% (w/v) skimmed milk TBS-T solution were supplemented with 1:2000 monoclonal, horseradish peroxidase (HRP)-conjugated α-poly-histidine antibody (murine, Sigma Aldrich, St. Louis, USA). The antibody solution was then added onto the membrane and incubated in the solution over night at 4°C shaking at 60 rpm. The membrane was then washed three times in 10 mL TBS-T for 5 min at 4°C shaking at 60 rpm. For visualisation, 3 mL TMB Enhanced One Component HRP Membrane Substrate (Sigma-Aldrich, St. Louis, USA) was poured on the membrane and incubated at room temperature for 10 min shaking at 60 rpm. Then the reaction was stopped by pouring approximately 50 mL TBS-T on the membrane. Lastly, images were taken.

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NanoLuc luciferase assay

For the measurements of the secretion of HiBiT(pep86)-tagged proteins the Nano-Glo® HiBiT Extracellular Detection System (Promega, Madison, USA) was utilised. First the Nano-Glo® HiBiT Extracellular Reagent was prepared freshly by mixing the LgBiT Protein 1:100 and the Nano-Glo® HiBiT Extracellular Substrate 1:50 into an appropriate volume of room temperature Nano-Glo® HiBiT Extracellular Buffer in a new tube by inversion. Per measured sample 100 µL of the reagent was prepared. After protein production, 100 µL of the cultures were transferred into a well of a 96 well assay plate (Corning Incorporated, Corning, USA; black plate, clear bottom with lid), mixed with 100 µL of the Nano-Glo® HiBiT Extracellular Reagent and incubated for 10 min at room temperature. The following luminescence measurements were conducted in TecanSpark plate reader with an integration time of 1000 ms. Furthermore, a 100 µL LB medium control and 100 µL of a cell lysate were measured as well. The cells were lysed via bead beating, but normalised to and OD600 of 1 and with only 3 µL of 50 mg/mL lysozyme used.

Reporter gene assay

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To evaluate the relative strength and regulatory behaviour of different promotor and RBS combinations, a super folder Green Fluorescent Protein (sfGFP-) based reporter gene assay was performed.

Strain cultivation and induction

500 µL LB overnight cultures (0.0005% (w/v) chloramphenicol) of B. subtilis carrying reporter gene expression constructs were used to inoculate 5 mL MOPS-based MCSE medium (0.0005% (w/v) chloramphenicol) to an OD600 of 0.05. Cultures were grown at 37°C under agitation at 120 rpm until mid-exponential phase was reached (OD600 0.4-0.6) and were induced with 0.002% (w/v) bacitracin, 0.0003% (w/v) bacitracin or 0.3% (w/v) xylose, respectively. 400 µL samples were taken at timepoints 0, 30 min post-induction and 60 min post-induction for fluorescence analysis. For comparability, constitutive promotors were measured at the same timepoints without prior induction.

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Fluorescence microscopy

For quantitative assessment and single cell analysis of sfGFP production, 1 µL of sample was pipetted onto a 1.5% (w/v) agarose pad on a microscope slide. Alongside, the single-cell heterogeneity and expression patterns were examined. Image acquisition was conducted using a Leica DMi 8 platform (Leica HI Plan 100x/1,25 OIL, Leica DFC9000 GT camera) before and after induction (30 min, 60 min). To allow for comparative analysis, the exposure time, gain and illumination settings were kept constant.

Image analysis

Fluorescence images acquired in fluorescence microscopy were analysed utilising Leica Application Suite X (LAS X) software (v. 3.10.1.29575) and ImageJ software (v. 1.54p). Representative fields were selected using the rectangle tool in such a way that each contained a single cell and kept constant. Cell boundaries were identified by comparison of the corresponding brightfield and fluorescence image. Then, regions of interest (ROIs) were transferred from image to image using the ROI manager in ImageJ. A background region, located in an area without cells, was selected as a blank and used to subtract the background pixel intensity from the measured mean grey value. The mean grey value reflects the average pixel intensity or average fluorescence intensity, respectively, of the selected region. Only cells that were completely within the brightfield image and fully enclosed by the selected rectangle were included in the analysis. In addition, cells that showed abnormal visual contrast (e.g. dying cells) were excluded based on visual inspection of the brightfield image. The remaining cells were analysed for their mean grey value, representing the fluorescence intensity. Data processing was conducted using Microsoft Excel (v. 2506) and GraphPad Prism (v. 8.3.0).

