iGEM 2025 — Team jgu-mainz

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{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents our daily activities, experiments, discussions, and decisions.{% endblock %}
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# Notebook

Explore our detailed lab documentation — week by week and experiment by experiment.

## Labbook 1

This labbook documents our experimental steps and notes of the **design** and **construction** of our first synthetic pyriform silk constructs. Moreover, various production attempts with and without secretion and the detection via the **split NanoLuc assay** are documented in here.

[View PDF](https://static.igem.wiki/teams/5673/src/notebook/2025-10-04-in-silico-construction-of-a-synthetic-pysp1-construct-and-its-production-in-b-subtilis-w168-withoutappendix-komprimiert.pdf)

## Labbook 2

This labbook documents our experimental steps and notes for the **evaluation of promoter-RBS combinations** and **intracellular spidroin production**

[View PDF](https://static.igem.wiki/teams/5673/src/notebook/labbook-evaluation-of-promoter-rbs-combinations-and-intracellular-spidroin-production-upload.pdf)

## Labbook 3

This labbook documents our experimental steps and notes of our **multi-copy** pyriform silk production attempts

[View PDF](https://static.igem.wiki/teams/5673/pdf/2025-10-04-spidroin-production-and-detection-in-plimo1.pdf)

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{% block title %}Engineering Success{% endblock %}
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# **Engineering**

Here you can find an overview over our engineering process!

![DBTL Cycle Pyricon](https://static.igem.wiki/teams/5673/src/engineering/dbtl-cycle.webp)

---

## **Key Achievements**

---

* Completed **7 full DBTL cycles** to tackle the production of synthetic pyriform silk in *B. subtilis* and assess a first prototype with more available biopolymers.
* Established ***Pyricloning***, a **PCR-free, modular cloning system for repetitive genes**, combining elements from RFC25 with the MoClo framework RFC1000.
* Successfully assembled and validated **repeat oligomers** up to an octamer and constructed **full CDS modules**.
* Identified **P*liaI* + st11** as the strongest promoter–RBS pair for **robust expression**, with **st7** offering useful **delayed kinetics**.
* Compared **plasmid-based vs. chromosomal integration**, revealing tradeoffs between yield and stability.
* Implemented detection strategies (HiBiT tagging) to **reveal low-level spidroin expression**.
* Found that **translation and secretion**, rather than transcription, are the **main bottlenecks** for pyriform silk production in *B. subtilis*.

## **The Challenge**

---

Spider silk genes like *pysp1* are highly complex, due to their large size and extreme repetitiveness, leading to great difficulties when it comes to cloning. Moreover, their expression in foreign hosts is challenging, as the translation of the mRNA comes with high stress for the respective cellular machinery, like tRNA depletion (Ramezaniaghdam *et al.*, 2022). Therefore, it requires precise regulatory tuning and ideally secretion to minimise cellular stress and proteolytic degradation. Unlike dragline silk, little work has been done on pyriform silk, making it an underexplored frontier for biomaterial production with high potential in biomineralisation applications like MICP-based biocements.

Our goal was to engineer a synthetic pyriform silk genes in the production platform in *B. subtilis*. This host offered advantages such as efficient secretion machinery, GRAS status, and genetic tractability (Zhang *et al.*, 2020; de Souza *et al.*, 2021; Radeck *et al.*, 2013).

However, challenges included:

* Cloning and expressing highly repetitive sequences without recombination.
* Selecting regulatory elements that allowed tuneable and stable expression.
* Overcoming translation, folding and secretion bottlenecks inherent to spidroins.

Through iterative DBTL cycles, we systematically overcame known challenges with each iteration step: from gene design and assembly to regulation, copy number, secretion and detection.

## **Initial Considerations**

---

We selected pyriform silk as the focus of our project because of its unique role in spiders: it forms a microframework onto which other silk proteins can harden (Greco *et al.*, 2020). This property directly parallels the requirements of MICP-based biocementation, where interstitial spaces often remain unfilled when calcification is driven metabolically by bacteria. By introducing a structural framework, pyriform silk could help overcome this bottleneck.

In reviewing prior iGEM work, including the [iGEM BOKU-Vienna 2022](https://2022.igem.wiki/boku-vienna/) project that pursued MaSp1 based on literature, we found that their design choices did not readily support flexible assembly of synthetic silk constructs, an important requirement for iterative troubleshooting for high-yield expression and secretion campaigns. For our specific biocementation objective, MaSp1 (dragline) also seemed less aligned with the application than pyriform silk, which is tailored for surface attachment and naturally provides a micro-framework that, in our hypothesis, could be conducive to biomineralisation. Our consultation with [Dr. Florian Menzel](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#:~:text=Dr.%20Florian%20Menzel) supported this direction, as his insights into the functional differences between silk types reinforced our choice to focus on pyriform silk as a more suitable candidate for biocement applications. Furthermore, we consulted with [Prof. Dr. Kevin Paine](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#:~:text=Prof.%20Dr.%20Kevin%20Paine) to better understand the challenges inherent to biocement production. His expertise affirmed that our approach addresses critical limitations in current strategies, thereby strengthening the rationale for our design. Accordingly, we oriented our design toward modular, secretion-oriented pyriform constructs and a compatible expression strategy.

In addition, pyriform silk is thought to self-aggregate into small fibers, which would greatly simplify downstream fiber formation and processing (Wang *et al.*, 2019).

As a template, we chose *pysp1* from *Araneus ventricosus*, one of the two fully annotated pyriform silk genes. This gene is significantly smaller than the corresponding version in *Argiope argentata*. Working with the smaller gene allowed us to design shorter constructs, thereby easing handling, cloning, and overall workflow efficiency.

---

## **Engineering success**

Iteration 1 - Gene design, repeat oligomer strategy, and CDS assembly

Design 1

In our first cycle, we aimed to generate functional parts from the *pysp1* template that could be used to construct synthetic pyriform silk genes. To achieve this, we first assessed the structural composition of PySp1 using multiple AlphaFold v3 predictions (Abramson *et al.*, 2024). After consulting [Dr. Florian Hof](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#:~:text=Dr.%20Florian%20Hof) and [Prof. Dr. Lukas Stelzl](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#:~:text=Prof.%20Dr.%20Lukas%20Stelzl) on AlphaFold predictions and disordered proteins, we concluded that the modularity of PySp1 corresponds to its domains and DNA sequences.

Based on this, we divided *pysp1* into three modular parts, NTE (N-terminal element), RPE (repeat element), and CTE (C-terminal element), that could later be assembled into synthetic pyriform silk genes. The sequences were codon-harmonised for *B. subtilis* W168 to reduce tRNA depletion and preserve co-translational folding pauses, thereby increasing the likelihood of producing a functional PySp1 protein.

A key challenge in assembling highly repetitive DNA sequences is that PCR-based methods often fail due to mispriming and polymerase slippage. To avoid these issues, we deliberately designed our cloning strategy to be PCR-free. Hence, we developed *Pyricloning*, a modular cloning strategy combining elements of RFC25 and the RFC1000 MoClo framework to generate higher-order repeat oligomers without compromising flexibility in downstream assembly. After discussing our approach with [Dr. Farley Kwok van Giezen](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#:~:text=Dr.%20Farley%20Kwok%20van%20der%20Giezen) and considering alternative methods for assembling repetitive sequences, we added specific restriction enzyme recognition sites to our parts.

![figure 1](https://static.igem.wiki/teams/5673/src/engineering/schematic-summary-of-pyricloning.webp)

***Figure 1: Schematic summary of “*Pyricloning*”**. The figure illustrates the stepwise construction of repeat oligomers and full-length coding sequences using the *Pyricloning* system.*

Build 1

We implemented *Pyricloning* to iteratively construct repeat oligomers from dimers up to octamers. These oligomers were then used to assemble CDS constructs with varying repeat numbers.

Test 1

Restriction digests, agarose gel electrophoresis, and sequencing validated our assembly strategy, with distinct bands corresponding to different repeat oligomers (Figure 2) and CDS constructs (Figure 3). Additionally, we computationally confirmed that codon harmonisation produced a distribution closer to the native profile by comparing codon usage in our sequences with that of *B. subtilis* W168, and contrasting it with the wild-type *A. ventricosus* codon usage.

![figure 2](https://static.igem.wiki/teams/5673/src/engineering/pyricloning-enables-the-seamless-construction-of-oligomers-from-repetitive-sequences.webp)

***Figure 2: *Pyricloning* enables the seamless construction of oligomers from repetitive sequences.** All plasmids were digested with BbsI and SapI for 1 h at 37 °C. The empty repeat assembly vector served as control. Arrows indicate the expected positions of the fragments.*

 ![figure 3](https://static.igem.wiki/teams/5673/src/engineering/successful-generation-of-a-synthetic-pyriform-silk-gene-called-sd2-with-a-repeat-tetramer-using-the-modular-cloning-system.webp)

***Figure 3: Successful generation of a synthetic pyriform silk gene called SD2 with a repeat tetramer using the modular cloning system.** All plasmids were digested with BsaI for 1 hour at 37 °C. The empty entry vector served as control. Arrows indicate the expected positions of the fragments.*

Learn 1

*Pyricloning* enabled robust, PCR-free construction of repeat oligomers up to octamers (and, in theory, beyond), which could be seamlessly integrated into complete CDS constructs. However, we observed occasional recombination events during repeat generation, likely due to using a *recA*+ *E. coli* strain rather than issues with the assembly strategy itself. These artifacts did not appear in later assemblies, but the experience prompted us to carefully validate construct sizes after each cloning step.

Codon harmonisation produced a distribution closer to the native sequence, which should reduce the risk of tRNA depletion and misfolding during translation. While we cannot directly quantify its effects, literature suggests that harmonisation increases the likelihood of successful protein expression, thus as an advantage we expect to benefit in future production attempts.

Iteration 2 - Regulatory element evaluation (promoters & RBSs)

Design 2

In recombinant spidroin production, the precise control of transcription and translation is crucial for success and overcoming common challenges such as low yields and the accumulation of insoluble protein, both frequently linked to limitations in transcription and translation (Connor, Zha & Koffas, 2024). To strengthen our approach, we consulted [Dr. Georg Fritz](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#:~:text=Dr.%20Georg%20Fritz) regarding the MoClo strategy. He provided valuable guidance on how to efficiently utilize this methodology, offering feedback and resources such as detailed guides and plasmid materials to optimize our workflow.

Therefore, prior to assembling complete transcription units with our CDS constructs, we evaluated transcriptional and translational regulatory elements to identify combinations that balance protein yield with cellular stress. We selected candidate promoters (P*xylA*, P*bceA*, P*liaI*, P*hpaII*) and ribosome binding sites (st4, st7, st11, wk8) that are well-characterized in *B. subtilis*. As a reporter, we used *sfgfp* to quantify expression as the protein matures rapidly with a folding time of 6 min in *E. coli* and is highly stable, thereby producing consistent fluorescence signals (Pédelacq *et al.*, 2006; Shields *et al.*, 2019).

 ![figure 4](https://static.igem.wiki/teams/5673/src/engineering/reporter-constructs.webp)

***Figure 4: Overview of the generation of reporter constructs.** P = promoter, RBS = ribosome binding site, Ter = terminator. A) Schematic of the reporter construct design including the specific 4 bp overhangs for the directional assembly of the required basic parts. B) Composition of level 0 basic parts for reporter constructs in the evaluation of the strength of different promoters. RBS st11, reporter gene *sfgfp* and terminator L3S1P47 were combined with either P*xylA*, P*bceA*, P*liaI* or P*hpaII*, respectively. C) Set of level 0 parts for the assembly of reporter constructs in evaluating translational efficiency of different RBSs. The promoter P*liaI*, reporter gene *sfgfp* and terminator L3S1P47 were assembled with either RBS st4, st7, st11 or wk8.*

Build 2

We assembled various reporter transcription units (promoter–RBS–*sfgfp*–terminator) using the MoClo RFC1000 framework. To ensure reliable comparisons, the constructs were genomically integrated in a single-copy format.

Test 2

Reporter strains were analysed using plate-reader fluorescence measurements and single-cell fluorescence microscopy. Both static end-point and kinetic measurements were performed, with time courses extending up to 60 min post-induction.

Learn 2

We observed a broad dynamic range of expression strengths across different promoter–RBS combinations, providing the flexibility to fine-tune constructs for future production attempts. P*liaI* emerged as the strongest inducible promoter, while P*xylA* displayed the weakest activation. Among the RBSs, st11 supported rapid and robust translation, whereas st7 exhibited slightly slower kinetics, which may reduce cellular stress during expression of repetitive proteins. These results formed the basis for promoter and RBS choices in subsequent spidroin transcription units.

Iteration 3 - Transcription unit assembly and genomic integration (Level-1 → Level-M/2 → *amyE*)

Design 3

Building on the results of the second cycle, we assembled complete transcription units (TUs) with our CDS constructs. To achieve high yields, we chose the strong promoter P*liaI*. As RBS, we selected st11, aiming for high yields while accounting for stress with a st7 variant with slower kinetics.

The Level-1 transcription units were then assembled into Level-M constructs containing homologous flanks for the *amyE* locus of *B. subtilis* W168 and a chloramphenicol resistance cassette for selection.

 ![figure 5](https://static.igem.wiki/teams/5673/src/engineering/design-of-the-first-transcription-units-used-for-synthetic-pyriform-silk-production.webp)

***Figure 5: Design of the first transcription units used for synthetic pyriform silk production. .** Level 1 showcases the framework used to build the transcription unit, while level M/2 depicts the actual transcription units build for the genomic integration into *B. subtilis* W168.*

Build 3

Because our CDS constructs from *Pyricloning* were already compatible with the RFC1000 MoClo framework, we were able to directly assemble level-1 TUs with our previously build CDS. Our first TUs contained CDS with monomeric RPEs to assess the production of the simplest constructs. Later we also build CDS with tetrameric and octameric RPEs, which were also used to assemble functional TUs. Successfully crafted level-1 transcription units were then used for Level-M (2) assemblies, which were subsequently integrated into the *B. subtilis* W168 genome via homologous recombination at the *amyE* locus.

Test 3

We validated the Level-1 and Level-M assemblies by restriction digests, agarose gel electrophoresis, and whole-plasmid sequencing. Successful genomic integration was confirmed using a starch hydrolysis assay: colonies with disrupted *amyE* no longer degraded starch, and indeed, colonies without hydrolysis halos indicated correct integration.

 ![figure 6](https://static.igem.wiki/teams/5673/src/engineering/successful-generation-of-a-complete-transcription-unit-including-a-synthetic-pyriform-silk-gene-construct-with-a-repeat-tetramer-via-moclo.webp)

***Figure 6: Successful generation of a complete transcription unit including a synthetic pyriform silk gene construct with a repeat tetramer via MoClo.** All plasmids were digested with SapI and NgoMIV for 1 h at 37°C. The empty entry vector served as control. Arrows indicate the expected positions of the fragments.*

 ![figure 7](https://static.igem.wiki/teams/5673/src/engineering/successful-generation-of-a-level-m-transcription-unit-via-moclo-for-the-genomic-integration-into-the-b-subtilis-w168.webp)

***Figure 7: Successful generation of a level M transcription unit via MoClo for the genomic integration into the *B. subtilis* W168.** All plasmids were digested with BsaI and SacI for 1 h at 37°C. The empty entry vector served as control. Arrows indicate the expected positions of the fragments.*

 ![figure 8](https://static.igem.wiki/teams/5673/src/engineering/successful-integration-of-level-m-tus-into-the-amye-locus-of-b-subtilis.webp)

***Figure 8: Successful integration of level M TUs into the *amyE* locus of *B. subtilis* W168, disrupting starch metabolism.**  A) The left and right homology flanks targeting the *amyE* locus of *B. subtilis* W168 recombined with the respective sites in the *amyE* gene, resulting in the genomic integration of the transcription unit. Colonies with a positively integrated transcription unit into the *amyE* locus lost the ability to hydrolyse starch, leading to the preservation of starch around those colonies. By staining the starch agar plates with Lugol solution (0.33% iodine) the remaining starch could be visualised.*

Learn 3

This cycle demonstrated that our cloning strategy was both robust and efficient for assembling synthetic pyriform silk genes into functional transcription units. The constructs proved stable across multiple assembly steps and integration events. These results also gave us confidence that the approach could be quickly adapted to address future challenges, allowing us to proceed with the first production tests using integrated spidroin constructs.

Iteration 4 - Intracellular spidroin production

Design 4

For our first production attempts, we tested two constructs driven by the P*liaI* promoter. Each carried a CDS with a monomeric RPE, the L3S1P47 terminator, and a hexahistidine tag for purification and Western blot detection. Since our earlier *sfgfp* reporter tests showed a robust and strong translation initiation for both the st11 and st7 RBS, we chose them to explore the impact of translational fine-tuning on expression of repetitive proteins.

In addition, we designed the cultivation process to probe the impact of growth conditions on spidroin production. Specifically, we compared two different media (LB and 2×YT) to test whether nutrient availability affected expression. We also reduced the cultivation temperature from 37°C to 18°C after induction in one batch to slow metabolism and protein biosynthesis, aiming to alleviate cellular stress associated with spidroin production.

Build 4

The transcription units were genomically integrated into the *amyE* locus of *B. subtilis* W168, and production was tested in both LB and 2×YT medium.

Test 4

After cultivation, cultures were harvested, and the pellets processed for SDS–PAGE and Western blot analysis. No spidroin was detectable with either method.

 ![figure 9](https://static.igem.wiki/teams/5673/src/engineering/spidroin-production-in-lb-and-2xyt-medium-was-not-detectable-in-sds-page-and-western-blot.webp)

***Figure 9: Spidroin production in LB and 2xYT medium was not detectable in SDS-PAGE and Western Blot.**  Whole cell lysate from protein production cultures of recombinant *B. subtilis* carrying the SPM constructs grown in different conditions. Odd numbers: *B. subtilis* P*liaI*-st11-SPM-His6-L3S1P47. Even numbers: *B. subtilis* P*liaI*-st7-SPM-His6-L3S1P47. Culture conditions: samples 1 & 2: LB medium at 37°C; samples 3 & 4: LB medium at 18°C; samples 5 & 6: 2xYT medium at 18°C. Controls: WT = induced *B. subtilis* wild-type; C = control, mNeonGreen-His6. Green arrows indicate the expected migration of SPM, whereas the red arrows indicate the protein band of mNG-His6. SPM = spidroin monomer A) SDS-PAGE of *B. subtilis* carrying the SPM constructs grown in different conditions (see above) in a 12% SDS-gel. After electrophoresis, the SDS-gel was washed with ddH2O and stained with Coomassie blue after fixation. B) Western Blot of *B. subtilis* carrying the SPM constructs grown in different conditions (see above). A 12% SDS-gel was subjected to electroplotting onto an activated polyvinylidene fluoride (PVDF) membrane. Thereafter, the membrane was blocked and incubated with HRP-conjugated α-poly-histidine antibody. After washing, visualisation took place using TMB Enhanced One Component HRP Membrane Substrate.*

Learn 4

Intracellular expression of spidroin constructs proved insufficient regardless of RBS or induction strength. Possible explanations include proteolytic degradation triggered by cytoplasmic stress or expression levels below the detection limits of standard methods. These findings prompted us to adopt a new strategy: increasing gene dosage with a multi-copy approach and using secretion signal peptides to bypass intracellular stress and proteases.

Iteration 5 - Multi-copy expression and secretion strategy

Design/ Build 5.1

Guided by our consultation with [Dr. Jolanda Neef](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#:~:text=Dr.%20Jolanda%20Neef), we implemented her input by converting the pBSMuL1 vector into a MoClo-compatible entry vector containing an *mrfp1*Δ*lac*O red/white selection cassette to enhance gene dosage and enable multi-copy expression in *Bacillus subtilis* W168. To test the functionality, we first cloned *sfgfp* constructs under two promoters, P*hpaII* (constitutive) and P*xylA* (previously described as inducible).

 ![figure 10](https://static.igem.wiki/teams/5673/src/engineering/design-of-the-transcription-units-used-for-evaluation-of-our-multi-copy-approach.webp)

***Figure 10: Design of the transcription units used for evaluation of our multi-copy approach.**  Level 1 showcases the framework used to build the transcription unit, while level 1 plasmid depicts the actual transcription units build for the transformation into *B. subtilis* W168.*

Test 5.1

After transforming *B. subtilis* W168, we evaluated expression and growth using plate-reader assays and fluorescence microscopy.

 ![figure 11](https://static.igem.wiki/teams/5673/src/engineering/bacterial-growth-and-fluorescence-signals-under-constitutive-promoter-regulation.webp)

***Figure 11: Bacterial growth and fluorescence signals under constitutive promoter regulation, P*xylA* and P*hpaII*.** In A, the growth curves of *B. subtilis* W168 harbouring the P*xylA*/P*hpaII*-m*sfgfp*-assemblies in MCSE-medium are shown. B: Promoter activities expressed as fluorescence/OD600 (WT-corrected). The OD600 graph is a representative of a total of two replicates. The fluorescence graph shows the mean and standard deviation of two biological replicates.*

Learn 5.1

The system functioned as expected. Nevertheless, against the expectations, P*xylA* behaved as constitutive in the multi-copy plasmid context, likely because the cognate repressor is encoded only in the chromosome and was insufficient to control the large number of promoter copies. Based on this observation, subsequent designs employed P*liaI*.

Design/ Build 5.2

After confirming *sfgfp* expression, we applied the strategy to pyriform silk constructs. Literature review revealed that secretion signals vary in performance depending on the target protein. Following advice from [Prof. Dr. Thomas Wiegert](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#:~:text=Prof.%20Dr.%20Thomas%20Wiegert), we incorporated the ss*yoaW* system, which includes a StrepII–SUMO tag for enhanced folding, alongside ss*lipA* from the pBSMuL1 vector for comparison.

We designed four new constructs: monomeric and tetrameric RPE variants, each with either ss*yoaW* or ss*lipA*, all driven by P*liaI*. These constructs were assembled in our new vector and transformed into *B. subtilis* W168.

 ![figure 12](https://static.igem.wiki/teams/5673/src/engineering/design-of-the-transcription-units-used-for-synthetic-pyriform-silk-production-in-our-multi-copy-approach.webp)

***Figure 12: Design of the transcription units used for synthetic pyriform silk production in our multi-copy approach.**  Level 1 showcases the framework used to build the transcription unit, while level 1 plasmid depicts the actual transcription units build for the transformation into *B. subtilis* W168.*

Test 5.2

Expression was evaluated in LB cultures, with samples analysed via SDS–PAGE and Western blot across multiple cellular fractions and the supernatant.

 ![figure 13](https://static.igem.wiki/teams/5673/src/engineering/protein-gels-of-five-different-fractions-of-the-b-subtilis.webp)

***Figure 13: Protein gels of five different fractions of the *B. subtilis* W168 WT and *B. subtilis* W168 carrying spider silk constructs showed no clear band for spider silk proteins, nor did Western Blot.**  For each fraction (whole cell lysate, secreted proteins, lysate after beadbeating, cytoplasmic proteins and cell debris) two SDS-PAGEs were run followed by performing with one a Western Blot, utilising a monoclonal anti-poly-histidine peroxidase antibody, 1:2000, and a substrate solution for colorimetric protein detection. The purple arrow indicates the height of the spidroin-tetramer, the blue one the height of the spidroin monomer and the green one the GFP-His positive control. L= molecular weight marker, 1= non-induced WT, 2= induced WT, 3= ss*yoaW*-monomer, 4= ss*yoaW*-tetramer, 5=ss*lipA*-monomer, 6=ss*lipA*-tetramer and C= control protein with a His-tag. 10% SDS gel.*

Learn 5.2

Despite increased gene dosage and secretion tags, no detectable pyriform silk was observed in either intracellular or extracellular fractions. After consulting [Prof. Dr. Tracy Palmer](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#:~:text=Prof.%20Dr.%20Tracy%20Palmer), we hypothesised that overexpression of such a large, complex protein in a multi-copy context overwhelmed the Sec secretion system, triggering protease upregulation and degradation. The high proportion of unfolded regions in spidroins makes them particularly vulnerable to proteolysis.

This outcome highlighted the need for more sensitive detection methods and guided us toward exploring the split NanoLuc assay in the following cycle.

Iteration 6 - Single-copy secretion and sensitive detection with HiBiT (pep86) tag & Nano-Glo® assay

Design 6

Following valuable feedback from [Prof. Dr. Tracy Palmer](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#:~:text=Prof.%20Dr.%20Tracy%20Palmer), we decided to take a step back and return to a single-copy genomic integration approach. This time, we designed constructs carrying secretion signal sequences to facilitate protein export without overwhelming the Sec pathway, which is essential for *B. subtilis* viability (Ling *et al.*, 2007; Souza *et al.*, 2021).

In parallel, we explored the split NanoLuc assay (Yang *et al.*, 2024; Pereira *et al.*, 2019) for highly sensitive detection and consulted [Dr. Margarethe Schwarz (Promega)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#:~:text=Dr.%20Margarethe%20Schwarz%20-%20Promega) regarding the Nano-Glo® HiBiT Extracellular Detection System. Based on these discussions, we designed new constructs that included a small C-terminal tag, pep86 (11 amino acids), which complements Promega’s LgBiT fragment to form an active luciferase. Combined with the N-terminal ss*yoaW* secretion signal provided by [Prof. Dr. Thomas Wiegert](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#:~:text=Prof.%20Dr.%20Thomas%20Wiegert), this setup offered a more sensitive detection method compared to traditional Western blots.