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Plate reader measurement

To evaluate the fluorescence before and after induction, 100 µL of culture from B. subtilis carrying reporter gene constructs was pipetted into single wells of a black 96-well plate with black walls and bottom (Sarstedt, Nümbrecht, Germany). End-point fluorescence measurements were conducted using a TECAN Spark® multimode microplate reader (Tecan group, Männedorf, Switzerland). Measurements were performed before (t = 0 h) and after induction (t = 30 min; 60 min) with excitation at λex = 475 nm and emission at λem = 520 nm. Prior to the measurement, an orbital shaking step of 60 s took place. All conditions were tested in technical duplicates and biological triplicates unless stated otherwise. Fluorescence values were corrected by subtracting the autofluorescence from the medium and B. subtilis W168 wild-type. To account for differences in cell density, fluorescence values were normalised to the optical density at 600 nm. Raw data were processed in Microsoft Excel (v. 2506) and GraphPad Prism (v. 8.3.0).

Biocementation methods

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Xanthan production

First a 5 mL LB culture was inoculated with a single Xanthomonas campestris colony and incubated at 30 °C while shaking (120 rpm) overnight. This preculture was used to inoculate a 200 mL xanthan production medium (sucrose 25 g/L, MgSO4 * 7× H2O 0.1 g/L, K2HPO4 1.5 g/L, citrate 0.5 g/L, yeast extract 5 g/L, glutamate 1.5 g/L, pH 7.0) culture the next day. Incubation took place for 11 days at 30 °C while shaking (120 rpm). Then, the supernatant was split from the cells by centrifugation at 3,500 x g for 15 min and transferred into a beaker. Next, 10 mg KCl and 4 mL 95% (v/v) ethanol per mL supernatant were added. After gently mixing the solution, xanthan precipitation took place over night at room temperature while standing still.

The next day, leftover supernatant was discarded and the precipitated xanthan solved in as little deionised water as possible. Afterwards, the solution was transferred into a dry part of SnakeSkin™ Dialysis Tubing (7 kDa molecular weight cut-off, Thermo Fisher Scientific, Waltham, USA) and dialysed in 5 L deionised water for 18 h. Then, the dialysis tube was transferred into fresh 5 L deionised water and dialysis continued for another 4 h. The obtained solution inside of the tubing was then transferred into a flat container and frozen at -20 °C. Finally, the frozen solution was lyophilised and stored at room temperature until further usage.

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Biocementation

Fresh YA medium (50 mM Tris-HCl pH 7.8, 2.32 mg/L MnSO4 * 4× H2O, 123 mg/L MgSO4 * 7× H2O, 2 g/L yeast extract, 100 mM sodium acetate) was inoculated with single colony of S. silvestris and incubated at 30 °C while shaking (120 rpm) over night. The next day, the culture was harvested by spinning it down at 4,000 x g for 10 min. Meanwhile, fresh YAC medium (50 mM Tris-HCl pH 7.8, 2 g/L yeast extract, 100 mM calcium acetate, 2.32 mg/L MnSO4 * 4× H2O, 123 mg/L MgSO4 * 7× H2O) was prepared and part of it supplemented with 0.5-1% (w/v) xanthan prepared earlier. Next, the cells were resuspended to a final OD600 of 2.7 in the respective media. The wells (2.6 cm width, 4.9 cm length, 1.1 cm depth) of a silicon form were filled with 12.5 g quartz sand (0.1-0.5 mm particle size, SAKRET®, Berlin, Germany) each and then supplemented with 5 mL of the cell suspensions with either 0%, 0.5% or 1% (w/v) xanthan. Also, 5 mL YAC medium with 1% (w/v) xanthan but without cells was added to one of the sand wells as control. Fresh YAC medium was added to the wells after distinct time and drying intervals and is indicated in the respective results part.

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