 ![figure 14](https://static.igem.wiki/teams/5673/src/engineering/design-of-the-transcription-units-used-for-synthetic-pyriform-silk-production-and-secretion-in-our-final-approach.webp)

***Figure 14: Design of the transcription units used for synthetic pyriform silk production and secretion in our final approach.**  Level 1 showcases the framework used to build the transcription unit, while level M/2 depicts the actual transcription units build for the genomic integration into *B. subtilis* W168.*

 ![figure 15](https://static.igem.wiki/teams/5673/src/engineering/schematic-illustration-of-the-nano-glo-hibit-extracellular-detection-system.webp)

***Figure 15: Schematic illustration of the Nano-Glo® HiBiT Extracellular Detection System.**A genomically integrated transcription unit encoding a recombinant protein fused to the small pep86 tag (also called HiBiT, 11 amino acids) is expressed in bacterial cells. If the fusion protein is secreted into the extracellular medium, pep86 can associate with the larger LgBiT fragment (156 amino acids), which is added to the supernatant along with the luciferase substrate, furimazine. Upon complementation of pep86 and LgBiT, an active luciferase enzyme is reconstituted, producing a luminescent signal proportional to the amount of secreted protein.*

Build 6

We generated three new constructs ([BBa\_255CP6QO](https://registry.igem.org/parts/bba-255cp6qo), [BBa\_25VXKHEZ](https://registry.igem.org/parts/bba-25vxkhez), [BBa\_25AE0AHE](https://registry.igem.org/parts/bba-25ae0ahe)) containing a monomeric, tetrameric, or octameric RPE. Each construct included the P*liaI* promoter, RBS st11, the N-terminal ss*yoaW* signal peptide, and the C-terminal pep86 tag, as discussed with [Prof. Dr. Tracy Palmer](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#:~:text=Prof.%20Dr.%20Tracy%20Palmer). These were genomically integrated into *B. subtilis* W168.

Test 6

We cultivated LB cultures of all three strains and analysed the supernatant and cell pellets. Protein detection was carried out using SDS–PAGE (Figure 16A), Western blot (Figure 16B), and the newly adapted split NanoLuc assay (Figure 17) with Promega’s Nano-Glo® HiBiT Extracellular Detection System.

 ![figure 16](https://static.igem.wiki/teams/5673/src/engineering/synthetic-spider-silk-proteins-could-not-be-detected-via-sds-page-or-western-blot.webp)

***Figure 16: Synthetic spider silk proteins could not be detected via SDS-PAGE or Western blot.**  Whole cell lysate samples from different production strains (TUC14/16/18) were tested. Additionally, an uninduced (unind.) and an induced (ind.) wild-type control were tested as well. Also, His-tagged m*sfgfp* was used as control (ctrl.) for the Western blot. A) SDS-PAGE of the different strains using a 10% SDS gel. Color Prestained Protein Standard, Broad Range (10-250 kDa) was used as ladder. B) A non-stained 10% SDS gel was used the Western Blot on a polyvinylidene fluoride membrane. A anti-hexahistidine-peroxidase-antibody was used together with the colorimetric TMB Enhanced One Component HRP Membrane Substrate for visualisation. The arrows indicate the expected heights of the different constructs from the different transcription units (green arrow TUC14/SD4 – 102 kDa, pink arrow TUC16/SD5 – 165 kDa, blue arrow TUC18/SD6 – 249 kDa) and the His-tagged *sfgfp* control (yellow arrow, 26.8 kDa).*

 ![figure 17](https://static.igem.wiki/teams/5673/src/engineering/endpoint-luminescence-measurements-of-production-cultures.webp)

***Figure 17: Endpoint luminescence measurements of production cultures.**  Measurements were performed in TecanSpark plate reader with 1000 ms integration time. Data accompanied by error bars are based on two replicates, while those without error bars represent single measurements. For the TUC18 cell pellet no data was obtained due to technical reasons. Samples were blanked with a LB medium control. Values were normalised to an OD600 of 1. Strains were induced with 20 µg/ml bacitracin.*

Learn 6

Using the sensitive split NanoLuc assay, we finally detected small amounts of pyriform silk protein, unlike with SDS–PAGE or Western blots. This confirmed that *B. subtilis* W168 is capable of producing pyriform silk, but also highlighted the need for significant optimisation in host engineering and downstream processing. Unfortunately, the yields remain far too low for biocementation prototyping.

From further discussions with [Prof. Dr. Tracy Palmer](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#:~:text=Prof.%20Dr.%20Tracy%20Palmer) and [Dr. Florian Hof](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#:~:text=Dr.%20Florian%20Hof), our next steps include:

* Integrating the constructs into *B. subtilis* strains lacking extracellular proteases (e.g., MidiBacillus).
* Adding protease inhibitors post-harvest to reduce degradation.
* Engineering strains with enhanced availability of the tRNAs most required by our synthetic spidroin and increased levels of Sec pathway components.

Initial attempts to integrate the constructs into midi *Bacillus* failed, as cells underwent rapid lysis after cloning. We also see high potential in medium optimisation, but due to time constraints within the iGEM competition, further investigation and optimisation were not possible.

Iteration 7 - Biocementation prototyping with xanthan

Design/ Build/ Test 7

To evaluate the effect of biopolymers on microbially induced calcite precipitation (MICP)-based biocementation, we tested xanthan in a simple prototype setup that could later be adapted for spider silk once sufficient quantities are available. Based on the valuable feedback from [Prof. Dr. Kevin Paine](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#:~:text=Prof.%20Dr.%20Kevin%20Paine) and [Mr. Tim Gemünden](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#:~:text=Tim%20Gem%C3%BCnden), we specifically aimed for this setup of **precast biocementation blocks**, reflecting their advice to begin with small-scale, controlled applications before scaling toward larger structural use. We designed the screen to mimic a non-sterile static environment, comparable to a conventional concrete cast.

Xanthan was obtained by culturing *X. campestris* and purifying the secreted biopolymer. After lyophilisation, we directly used the product in our prototypes. To keep the setup simple and reproducible, we combined sand, pre-cultured bacteria, defined medium, and xanthan.

The biocementation process was then allowed to proceed for several days, during which the developing bricks were regularly supplied with fresh medium.

 ![figure 18](https://static.igem.wiki/teams/5673/src/engineering/bioconsolidated-sand-bricks-after-biocementation.webp)

***Figure 18: Bioconsolidated sand bricks after biocementation.**  Initial attempt of biocementation with S. silvestis. Images were taken 17 days after the initial inoculation. All xanthan concentrations shown are (w/v). A) Side view of the bioconsolidated sand bricks. B) Top view of the bioconsolidated sand bricks. C) Wells of the removed sand bricks.*

Learn 7

Our initial prototypes demonstrated that xanthan had a clear positive effect on both the mechanical properties and calcification of the bricks. This indicated that biopolymers can indeed benefit MICP-based biocementation. For future cycles we would like to change the biocementation towards a more complex but efficient process in flow cycle set up. Due to time constraints and limited resources, we were unable to pursue this in the wet lab before the end of the iGEM competition.

---

## References

Abramson, J., Adler, J., Dunger, J., Evans, R., Green, T., and Pritzel, A., *et al.* (2024) Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, doi: 10.1038/s41586-024-07487-w.

Greco, G., Wolff, J.O., and Pugno, N.M. (2020) Strong and Tough Silk for Resilient Attachment Discs: The Mechanical Properties of Piriform Silk in the Spider Cupiennius salei (Keyserling, 1877). Frontiers in Materials, doi: 10.3389/fmats.2020.00138.

Ling, L.F., Zi, R.X., Wei, F.L., Jiang, B.S., Ping, L., and Chun, X.H. (2007) Protein secretion pathways in Bacillus subtilis: implication for optimization of heterologous protein secretion. Biotechnology advances, doi: 10.1016/j.biotechadv.2006.08.002.

Pereira, G.C., Allen, W.J., Watkins, D.W., Buddrus, L., Noone, D., and Liu, X., *et al.* (2019) A High-Resolution Luminescent Assay for Rapid and Continuous Monitoring of Protein Translocation across Biological Membranes. Journal of molecular biology, doi: 10.1016/j.jmb.2019.03.007.

Ramezaniaghdam, M., Nahdi, N.D., and Reski, R. (2022) Recombinant Spider Silk: Promises and Bottlenecks. Frontiers in bioengineering and biotechnology, doi: 10.3389/fbioe.2022.835637.

Souza, C.C. de, Guimarães, J.M., Pereira, S.D.S., and Mariúba, L.A.M. (2021) The multifunctionality of expression systems in Bacillus subtilis: Emerging devices for the production of recombinant proteins. Experimental biology and medicine (Maywood, N.J.), doi: 10.1177/15353702211030189.

Wang, K., Wen, R., Jia, Q., Liu, X., Xiao, J., and Meng, Q. (2019) Analysis of the Full-Length Pyriform Spidroin Gene Sequence. Genes, doi: 10.3390/genes10060425
Yang, Y., Scott, A.A., Kneuper, H., Alcock, F., and Palmer, T. (2024) High-throughput functional analysis provides novel insight into type VII secretion in Staphylococcus aureus. Open biology, doi: 10.1098/rsob.240060.

Zhang, X., Al-Dossary, A., Hussain, M., Setlow, P., & Li, J. (2020): Applications of *Bacillus subtilis* Spores in Biotechnology and Advanced Materials, in: Applied and environmental microbiology, v. 86(17), doi: 10.1128/AEM.01096-20.

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{% endblock %}

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{% set header\_image = "https://static.igem.wiki/teams/5673/src/header/pipette-nah.webp" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Have a look at our Materials and Methods.{% endblock %}
{% block page\_content %}

# **Material and Methods**

Here we clearly state our experimental procedures and materials we used throughout our wet lab period!

---

## **Bacterial Cultivation**

---

### *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**

---

### 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.

---

### 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.

---

### 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.

---

### 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**

---

### 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.

---

### 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).

---

### 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 (P*xylA*, P*bceA*, P*liaI* and P*hpaII*), 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. --- 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. --- 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. --- Bacterial transformationTransformation 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**

---

### 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.

---

### 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.

---

### 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.

---

### 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**

---

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.

---

### 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).

---

### 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**

---

### 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.

---

### 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.

## **Documents**

---

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[open PDF in new tab](https://static.igem.wiki/teams/5673/pdf/plasmids-primers-and-strains.pdf).

## **References**

* Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, C. A., 3rd, & Smith, H. O. (2009): Enzymatic assembly of DNA molecules up to several hundred kilobases, in: Nature methods, V. 6(5), pp. 343–345, doi: 10.1038/nmeth.1318.
* Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., and Ronneberger, O., et al. (2021) Highly accurate protein structure prediction with AlphaFold. Nature, doi: 10.1038/s41586-021 03819-2.
* Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., & Erlich, H. (1986): Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction, in: Cold Spring Harbor symposia on quantitative biology, V. 51(1), pp. 263–273, doi: 10.1101/sqb.1986.051.01.032.
* Pettersen, E.F., Goddard, T.D., Huang, C.C., Meng, E.C., Couch, G.S., Croll, T.I., Morris, J.H., and Ferrin, T.E. (2021) UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein science : a publication of the Protein Society, doi: 10.1002/pro.3943.
* Sanger, F., Nicklen, S., & Coulson, A. R. (1977): DNA sequencing with chain-terminating inhibitors, in: Proceedings of the National Academy of Sciences of the United States of America, V. 74(12), pp. 5463–5467, doi: 10.1073/pnas.74.12.5463.
* Sekiguchi, J., Takada, N., & Okada, H. (1975): Genes affecting the productivity of alpha-amylase in Bacillus subtilis Marburg, in: Journal of bacteriology, v. 121(2), pp. 688–694, doi: 10.1128/jb.121.2.688-694.1975.
* Waterhouse, A.M., Procter, J.B., Martin, D.M.A., Clamp, M., and Barton, G.J. (2009) Jalview Version 2--a multiple sequence alignment editor and analysis workbench. Bioinformatics (Oxford, England), doi: 10.1093/bioinformatics/btp033.
* Weber, E., Engler, C., Gruetzner, R., Werner, S., Marillonnet, S. (2011): A Modular Cloning System for Standardized Assembly of Multigene Constructs, in: PLoS ONE, v. 6(2), doi: 10.1371/journal.pone.0016765.

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{% extends "layout.html" %}
{% set header\_image = "https://static.igem.wiki/teams/5673/src/header/lab-work.webp" %}
{% block title %}Results{% endblock %}
{% block lead %}The core of our project and wet lab!{% endblock %}
{% block page\_content %}

# Results

Welcome to the core of our research quest!

---

## **Key Results**

[![DNA icon](https://static.igem.wiki/teams/5673/src/results/highlights/pyricloning.webp)

### "*Pyricloning*"

* PCR-free framework reliably assembled **repeat oligomers and full CDS modules**
* Provides a **scalable, genome-integrable assembly standard** for repetitive genes](#Pyricloning)

[![Spider silk icon](https://static.igem.wiki/teams/5673/src/results/highlights/spider-silk-production.webp)

### Spider Silk Production

* Iterative engineering confirmed **spidroin expression** and limited secretion
* Not gel-detectable but **NanoLuc/HiBiT assay** shows functional, **low-level production** in *B. subtilis* W168](#SpiderSilk)

[![Warning icon](https://static.igem.wiki/teams/5673/src/results/highlights/bottlenecks.webp)

### Bottlenecks

* **Ribosome stalling** in repeat-rich regions and **depletion tRNA pool** identified
* Premature folding + rapid degradation remain key hurdles](#Bottlenecks)

[![Biocementation icon](https://static.igem.wiki/teams/5673/src/results/highlights/bio-cementation.webp)

### Biocementation

* **Proof of concept** with **xanthan** in biocementation
* Reliably showed **improved material properties**](#Biocementation)

[![Rocket icon](https://static.igem.wiki/teams/5673/src/results/highlights/next-steps.webp)

### Next Steps

* **Translational resource engineering** (co-expression of needed tRNAs, moderated initiation)
* **Protease-reduced** backgrounds + **chaperone assistance** (DnaK/DnaJ/GroEL, PrsA)
* Amino acid supplementation + expanded signal peptide screening](#NextSteps)

---

## 🧬"*Pyricloning*"

This results section reports our progress toward a practical route for recombinant pyriform spider silk and the creation of a biocement, from gene design to detection and application prototypes. To enable rapid assembly of long, repeat rich sequences, a PCR free workflow, "*Pyricloning*", was developed that combines RFC25 repeat oligomerisation with MoClo hierarchy for constructing single copy, genomically integrable expression units and multi repeat variants up to an octamer.

Design - Pyriform spider silk - choosing the right protein

## Design - Pyriform spider silk - choosing the right protein

Background Information - The Glory of Spider silk and the bacterial Workhorse *Bacillus subtilis*

### Spider silk types

Spider silk is considered to be one of biomaterials of the future, which is already used to solve modern problems in innovative and sustainable ways (Fang *et al.* 2025; Pulkkis 2020; Ramezaniaghdam *et al.* 2022; Spiess *et al.* 2010). Being stronger than most kind of steels or Kevlar, ultra-light weight, biodegradable and highly elastic at the same time makes dragline silk a highly promising candidate for biotechnological innovations (Gosline *et al.* 1999; Vollrath und Knight 2001). Furthermore, applications of spider silk span a broad range of industries and branches like biomedicine, aerospace and biosensors to name just a few (Ramezaniaghdam *et al.* 2022).
To date most of the research in this area has focused solely on dragline silk of spiders, which is known for its extreme tensile strength, even though spiders possess multiple other types of silk as well, which they use for specialized tasks as shown in Figure 1. These different silk types possess different properties, potentially useful in different applications.

![figure 1](https://static.igem.wiki/teams/5673/src/results/overview-silk-types.webp)

***Figure 1: Different types of spider silk and their respective usage.** Spiders use a variety of silks for specialised tasks, e.g. dragline silk with a high tensile strength as framework of the web, while aggregate silk acts as a glue to catch prey. Other types of silk and their respective function are shown in the figure. From (Ramezaniaghdam, Nahdi & Reski, 2022).*

Pyriform silk, which is also called the attachment cement of spiders, is used for anchoring the web to surfaces and to join different silk types together. This silk type consists of two spider silk proteins (which are also called spidroins), a dry fibre (pyriform silk protein 1 (PySp1) and a wet glue (pyriform silk protein 2 (PySp2); Geurts *et al.* 2010; Wolff *et al.* 2015). The dry fibre forms a micro framework, as shown in Figure 2, in which the glue hardens (Greco *et al.* 2020).

![figure 2](https://static.igem.wiki/teams/5673/src/results/sem-attachment-disc.webp)

***Figure 2: Scanning electron microscopy (SEM) image of the median part of the attachment disc.** PySp1 spidroins are spun into a matrix in which PySp2 can harden to form the attachment discs. The quality of the image was upscaled by AI. Adapted from Greco *et al.* 2020.*

Such an organized matrix could substantially improve construction materials, for example innovative cements based on MICP-driven biomineralization, by supplying additional nucleation sites, creating surfaces for bacterial colonization, and minimizing gaps between particles.
Until now, only two complete coding sequences (CDS) of pyriform 1 silk genes (*pysp1*) have been annotated (*Araneus ventricosus*, Accession number: MH376748 (Wang *et al.* 2019) and *Argiope argentata* Accession number: KY398016.1 (Chaw *et al.* 2017)). Other submissions in the NCBI GenBank database consist only of partial fragments or unvalidated whole-genome shotgun entries (NCBI GenBank, as of April 15, 2025). Both genes are divided into five distinct domains, as illustrated for the *pysp1* from *A. ventricosus* in Figure 3. Since the pyriform silk gene from *A. ventricosus* is significantly smaller (11,935 bp) than that of *A. argentata* (17,280 bp) it was chosen as template for further work in this project.

![figure 3](https://static.igem.wiki/teams/5673/src/results/schematic-pysp1.webp)

***Figure 3: Schematic representation of the *pysp1* gene from *Araneus ventricosus*.** The gene consists of an N-terminal signal peptide (SP) included in the non-repetitive N-terminal domain (NTD). A long (765 bp) N-terminal linker region (N-linker) connects the NTD to the central repeat region, which consists of 16 highly similar repeat units. A second linker region (C-linker) connects the repeat region to the non-repetitive C-terminal domain (CTD). Elements are not drawn to scale.*

### *Bacillus subtilis* as ideal expression host

*B. subtilis* was selected as ideal expression host as inclusion body formation is less frequently observed compared to *E. coli*; its efficient secretion systems (Zhang *et al.*, 2020); its genetic tractability (Radeck *et al.*, 2013); it is exotoxin and endotoxin-free and generally recognised as safe (GRAS; de Souza *et al.*, 2021); many tools and strategies are available for its genetic and metabolic engineering (Cruz Ramos *et al.*, 2000; Radeck *et al.*, 2013; Newman *et al.*, 2020; Brockmeier, Wendorff & Eggert, 2006); can be grown on inexpensive media (Zhang *et al.*, 2020). Typical approaches for stable heterologous protein production in *B. subtilis* often include the creation of single-copy transformants. For genomic integration, the amyE locus was selected because it is non-essentiality and ease for selection against successful integrants.

Design of the synthetic pyriform silk gene

To overcome the challenges of cloning a highly repetitive gene, the design and construction of a simplified synthetic pyriform silk gene was aimed at. First, the *pysp1* gene sequence from *A. ventricosus* was analysed and split into three domains to ease downstream workflows and cloning. All three domains were harmonized towards the codon usage of *B. subtilis* W168, possibly circumventing tRNA depletion and the loss of translation pauses, some of which may be important for correct protein folding. Consequently, this might have increased the chance of obtaining correctly folded protein and better yields. A modular design of the single domains, including the utilization of MoClo and RFC25 cloning standards and corresponding restriction enzymes, allowed the generation of constructs with increasing complexity.

### AlphaFold predictions hypothesis domain related structural independency

To date, no complete resolved structures of pyriform silk proteins have been published. To gain a better understanding of the protein′s structure, we used AlphaFold v3 (Abramson *et al.* 2024) to generate structure predictions of synthetic constructs with varying numbers of repeat units. The impact of increasing repeat numbers on the protein structure was subsequently evaluated in ChimeraX (Pettersen *et al.* 2021). In Figure 4, the predictions of synthetic pyriform silk proteins with one, four and eight repeat units are shown.

![figure 4](https://static.igem.wiki/teams/5673/src/results/alphafold-pyssp1.webp)

***Figure 4: AlphaFold structure predictions of different synthetic *pysp1* constructs.** Structures were predicted with AlphaFold3 server (standard settings) and visualised in ChimeraX. The same colour code as in Figure 3 was applied for the different domains with different blue shades for the single repeat units. N- and C- terminus are marked with N and C, respectively. D) Structures from B and C were rotated 90 ° toward the front.*

Predictions indicate that all domains, except the linkers (lime green and magenta), form α-helical bundles connected by unfolded regions. These unfolded regions contain poly-proline stretches, likely keeping them unstructured. The N-linker forms a single long α-helix around which repeat units fold, a feature that becomes more prominent with increasing repeat numbers (Figure 4C-D). The C-linker is mostly unfolded: while partial α-helices appeared in constructs with one and four repeats, it remained unfolded in the eight-repeat construct (Figure 4A-C). In all cases, a short α-helix within the C-linker consistently integrated into the α-helical bundle of the C-terminal domain. Importantly, varying repeat numbers did not alter the base structure but led to more globular assemblies (Figure 4A-C). Since hypothesized structures showed no major organizational differences, we decided to redesign the *pysp1* gene into 3 parts (Figure 5), which could be flexibly reassembled into simplified synthetic pyriform silk genes, from one to theoretically infinitely repeats instead of the wild type’s sixteen.

![figure 5](https://static.igem.wiki/teams/5673/src/results/schematic-synpysp1-elements.webp)

***Figure 5: Schematic representation of the synthetic pyriform silk gene elements.** The base gene structure was modified towards the three new segments. The two N- and C-terminal domains were summarised as NTE and CTE, respectively. The RPE contains all the repeat monomer units of a construct ranging from one to eight units. The initial synthesised RPE contained only one repeat monomer unit. Elements are not drawn to scale. Illustration was created with BioRender.*

These 3 parts were further refined for a smooth assembly into a complete synthetic pyriform silk gene as described in the following chapters.

Modular assembly via “*Pyricloning*” allows flexible, seamless and fast construction of genomically integrable pyriform silk production units

The construction and cloning of highly repetitive genes present significant challenges. Flexible assembly techniques like Gibson Assembly are impractical, since they require long single stranded DNA overhangs, which would pose a risk for undesired recombination or hybridisation (Torella *et al.* 2014). Standard cloning methods based on restriction digestion and ligation lack the scalability, flexibility and modularity required to rapidly assemble large synthetic operons or expression units with fine-tuned architectures (Marillonnet & Grützner 2020). Moreover, PCR-based strategies are often error-prone and inefficient when applied to highly repetitive sequences (Hommelsheim *et al.* 2014).

To overcome these limitations, we developed a novel PCR free cloning strategy termed *Pyricloning*. This approach combines elements of the type IIP-restriction-based assembly standard RFC25 (Grünberg *et al.* 2009) for repeat oligomer generation with the type IIS-dependent MoClo (Modular Cloning) assembly system (Weber *et al.* 2011), providing a flexible and robust framework for DNA assemblies.

The RFC25 BioBrick standard (also known as Freiburg Standard) is a refinement of the original BioBrick assembly method, developed by the 2007 iGEM team from Freiburg (Grünberg *et al.* 2009). It enables in-frame fusion of protein coding sequences, ideal for the buildup of repeat oligomers. It employs AgeI and NgoMIV sites flanking each part, in this case the repeat elements (Figure 6). Here the AgeI and NgoMIV sites are between the coding region and the SapI sites, which are needed for the MoClo assembly. These create compatible overhangs, preserving the reading frame during ligation and leaving a stable but non-cleavable AgeI/NgoMIV scar. Each scar encodes two amino acids (threonine, glycine), but the prefix and suffix remain intact, allowing iterative extensions without frame shifts or stop codons.

![figure 6](https://static.igem.wiki/teams/5673/src/results/schematic-summary-of-pyricloning.webp)

***Figure 6: Schematic summary of “*Pyricloning*”.*** The figure illustrates the stepwise construction of repeat oligomers and full-length coding sequences using the *Pyricloning* system. Initial repeat units were assembled into the opened level -1 pRPAV vector using elements from both the RFC25 (NgoMIV, AgeI) and MoClo (SapI) standards, enabling the formation of higher-order oligomers. These oligomers were then combined into a complete coding sequence including NTE and CTE domains. The resulting CDS could be used in level 1 MoClo assemblies to generate transcription units leveraging BsaI (not shown here). Color-coded blocks represent the DNA parts, restriction enzyme recognition sites and their respective overhangs, which enabled a specific assembly order. The type IIS-restriction enzymes SapI and BsaI cut outside their recognition sites, generating specific 3/4 bp overhangs. In contrast, the type IIP-restriction enzymes AgeI and NgoMIV cleave within their recognition sites and produce compatible overhangs that, once ligated, form a scar sequence no longer recognised by either enzyme. Elements are not drawn to scale.

In *Pyricloning*, RPE vectors are digested in two parallel reactions, one with SapI/NgoMIV and the other with SapI/AgeI. This generates two repeat units with defined overhangs. After enzyme inactivation, a SapI-digested entry vector and T4 ligase are added. The specific overhangs ensure directional ligation: the NgoMIV-flanked fragment integrates at the vector’s left site, while the AgeI-flanked fragment ligates to the right. The resulting AgeI/NgoMIV scar stabilises the fusion, yielding a repeat dimer.

This dimer can be recursively combined with monomers or other dimers to generate higher-order oligomers (trimers, tetramers, etc.). Ultimately, assembled repeat blocks are integrated into complete coding sequences (CDS) through the MoClo hierarchy (RFC1000), enabling seamless progression toward functional pyriform silk expression units.

To be able to express synthetic CDS (level 0), a full expression construct (level 1) consisting of a promoter, a ribosome binding site and a terminator needed to be formed around them, enabling ideally controllable transcription. For the assembly of the different constructs across the levels, the respective parts together with a suitable entry vector were combined in a one-tube MoClo reaction.

The entry vectors for all MoClo reactions, as well as all other MoClo parts, which were not generated by us, were kindly provided by [Georg Fritz](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D#:~:text=Dr.%20Georg%20Fritz) (University of Western Australia). Next we edited our sequences with respect to the codon usage bias to increase our chances of obtaining spider silk proteins later.

Codon harmonisation approximates the distance of codon usage and codon frequency

Heterologous protein production can be challenging, particularly for complex proteins such as spider silk. Their repetitive nature leads to a high abundance of specific amino acids. Especially serine (937 amino acids – 23.6%), alanine (709 amino acids – 17.8%) and glutamine (539 – 13.6%) alone make up over 50% of the wild-type protein’s amino acid composition. To minimize resulting risks like tRNA depletion, misfolding or degradation, codon harmonization was employed to all three parts of the synthetic pyriform silk gene, aligning it with the codon usage of *B. subtilis* W168.

Info Box - Why does codon harmonisation matter?

#### How codon usage bias affects recombinant protein production

Codon usage bias significantly affects heterologous protein production in microbial systems. Different organisms prefer certain synonymous codons to encode the same amino acid (Gustafsson *et al.* 2004). When genes with a codon usage profile that differs significantly from the host’s are expressed, translation can be hampered by insufficient levels of the corresponding tRNAs (Angov *et al.* 2011). As illustrated in Figure 7, the presence of rare codons (left panel) can slow down translational speed. In the case shown in the left panel, only a few correct tRNAs are available to bind the ribosome, while many incorrect tRNAs can compete for the same site. In contrast, a frequently used codon has a much larger tRNA pool, increasing the chances of correctly binding to the ribosome (right panel; Ikemura 1981).

![figure 7](https://static.igem.wiki/teams/5673/src/results/impact-codon-usage.webp)

***Figure 7: Impact of codon usage bias on translation efficiency.** Little used codons (left) have a low tRNA pool, resulting in a slow incorporation into the ribosome due to increased competition among the different tRNAs for ribosome binding. Frequently used codons (right) have a larger tRNA pool, which leads to a faster incorporation. Grey tRNAs represent any other tRNA present in the cell.*

Codon optimisation seeks to adapt rare codons to the host’s preferred synonyms, improving compatibility with its translational machinery (Plotkin und Kudla 2011). It is important to note that increased translation speed due to codon optimisation is not universally beneficial. In some cases, slower translation at specific regions – caused by rare codons – can assist in proper co-translational folding of complex proteins (Angov *et al.* 2011). This is exemplified in Figure 8. As it takes more time for the tRNA to come to the ribosome in a rare codon situation, the translation speed is slowed down, allowing the already formed peptide chain to fold into a defined structure before other domains are translated (Liu 2020). To account for this effect in a heterologous system, the gene’s codon frequencies can be adapted to the codon usage of the host organism. By rep*lacI*ng rare codons with synonymous codons that are also rarely used in the host, one can approximate the native translation speed and maintain co-translational folding (Angov *et al.* 2011).

![figure 8](https://static.igem.wiki/teams/5673/src/results/codon-usage-translation-dynamics-and-protein-folding.webp)

***Figure 8: Impact of codon usage on translational dynamics and protein folding.** The speed of translation is directly affected by the distribution of differently used codons. Frequently used codons, with a high tRNA pool result in a faster translation speed, whereas rare codons with small tRNA pools lead to slower translation speeds or even pauses. In some cases, the translation speed can is important for proper protein folding, giving domains time to form before other parts of the protein are translated.*

Summarized, codon optimization can alleviate translational inefficiencies by adapting the gene sequence to reflect the tRNA availability of the host, but it does not fully address downstream problems such as misfolding, aggregation, or degradation (Gustafsson *et al.* 2004). For complex proteins, like spidroins with modular repeat structures, intracellular folding may fail or tRNA pools of highly used codons deplete, resulting in inclusion body formation and a loss of functional protein (Ramezaniaghdam *et al.* 2022).

#### Addressing Codon usage bias with Codon Harmonisation

As already mentioned rare codons can induce ribosomal pauses which facilitate co-translational folding of complex domains (Liu 2020). However, heterologous gene expression often introduces mismatches between donor and host codon usage, as seen in *Bacillus subtilis*, leading to low yields or misfolded proteins (Gustafsson *et al.* 2004).
Codon harmonization represents a more nuanced strategy then codon optimization. Instead of uniformly rep*lacI*ng rare codons with frequent ones in the host, harmonization aims to preserve the donor’s codon distribution relative to host usage (Angov *et al.* 2011). Rare codons in the donor are substituted with similarly rare codons in the host, while frequent donor codons are aligned with frequent host codons (Parvathy *et al.* 2022). This approach retains local pauses critical for folding while preventing depletion of tRNA pools (Hanson and Coller 2018; Quax *et al.* 2015).
Despite these advantages, harmonisation is technically demanding. Accurate codon usage profiles from both donor and host are required, which can be difficult without well-annotated genomes. Available bioinformatics tools for harmonisation often require specific input formats and may not function reliably (Willems *et al.* 2023). Furthermore, unintended biases can be introduced if codon redistribution disrupts subtle regulatory elements within mRNA.
In conclusion, codon usage directly impacts translational efficiency, folding dynamics, and host cell fitness (Gustafsson *et al.* 2004; Quax *et al.* 2015). While traditional optimisation enhances yields, it ignores the roles of rare codons in folding and translation regulation (Angov *et al.* 2011). Codon harmonisation, though more complex to implement, offers a refined strategy that preserves native translational behaviour while adapting constructs to the host. This is especially promising for aggregation-prone or structurally complex proteins, such as spider silk, where folding pausing mechanisms are essential (Parvathy *et al.* 2022). With ongoing improvements in bioinformatics, harmonisation may soon become a practical tool for synthetic biology and industrial biotechnology applications (Willems *et al.* 2023).

To assess differences in the codon usages (CU) between the donor organism (*A. ventricosus*) and the host organism (*B. subtilis* W168), codon usage tables were generated for both species. For *B. subtilis* W168, the annotated reference genome (ASM904v1) was utilised for the extraction of the codon usage. Although a reference genome exists for *A. ventricosus*, it is poorly annotated, with most of the entries consisting of hypothetical or predicted genes. This made it unsuitable for generating a reliable codon usage table. Furthermore, as a eukaryote, *A. ventricosus* contains introns within its genes, which would distort codon usage analysis on genomic sequences.

A screen of the NCBI GenBank database yielded 28 available cDNA entries for *A. ventricosus*. After sorting out redundant and synthetic entries, 14 cDNA sequences were selected for further analysis. To obtain the coding regions, open reading frames were extracted from these sequences and used to generate the *A. ventricosus* codon usage table.

These codon usage tables were then compared to identify codons with similar or differing usage patterns between the two organisms. This section illustrates the harmonisation process using proline codons as an example, although the same process was applied to all amino acids. Proline codon usage is shown in bars 1 and 2 of Figure 9 for *A. ventricosus* and *B. subtilis* W168, respectively. While the usage of the CCU codon is nearly identical, making the difference negligible, the codons CCC, and especially CCA and CCG, exhibit more substantial differences. These discrepancies are critical to address for ensuring proper protein production and folding.

To gain a better understanding of the relationship between host codon usage and codon frequency (CF) – defined as the relative occurrence of a specific codon for a given amino acid in the wild-type gene – the codon frequency of the wild-type sequence was extracted and compared to codon usage data. Bar 3 of Figure 9 illustrates the proline codon frequency in the wild-type gene, which closely mirrors the codon usage pattern observed in *A. ventricosus*, albeit with a slightly higher frequency of CCG, CCA, and CCC, and a lower occurrence of CCT. In our project, differences between codon usage and codon frequency are referred to as "distances." These distances for the proline codons - between the wild-type gene and *A. ventricosus* (bar 5), and between the wild-type gene and *B. subtilis* W168 (bar 7) - are clearly distinct. As with codon usage, a substantial discrepancy in distances is evident.

To design a well-harmonised synthetic pyriform gene for expression in *B. subtilis* W168, the goal was to achieve distance values comparable to those shown in bar 5. Thus, the distance between the codon frequency of the harmonised synthetic pyriform gene and the codon usage of *B. subtilis* W168 was approximated towards the wild-type situation. This might have led to a more proportional distribution of the burden on tRNA pools, while maintaining distances that may be involved in regulating translation speed through translation pauses.

![figure 9](https://static.igem.wiki/teams/5673/src/results/harmonization-pro-codon.webp)

***Figure 9: Harmonisation of the proline codons in the synthetic pyriform silk gene** The bars are colour-coded to represent the different codons CCG (pink), CCA (green), CCC (orange) and CCT (blue). “OPT” denotes a hypothetical codon-optimised pyriform silk gene in which all proline codons were changed to CCG. Since the distances are portrayed in absolute values, their sum exceeds 1 in bar 8. RU = relative units.*

In the case of proline codons, the simplest way to achieve a harmonised sequence with close to native distances, was to switch the CCC codons to CCA, the CCA codons to CCG and the CCG codons to CCC. Additionally, one CCG codon (codon 576) was changed to CCU to better match codon usage-to-frequency distance. These modifications resulted in new proline codon frequencies, shown in bar 4. As a result, the corresponding distances closely resembled those of the wild-type gene, as depicted in bar 6.

In contrast, bar 8 shows the distances of a hypothetical codon optimised synthetic pyriform gene, in which all proline codons were switched to CCG – the most used proline codon of *B. subtilis* W168. This resulted in even greater distances compared to the wild-type gene in *B. subtilis* W168 (bar 7), with a total value greater than one (distances were calculated as absolute values).

For amino acids where switching codons was not viable due to the different codon usage of *A. ventricosus* and *B. subtilis* W168, and single codons needed to be addressed manually, we wrote a programme to facilitate this step. In brief, the used code calculated the distance of each codon along the sequence and then highlighted it, if a certain distance threshold was surpassed. Thereby, codon hotspots and single codons with high distances could be directly identified. If an effect on translation of these hotspots could be ruled out, for example, if the codons were used far less than the average codon usage of *A. ventricosus* (which would also yield a high distance), they were distributed across synonymous codons to approximate the native distances, thus lowering the burden on single tRNA pools. Conversely, when codons were used far more frequently than the average, they were manually substituted one by one to best match the wild-type distances and preserve their relative distribution across the sequence.

However, as mentioned earlier, this process was only necessary if the codon usage pattern for an amino acid differed a lot between *A. ventricosus* and *B. subtilis*. In cases like CCU no adaptations were made, as the distances were already similar. Finally, the distances of the synthetic and the wild-type *pysp1* were approximated across all codons, similar to Figure 9 bar 5 and 6, circumventing problems like tRNA depletion and protein misfolding.

Overall, the harmonisation of the synthetic pyriform silk gene minimised discrepancies in codon usage and codon frequency between the host organism and the synthetic gene construct, while preserving a close-to-native distribution of tRNA demand. This approach should increase the chances of obtaining sufficient expression levels and proper protein folding during heterologous production in *B. subtilis*.

Generation of synthetic spider silk genes

## Generation of synthetic spider silk genes

Now we could start building up our constructs with our very own designed cloning standard, which combined the MoClo RFC1000 standard and the BioBrick RFC25 standard.

Repeat Oligomer generation

First, multiple repeat oligomers were generated. For this purpose, a dedicated plasmid called pRPAV (repeat assembly vector) was designed, featuring an mRFP cassette as visual marker for empty vectors, a kanamycin resistance cassette for selection, and recognition sites for the MoClo enzyme SapI with predefined overhangs for repeat unit integration as shown in Figure 10A. As shown in Figure 10B the repeat oligomers were gradually built up from the monomer utilizing the same reaction protocol as described previously. Figure 10C illustrates a control digest (BbsI/SapI) validating the assembled repeat oligomers.

![figure 10](https://static.igem.wiki/teams/5673/src/results/pyricloning-enables-the-seamless-construction-of-oligomers-from-repetitive-sequences.webp)

***Figure 10: *Pyricloning* enables the seamless construction of oligomers from repetitive sequences.** Arrows in A) and B) indicate orientation of the SapI. A) Schematic representation of the repeat assembly vector (pRPAV). The mRFP cassette contains 4 BbsI cutting sites, resulting in five small fragments if cut by the BbsI and SapI. B) Schematic representation of the repeat oligomer buildup. By digesting the repeat monomer in two different reactions with the indicated restrictions enzymes a repeat dimer can be ligated into the pre-opened (SapI) pRPAV vector. This process can be flexibly adapted to create the desired repeat oligomer as indicated. C) All plasmids were digested with BbsI and SapI for 1 hour at 37 °C. The empty repeat assembly vector served as control. Arrows indicate the expected positions of the fragments. The 1% (w/v) agarose gel was run in 1× TAE buffer (40 mM Tris pH 8.5, 20 mM acetic acid, 1 mM EDTA) at constant 130 V for 30 min and the 1 kb plus DNA ladder (L) from NEB was used for size comparison. GelRed® Nucleic Acid Gel Stain was used to stain the DNA. Images were taken in the E-BOX CX5 TS Edge UV gel documentation system. For visualisation the UV-image was colour inverted. Elements are not drawn to scale.*

The cut backbone was observed at ~2203 bp across all lanes, including the control (empty pRPAV vector). Distinct bands corresponding to a repeat monomer (655 bp), dimer (1,300 bp), trimer (1,945 bp), tetramer (2,600 bp), and octamer (5,200 bp) appeared at the expected heights as indicated by the green arrows. Thereby, the successful generation of repeat oligomers up to an octamer was validated.
Unexpected additional bands in the dimer, trimer, and octamer lanes suggest possible recombination events due to the use of a recA+ strain (NEB Turbo) for cloning. This is supported by the fact that these bands align with the expected sizes of repeat oligomers with lower repeat numbers. In the trimer lane, a faint band corresponds to the expected height of the repeat dimer. Similarly, in the tetramer lane, bands corresponding to the expected heights of the repeat trimer and dimer can be seen. In the octamer lane, multiple faint bands between the correct repeat band (5,200 bp) and the cut backbone might indicate the presence of tetra-, penta-, hexa-, and heptameric forms. Despite these artifacts, the majority of plasmids contained correctly assembled repeat oligomers, enabling the construction of synthetic pyriform silk genes with increasing repeat numbers.

CDS Generation

Next, we used these repeats to build different CDS of synthetic spider silk genes. Initially, a basic synthetic spidroin construct (level 0) consisting of the standard NTE, a monomeric RPE and the standard CTE, was assembled to prove the concept and evaluate the protocol. In Figure 11A the level 0 CDS assembly is shown as a schematic. First, SapI cuts out all the parts from their vectors and the *lacZ*α fragment from the entry vector. This allows the parts to be ligated in the entry vector in the specific order of NTE, RPE and CTE. While the SapI recognition sites of the entry vector are lost during the assembly, the BsaI recognition sites are maintained for further assemblies into full expression constructs (level 0). Following the same method, we successfully assembled a level 0 CDS construct with a tetrameric RPE, as depicted in Figure 11. Later, a level 0 CDS with an octameric RPE was assembled as well. Figure 11B shows the control digest (BsaI) of the synthetic pyriform silk gene construct with a tetrameric RPE, exemplifying the validation process for all assembled synthetic CDS (level 0).

![figure 11](https://static.igem.wiki/teams/5673/src/results/abb-11-new.webp)

***Figure 11: Successful generation of a synthetic pyriform silk gene with a repeat tetramer using the modular cloning system.** A) Schematic representation of the assembly reaction. Arrows indicate the orientation of the type IIS restriction enzymes. All of the three level -1 vectors and the level 0 entry vector were digested with SapI and ligated via T4 DNA ligase, resulting in the assembly of the level 0 CDS SD2. The parts assemble specifically according to the indicated colour code of the 3/4 bp overhangs (SapI/BsaI, respectively). While SapI restriction sites were lost during the assembly, the level 0 CDS maintained the BsaI restriction sites for further assembly into level 1 transcription units. Elements are not drawn to scale. B) All plasmids were digested with BsaI for 1 hour at 37 °C. The empty entry vector served as control. Arrows indicate the expected positions of the fragments. The 1% (w/v) agarose gel was run in 1× TAE buffer (40 mM Tris pH 8.5, 20 mM acetic acid, 1 mM EDTA) at constant 130 V for 30 min and the 1 kb plus DNA ladder (L) from NEB was used for size comparison. GelRed® Nucleic Acid Gel Stain was used to stain the DNA. Images were taken in the E-BOX CX5 TS Edge UV gel documentation system. For visualisation the UV-image was colour inverted.*

In both lanes the cut backbone (2243 bp, black arrow) appeared at the same height around 2,800 bp running higher than expected. Since the backbone band of the control lane ran higher than expected too, this observation was attributed to the gel and a bending of the ladder bands in the upper region of the gel. The band of the *lacZ*α fragment (602 bp, purple arrow) of the control digest appeared at the correct height around 650 bp, confirming that the digest worked as expected. Furthermore, a band in the lane of the CDS construct SD2 (spidroin 2) appeared around 4,400 bp corresponding to the correct CDS fragment (4406 bp, green arrow). Thereby, the correct and successful assembly of the synthetic CDS (level 0) with a NTE, a tetrameric RPE and a CTE was validated.
In table 1, all successfully created synthetic CDS (level 0) are listed. These level 0 CDS assemblies were then brought into a full level 1 expression construct.

Tab. 1: List of selected level 0 CDS constructs of synthetic pyriform silk genes generated in this study

| Description of CDS 1 | Composition 2, 3, 4, 5, 6 |
| --- | --- |
| SD1 | NTE-RPE monomer-CTE |
| SD2 | NTE-RPE tetramer-CTE |
| SD3 | NTE-RPE octamer-CTE |
| SD4 | yoaWSP-mNTE-RPE monomer-mCTE-pep86 |
| SD5 | yoaWSP-mNTE-RPE tetramer-mCTE-pep86 |
| SD6 | yoaWSP-mNTE-RPE octamer-mCTE-pep86 |

1SD = Spidroin construct. 2NTE = N-terminal element. 3RPE = Repeat element. 4CTE = C terminal element. 5SP\_*yoaW* = secretion signal peptide sequence from *yoaW*. 6pep86 = C terminal fusion-tag required for the NanoLuc assay.

In summary, we successfully established a modular and robust strategy for assembling synthetic spider silk genes by combining repeat oligomer generation with CDS assembly. The pRPAV system enabled the seamless, PCR-free buildup of repetitive elements in an iterative manner, allowing the reliable generation of oligomers up to the octamer level. Despite minor recombination artifacts, the majority of clones contained correctly assembled RPEs, which could be directly integrated into CDS constructs. By coupling these RPEs with the NTE and CTE, we demonstrated the modularity and flexibility of our cloning framework. Overall, this system offers a powerful advantage for the assembly of highly repetitive sequences, overcoming the limitations of conventional restriction ligation and PCR-based cloning methods and providing a scalable route via the RFC1000 framework to synthetic spider silk gene construction.
Having established the successful construction of the spidroin CDS modules, the next step was to evaluate regulatory element variants. Therefore, before building complete transcription units with our spidroin CDS, we systematically tested different combinations of promoters, ribosome binding sites, and terminators to determine the optimal expression strength for efficient spidroin production.

Evaluation of optimal Expression Cassettes

## Evaluation of optimal Expression Cassettes

In recombinant spidroin production, the precise control of transcription and translation is crucial for success, and overcoming common challenges such as low yields and the accumulation of insoluble protein, both frequently linked to limitations in transcription and translation (Connor, Zhu & Koffas, 2024). As promoters and RBSs are significant elements to tune the gene expression rate, we aimed to evaluate the transcriptional and translational activity of different promoter and RBS combinations and identify the optimal combination for efficient recombinant spidroin production in *B. subtilis*. To achieve this, we employed a fluorescence-based reporter gene assay to systematically and quantitatively compare promoter strengths and translational efficiencies across various RBSs. The Modular Cloning system was adapted for rapid and convenient construction of various expression constructs harbouring different combinations of promoters and RBSs fused to a reporter gene.

Creation of a *sfgfp* level 0 construct

Info Box - Why we selected *sfgfp* as reporter gene?

sfGFP is a well-suited reporter as the protein matures rapidly with a folding time of 6 min in *E. coli* and is highly stable, thereby producing consistent fluorescence signals (Pédelacq *et al.*, 2006; Shields *et al.*, 2019). For that reason, *sfgfp* was selected as reporter gene (Shaner *et al.*, 2013).

To be able to utilise *sfgfp* for the assembly with various promoters and RBSs using the MoClo system, the CDS must first be made compatible. As the MoClo system used is based on utilising restriction type IIS enzymes BsaI and SapI, the *sfgfp* sequence was first checked for unwanted recognitions sites of the latter enzymes. Fortunately, the CDS of *sfgfp* did not contain any BsaI or SapI recognition sites. Thereafter, the CDS of *sfgfp* was amplified using PCR with the primers SG1747 and SG1748. Each of the primers carried overhangs that comprised a SapI recognition sequence alongside with a four basepair sequence appropriate for directional assembly of the CDS in MoClo. Meaning, SG1747 comprised aATG, whereas SG1748 harboured GCTT. The 761 bp PCR product, the *sfgfp* CDS, was then cloned into a level 0 destination vector pICH41308 via a MoClo level 0 reaction. Colony PCR followed by Sanger sequencing validated the correct insertion of *sfgfp* into the vector (Figure 12).

![figure 12](https://static.igem.wiki/teams/5673/src/results/creation-sfgfp.webp)

***Figure 12: Creation of a *sfgfp* MoClo level 0 part.** L = Ladder. A) Schematic workflow of the *sfgfp* level 0 part generation. First the PCR amplification of *sfgfp*. Then the cloning of the *sfgfp* sequence into the level 0 destination vector pICH41308 via a level 0 MoClo reaction (see section 2.6.1). B) PCR product of the *sfgfp* CDS using primers harbouring overhangs that carry a SapI recognition sequence and designed 4 bp sequence for directional assembly in MoClo. C) cPCR verification of correct insertion of the *sfgfp* sequence (856 bp) in the destination vector pICH41308 using check primers SG1750 and SG1751. C = Control, empty level 0 vector pICH41308 harbouring a *lacZ* cassette (721 bp).*

Construction of Promoter-RBS-*sfgfp* fusion constructs

Before the screening of promoter strengths and RBS activities, four promoters of varying strength and regulation, together with four synthetic RBSs, were selected for systematic analysis. The promoters included three inducible promoters P*xylA*, P*bceA*, and P*liaI*, and the constitutive P*hpaII* (Bhavsar, Zhao & Brown, 2001; Ohki *et al.*, 2003; Toymentseva *et al.*, 2012; Zhang *et al.*, 2017). As synthetic RBSs, st4, st7, st11, and wk8 were chosen, while the terminator L3S1P47 was integrated based on a prior screening as the strongest available terminator (Newman *et al.*, 2020). All selected parts originated from the *B. subtilis* MoClo library of Newman *et al.*, except P*hpaII*, which was made available as MoClo part in our work (AG Fritz; Newman *et al.*, 2020).

In an additional modification we replaced the *lacZ* cassette of MoClo destination vectors with an *mrfp1*-based red/white selection cassette, eliminating the requirement for IPTG and X-Gal in the screening for successful transformants. Using Gibson assembly, new destination vectors were generated: pMMS0 (Level 0), pMMS1 (Level 1), and pMMSM1 (Level M1).

Reporter constructs followed the modular architecture described by Weber *et al.* (Figure 13A; Weber *et al.*, 2011). For promoter evaluations, transcriptional fusions of P*xylA*, P*bceA*, P*liaI*, or P*hpaII* with RBS st11, *sfgfp*, and L3S1P47 were assembled (Figure 13B). Conversely, RBS evaluation constructs employed P*liaI* with either st4, st7, st11, or wk8 combined with *sfgfp* and L3S1P47 (Figure 13C). MoClo level 1 reactions generated these transcriptional units in pMMS1, which were validated by colony PCR (cPCR).

To obtain single-copy genomic integrations, validated transcriptional units were subjected to level M cloning for insertion into the non-essential *amyE* locus (Dierksheide & Li, 2024). Level M constructs correspond to level 2 constructs in the RFC1000 standard and can be seen as equal. The assembly of integration constructs is described more in detail in sections below. Homology-flanked constructs were verified by cPCR (figure 13 E) and integrated into *B. subtilis* W168 (Figure 13D). Successful integrants were confirmed using a Lugol starch hydrolysis assay (Figure 13F), in which correctly disrupted *amyE* strains showed no halo formation, unlike the *B. subtilis* wild type.

The cloning strategy for various reporter constructs is depicted in the figure below (figure 13).

![figure 14](https://static.igem.wiki/teams/5673/src/results/overview-generation-reporter-constructs.webp)

***Figure 13: Overview of the generation of reporter constructs.** P = promoter, RBS = ribosome binding site, Ter = terminator. A) Schematic of the reporter construct design including the specific 4 bp overhangs for the directional assembly of the required basic parts. B) Composition of level 0 basic parts for reporter constructs in the evaluation of the strength of different promoters. RBS st11, reporter gene *sfgfp* and terminator L3S1P47 were combined with either P*xylA*, P*bceA*, P*liaI* or P*hpaII*, respectively. C) Set of level 0 parts for the assembly of reporter constructs in evaluating translational efficiency of different RBSs. The promoter P*liaI*, reporter gene *sfgfp* and terminator L3S1P47 were assembled with either RBS st4, st7, st11 or wk8. D) Schematic of an assembled level M vector harbouring a reporter construct flanked with *amyE* homology flanks and catr. The vector directs the integration of the reporter construct into the *amyE* locus of *B. subtilis* genome. E) Representative cPCR verification of the correct level M assembly of *amyE*-LF- catr-(P*hpaII*, st11, *sfgfp*, L1S3P47)-*amyE*-RF using check primers SG1955 and SG1956 (~3.5 kb). C = control, empty level M vector pMMSM1 (1242 bp). F) Representative verification of *B. subtilis* integration of level M reporter construct via Lugol starch hydrolysis assay. 1-6: Colonies of *B. subtilis* transformants with different level M reporter constructs; C = wild-type *B. subtilis* W168 with intact *amyE*.*

Ranking of different promoter strengths

To compare promoter strengths, *B. subtilis* strains carrying transcriptional fusions of selected promoters P*xylA*, P*bceA*, P*liaI* and P*hpaII* with st11-*sfgfp*-L3S1P47 were analysed following induction with the appropriate stimuli. Expression was monitored by end point fluorescence measurements in a plate reader (Figure 14A) and by fluorescence microscopy, both qualitatively and quantitatively via single cell mean fluorescence determination (Figure 14B–C).

Plate reader data showed a strong variability at t0 due to low OD600 values, reflected in large error bars. By 30 and 60 min post induction, P*liaI* and P*hpaII* consistently exhibited the highest fluorescence values, followed by P*bceA*, while P*xylA* remained at wild type levels. Fluorescence microscopy confirmed these findings. Notably, all inducible promoters were inactive prior to induction but produced clear *sfgfp* signals across all cells after induction, whereas the constitutive P*hpaII* promoter drove steady fluorescence throughout. Importantly, microscopy revealed an uniform promoter activity, with no detectable cell to cell heterogeneity.

The quantitative mean single cell fluorescence analysis at 60 min substantiated the bulk trends but additionally showed P*liaI* to be significantly stronger than P*hpaII*, a difference not resolved in plate reader data. P*bceA* showed intermediate fluorescence, while P*xylA*, although weakest, still displayed fluorescence above the wild type control. Statistical analysis confirmed the promoter ranking as P*liaI* > P*hpaII* > P*bceA* > P*xylA*, each significantly different from the next, with all tested promoters significantly stronger than the negative control.

![figure 14](https://static.igem.wiki/teams/5673/src/results/results-promoter.webp)

***Figure 14: The *liaI* promoter is the strongest among the promoters tested.** The promoter strength of the inducible promoters P*xylA*, P*bceA* and P*liaI* as well as the constitutive promotor P*hpaII* was evaluated using a reporter gene assay. Error bars represent standard deviation from the mean. Control = *B. subtilis* W168 wild-type A) Plot of corrected fluorescence intensities measured at 520 nm against the time. End-point fluorescence intensities of *B. subtilis* measured in a plate reader. n = 3. AFU = arbitrary fluorescence units. B) Scatter blot of mean single-cell fluorescence intensities of each promoter construct 1 h post-induction. n ≥ 50 cells. Significance: \*\*\*\* = p<0.0001, unpaired t-test with Welch’s correction. a.u. = arbitrary units. C) Representative composite fluorescence microscopy images of *B. subtilis* harbouring different promoter constructs at different time points before and after induction. Black scale bar = 10 µm.*

Evaluation of translational efficiencies of different synthetic RBSs

To assess translational efficiencies, *B. subtilis* strains carrying translational fusions of four synthetic RBSs (st4, st7, st11, wk8) with the promoter P*liaI*, reporter gene *sfgfp* and terminator L3S1P47 (Figure 15A) were analysed following bacitracin induction. Expression was monitored via end point plate reader fluorescence (Figure 15B) and fluorescence microscopy (Figure 15C–F).

As observed in the promoter evaluations, plate reader measurements showed high variability at t0 due to low OD600 values, with large error bars persisting at 30 min post induction. At both 30 and 60 min, fluorescence signals were only detected for st7 and st11 constructs, with st7 showing slightly higher bulk values. In contrast, fluorescence microscopy revealed that all RBS variants yielded detectable *sfgfp* fluorescence at 30 min, significantly above the negative control. Strongest induction was again seen for st7 and st11. Temporal differences were apparent: st11 reached near maximum fluorescence within 30 min, while st7 displayed delayed kinetics, reaching comparable levels only after 60 min. Accordingly, translational efficiencies at 60 min were ranked as st11, st7 > st4 > wk8.

![figure 15](https://static.igem.wiki/teams/5673/src/results/results-rbss.webp)

***Figure 15: Synthetic RBSs st7 and st11 exhibit high translational efficiency.** Qualitative and quantitative comparison of translational efficiencies of different synthetic RBSs (st4, st7, st11 & wk8). Error bars represent standard deviation from the mean. Significance: \*\*\*\* p<0.0001, unpaired t-test with Welch’s correction. Control = *B. subtilis* W168; a.u. = arbitrary units. A) Overview of the reporter constructs with different RBSs but the same promoter (P*liaI*), reporter gene (*sfgfp*) and terminator (L3S1P47). B) Plot of corrected end-point fluorescence intensities measured at 520 nm against the time. End-point fluorescence intensities of *B. subtilis* were measured in a plate reader. n = 3. C-E) Scatter blot of mean single-cell fluorescence intensities of different RBS constructs. Cells from three different fluorescence images were used for the measurement. n ≥ 50 cells. Quantification took place at 30 min (C) and 60 min (D) post-induction. E) Representative scatter plot of st7 construct at all time points of mean single cell fluorescence. F) Representative composite fluorescence microscopy images of *B. subtilis* harbouring different RBS constructs at different time points before and after induction. Black scale bar = 10 µm.*

Discussion - Assessment of optimal Expression construct

The precise control of gene expression is essential for efficient heterologous protein production (Hershey, Sonenberg & Mathews, 2012), including recombinant spidroins, where fine‐tuned expression improves yields while reducing metabolic burden (Connor, Zha & Koffas, 2024). In metabolic engineering, promoter–RBS libraries have been successfully deployed to balance transcription and translation (Mutalik *et al.*, 2013). To establish a framework for controlled gene expression in *B. subtilis*, we evaluated promoter strengths and ribosome binding site (RBS) activities using *sfgfp* as a MoClo-compatible reporter gene (Figure 12 and Figure 13).

### Bacitracin-inducible promoter P*liaI* as promising candidate for fine-tuned protein production in *B. subtilis*

Four promoters were tested, P*xylA*, P*bceA*, P*liaI*, and P*hpaII*. A clear ranking emerged, with P*liaI* > P*hpaII* > P*bceA* > P*xylA*, reflecting distinct transcription initiation strengths. At the pre-induction time point (t0), fluorescence microscopy confirmed the absence of *sfgfp* expression in inducible constructs, consistent with tight regulation during exponential growth. Following induction, all inducible promoters rapidly reached maximum activity within 30 minutes and maintained stable expression until 60 minutes, showing robust responsiveness to inducers. Notably, fluorescence microscopy revealed no cell-to-cell heterogeneity in expression, supported by uniform intensity distributions and a Shapiro-Wilk test for normality, indicating tight and reliable regulation. This aligns with previous analyses of inducible promoters including P*xylA*, P*bceA*, and P*liaI* (Radeck *et al.*, 2013; Fritz *et al.*, 2015; Ohki *et al.*, 2003; Toymentseva *et al.*, 2012). For instance, P*liaI* has been reported to display a strong activation within 10 min of induction (Toymentseva *et al.*, 2012).

By contrast, P*hpaII* produced fluorescence at all time points, consistent with its constitutive activity and previous reports (Guan *et al.*, 2015). A technical variation was observed in bulk fluorescence data at t0, likely resulting from normalisation artifacts at low OD600 (≈0.5). Here, even small measurement inaccuracies disproportionately affected calculated fluorescence values. In contrast, microscopy showed no expression, indicating that microscopy provides more reliable baseline measurements.

Both microscopy and plate reader analyses identified P*liaI* and P*hpaII* as the strongest promoters, though only single-cell quantification revealed a statistically significant strength difference in favour of P*liaI*. No difference between P*xylA* and the negative control was detected in plate reader assays. However, fluorescence microscopy confirmed a weak but significant P*xylA* activity. These results highlight the reduced sensitivity of bulk measurements compared to single-cell fluorescence quantification, which better captures weak expression events (Miyashiro & Goulian, 2007). Collectively, P*liaI* emerged as the most promising promoter for recombinant spidroin expression in *B. subtilis*, combining tight regulation with strong induction potential.

### st7 and st11 are promising RBSs for fine-tuned gene expression in *B. subtilis*

Translational efficiencies were assessed for four synthetic RBSs (st4, st7, st11, wk8). Bulk plate reader measurements again showed high variability at t0 due to low cell densities, but after induction, only st7 and st11 displayed fluorescence significantly above the control. Among them, st7 yielded slightly higher values than st11 in the bulk assay, suggesting a stronger translation.

Single-cell fluorescence analyses, however, provided a more detailed perspective. All four RBS constructs yielded measurable *sfgfp* expression at 30 min, with st7 and st11 markedly outperforming st4 and wk8. Importantly, st11 reached near-maximal expression within 30 min, while st7 exhibited delayed kinetics, reaching levels comparable to st11 only by 60 min. Statistical analyses confirmed significant differences between st7 and st11, supporting the robustness of these observations. Based on the fluorescence microscopy’s higher sensitivity compared to population-averaged assays, these data more reliably represent true translational differences (Miyashiro & Goulian, 2007).

The slower activation of st7 may derive from differences in Shine–Dalgarno complementarity, which strongly influences the ribosome recruitment efficiency, or from inhibitory mRNA secondary structures near the RBS that reduce the accessibility for translation initiation (Rodnina, 2016; Hershey, Sonenberg & Mathews, 2012; Englund, Liang & Lindberg, 2016). Even subtle differences in secondary structure stability can substantially lower translation rates (Rodnina, 2016).

![figure 16](https://static.igem.wiki/teams/5673/src/results/complementarity-shine-delgarno-sequence.webp)

***Figure 16: Complementarity of Shine-Dalgarno sequence to anti Shine-Dalgarno sequence affects translation initiation rate.** (in part adapted from Yang *et al.*, 2016). SD-site = Shine-Dalgarno sequence on mRNA; anti-SD-sequence = anti Shine-Dalgarno sequence of 16s rRNA.*

Taken together, st11 emerges as the most effective RBS for rapid and robust protein production in *B. subtilis*. However, the delayed but strong activity of st7 presents a valuable alternative when a slower translation initiation rate may be advantageous for correct protein folding which is a common challenge in recombinant production of complex or aggregation-prone proteins (Rosano & Ceccarelli, 2014).

### Conclusions from Expression construct screening

This subproject confirms the importance of systematically evaluating regulatory elements at the transcriptional and translational levels. The promoter evaluation identified P*liaI* as the strongest and most tightly regulated candidate for high-level heterologous expression in *B. subtilis*. Similarly, comparative testing of RBS variants highlighted st11 and st7 as functional and strong, but with distinct kinetic behaviours affecting timing of protein accumulation. Together, these findings provide a foundation for constructing finely tuned expression systems tailored to recombinant spidroin production in *B. subtilis*.

Synthetic spider silk gene cassette build up and genomic integration

## Synthetic spider silk gene cassette build up and genomic integration

Based on the established cloning strategy “*Pyricloning*”, we next assembled multiple functional transcriptional units of different synthetic *pysp1* constructs to evaluate the functionality of our modular approach. Building on the results from the reporter gene assay, we focused particularly on the strong promoter P*liaI* in combination with the synthetic ribosome binding sites st7 and st11, which had shown the highest and most consistent activity. These constructs formed the basis for our first production attempts, allowing us to compare the effects of transcriptional strength and translation efficiency on *pysp1* expression.

In addition, we also constructed three other transcription units based on the same findings, which served as backup constructs and validation tools to verify successful Level 1 assemblies. Due to their simpler composition, these units allowed us to confirm the robustness and efficiency of our cloning strategy while in parallel assembling the production constructs.

Transcriptional unit Assembly

The first of these, transcription unit TUC1, was built with the well-characterized promoter P*xylA* (Heiss *et al.* 2016; Schmiedel *et al.* 1997), the strong synthetic ribosome binding site st11 (Salis *et al.* 2009), the SD1 coding sequence, and the strong synthetic terminator L3S1P47 (Chen *et al.* 2013). Identical reaction conditions were used as for the Level 0 assemblies, but with BsaI instead of SapI. Subsequently, transcription units containing the SD2 coding sequence were generated with either P*liaI* (TUC2) or P*xylA* (TUC3) as promoters, while all other elements remained identical to TUC1. As an example representative of all Level 1 transcription unit assemblies, Figure 17A illustrates the assembly of TUC3, and Figure 17 shows the corresponding control digest (SapI and NgoMIV) used to verify successful construction.

![figure 17](https://static.igem.wiki/teams/5673/src/results/abb-17-new.webp)

***Figure 17: Successful generation of a complete transcription unit including a synthetic pyriform silk gene construct with a repeat tetramer via MoClo.** A) Schematic representation of the assembly reaction. Arrows indicate the orientation of the type IIS restriction enzymes. All of the four level 0 vectors and the level 1 entry vector were digested with BsaI and ligated via T4 DNA ligase, resulting in the assembly of the level 1 transcription unit TUC3. The parts assemble specifically according to the indicated colour code of the 4 bp overhangs. While BsaI restriction sites were lost during the assembly, the level 1 transcription unit maintained the SapI restriction sites for further assembly into level M transcription units. Elements are not drawn to scale. B) All plasmids were digested with SapI and NgoMIV for 1 hour at 37°C. The empty entry vector served as control. Arrows indicate the expected positions of the fragments. The 1% (w/v) agarose gel was run in 1× TAE buffer (40 mM Tris pH 8.5, 20 mM acetic acid, 1 mM EDTA) at constant 130 V for 30 min and the 1 kb plus DNA ladder (L) from NEB was used for size comparison. GelRed® Nucleic Acid Gel Stain was used to stain the DNA. Images were taken in the E-BOX CX5 TS Edge UV gel documentation system. For visualisation the UV-image was colour inverted.*

A distinct band pattern correlating to the backbone fragments can be seen in both lanes. Since the *lacZ*α fragment (purple arrow) and one of the backbone fragments (black arrows) have almost the identical size (600 bp and 591 bp respectively), these two bands cannot be distinguished in the gel. However, the band appearing around 600 bp showed a strong signal indicating the presence of both bands. While the band pattern of the TUC3 lane missed such a strong signal around 600 bp, two additional bands around 3300 bp and 1600 bp indicated the presence of the transcription unit fragments (green arrows) at the correct heights. By this the successful generation of the level 1 transcription unit was confirmed.

Several level 1 transcription units with different promoters and synthetic CDS were generated during this project which are listed in table 2. Validation was done the same way as described above. Selected constructs were further used in the next step to assemble level M transcription units.

Tab. 2: List of selected level transcription units with synthetic pyriform silk genes assembled during this project

| Description of the transcription unit | Composition |
| --- | --- |
| TUC1 | P*xylA*-st11-SD1-L3S1P47 |
| TUC2 | P*liaI*-st11-SD2-L3S1P47 |
| TUC3 | P*xylA*-st11-SD2-L3S1P47 |
| TUC4 | P*liaI*-st11-SD1-L3S1P47 |
| TUC13 | P*xylA*-st11-SD4-L3S1P47 |
| TUC14 | P*liaI*-st11-SD4-L3S1P47 |
| TUC15 | P*xylA*-st11-SD5-L3S1P47 |
| TUC16 | P*liaI*-st11-SD5-L3S1P47 |
| TUC18 | P*liaI*-st11-SD6-L3S1P47 |

Assembly of level M constructs for genomic integration

With the successful assembly of level 1 transcription units, the next step was to add homology flanks for the genomic integration (level M) into *B. subtilis* W168. Consequently, level M transcription units, incorporating homology flanks for the *amyE* locus and a chloramphenicol resistance cassette were assembled as depicted in Figure 18A. In general, the same reaction conditions and principles as in level 0 were utilised, using different parts and entry vectors. Figure 18B presents the control digest (BsaI and SacI) of TUC2 level M, exemplary for all level M assemblies.

![figure 18](https://static.igem.wiki/teams/5673/src/results/figure-18-full.webp)

***Figure 18: Successful generation of a level M transcription unit via MoClo for the genomic integration into the *B. subtilis* W168.** A) Schematic representation of the assembly reaction. Arrows indicate the orientation of the type IIS restriction enzymes. All of the three level 1 vectors and the two level M vectors were digested with SapI and ligated via T4 DNA ligase, resulting in the assembly of the level M transcription unit TUC2. The parts assemble specifically according to the indicated colour code of the 4 bp overhangs. While SapI restriction sites were lost during the assembly, the level M transcription unit maintained the BsaI restriction sites, which can be used for control digests. The left and the right homology flank (LF/RF) targeted the *amyE* locus of B. subtilis W168, enabling genomic integration of the level M transcription unit. Moreover, the left homology flank also included a chloramphenicol resistance cassette (cmr) for selection after genomic integration. An end linker, which is a short non-coding DNA sequence, was required for the assembly into the entry vector. Elements are not drawn to scale. B) All plasmids were digested with BsaI and SacI for 1 h at 37°C. The empty entry vector served as control. Arrows indicate the expected positions of the fragments. The 1% (w/v) agarose gel was run in 1× TAE buffer (40 mM Tris pH 8.5, 20 mM acetic acid, 1 mM EDTA) at constant 130 V for 30 min and the 1 kb plus DNA ladder (L) from NEB was used for size comparison. GelRed® Nucleic Acid Gel Stain was used to stain the DNA. Images were taken in the E-BOX CX5 TS Edge UV gel documentation system. For visualisation the UV-image was colour inverted.*

Both the control and the TUC2 lane showed the backbone band (black arrows) at the expected heights around 5,000 and 4,600 bp, respectively. Also, all bands in the TUC2 lane appear at heights corresponding to the level M transcription unit fragments (green arrows). Since the repeat monomer sequence has a SacI recognition site, three of the repeat monomers were cut out by SacI, corresponding to the band (blue arrow) appearing around 650 bp. Moreover, the *lacZ*α fragment also contained a SacI recognition site, which, together with the BsaI recognition site, yielded a 244 bp fragment that was too faint to be visible in the control lane. As the expected and correct band pattern appeared for the TUC2 lane, while those bands could not be detected in the control, the assembly of the level M transcription unit was confirmed. To further verify the assembly process, the plasmid containing the TUC4 level M transcription unit was analysed through whole plasmid sequencing, which served as a representative example for all other level M transcription units. The resulting sequence confirmed that the assembly worked as expected.
Several level M transcription units were constructed during this thesis, listed in table 3, which were validated the same way as described above. These transcription units were next used for the genomic integration into *B. subtilis* W168.

Tab. 3: List of slected level 2/M transcription units habouring *pysp1* constructs and signal peptides

| Description of the level M transcription unit | Composition 7, 8 |
| --- | --- |
| TUC1 level M | *amyE* LF – cmr - TUC1- *amyE* RF |
| TUC2 level M | *amyE* LF - cmr - TUC2- *amyE* RF |
| TUC3 level M | *amyE* LF - cmr - TUC3- *amyE* RF |
| TUC4 level M | *amyE* LF - cmr - TUC4- *amyE* RF |
| TUC13 level M | *amyE* LF - cmr - TUC13- *amyE* RF |
| TUC14 level M | *amyE* LF - cmr - TUC14- *amyE* RF |
| TUC15 level M | *amyE* LF - cmr - TUC15- *amyE* RF |
| TUC16 level M | *amyE* LF - cmr - TUC16- *amyE* RF |
| TUC18 level M | *amyE* LF - cmr - TUC18- *amyE* RF |

7*amyE* LF is the left homology flank targeting the *amyE* locus of *B. subtilis* W168. 8  *amyE* RF is the right homology flank targeting the *amyE* locus of *B. subtilis* W168.

Genomic integration of level M transcription units

The last step prior to test heterologous protein production in *B. subtilis* W168 was the genomic integration. Figure 19A shows a schematic of the integration process leading to the loss of the ability to hydrolyse starch. For the transformation, *B. subtilis* W168 was cultured in Paris medium, in which the cells become naturally competent (Harwood 1990; Anagnostopoulos & Spizizen 1961). Plasmids containing the level M constructs were added to these cultures, followed by selection with chloramphenicol. Since the level M plasmids lacked a replication origin for *B. subtilis* W168, only cells that successfully integrated the constructs into their genome were able to survive. Integration of the level M constructs into the *amyE* locus was facilitated by the previously added homology flanks through two homologous recombination events. Because the native *amyE* locus enabled *B. subtilis* W168 to utilise starch as a carbon source, and the construct was targeted to this locus for integration, it was tested whether the transformed strains had lost the ability to metabolise starch. Clones that grew in the presence of chloramphenicol were restreaked on starch plates to assess their ability to hydrolyse starch. After one day of incubation, Lugol’s solution was added to detect the presence of starch and thereby confirm successful disruption of the *amyE* locus.

![figure 19](https://static.igem.wiki/teams/5673/src/results/successful-integration-of-tuc1-level-m-into-the-amye-locus.webp)

***Figure 19: Successful integration of TUC1 level M into the *amyE* locus of *B. subtilis* W168, disrupting starch metabolism.** A) The left and right homology flanks targeting the *amyE* locus of *B. subtilis* W168 recombined with the respective sites in the *amyE* gene, resulting in the genomic integration of the transcription unit. Since a chloramphenicol resistance cassette was introduced as well, screening for positively integrated clones via the supplementation of 5 µg/mL chloramphenicol was facilitated. B) Colonies with a positively integrated transcription unit into the *amyE* locus lost the ability to hydrolyse starch, leading to the preservation of starch around those colonies. By staining the starch agar plates with Lugol solution (0.33% iodine) the remaining starch could be visualised. While the wild type clearly showed a starch hydrolysis halo, multiple colonies had no such halo around them, which validated the positive integration into the *amyE* locus. The starch agar plates were cast without chloramphenicol, to include a wild-type control.*

As shown in Figure 19B, *B. subtilis* W168 wild-type colonies, which have an intact *amyE* locus, can hydrolyse starch, resulting in a clear halo around the colony. This halo forms because starch is degraded and cannot be stained by Lugol’s solution. In contrast, colonies with a properly integrated transcription unit in the *amyE* locus lose this hydrolytic ability. As the starch remains undegraded, Lugol’s solution stains the surrounding agar uniformly, and no halo is observed.
No starch degradation halos were observed for all C1 colonies as well as for C2 of TUC14 and TUC15 and for C3 of TUC13 and TUC16. Thereby, the successful integration of the respective transcription units into the genome of *B. subtilis* W168 was validated. Due to time constraints, the only other transcription unit assembled to level M, which was integrated into the *B. subtilis* W168 genome was TUC1. Depending on their design, these clones were used in various assays to evaluate whether *B. subtilis* W168 can produce and secrete the synthetic pyriform silk proteins.

First Spidroin production attempts in intracellular manner

## First Spidroin production attempts in intracellular manner

Cloning of synthetic *pysp1* monomer constructs for intracellular spidroin production

As we found earlier in the reporter gene assay that among the tested combinations of promoters and RBSs, the *liaI* promoter in combination with the synthetic RBSs st11 and st7 exhibited particularly strong and consistent activity. Two different RBSs were selected as st11 and st7 showed comparable maximal fluorescence 60 min after induction. However, time dependent differences were observed between these RBSs, which can be particularly useful for tuning the expression of the spidroin genes.

Tradionally, the intracellular production of spidroins in bacteria, insect cells or plants is pursued (Jin *et al.*, 2022). Building on these findings, the intracellular production of spidroin was in focus. Furthermore, as it is known that large, intrinsically disordered proteins tend to aggregate in the cytosol and pose significant challenges for heterologous production, we aimed to begin with the evaluation of the recombinant production of the simplest form of our synthetic *pysp1* construct. An exemplary construct design is shown in figure 20.

![figure 20](https://static.igem.wiki/teams/5673/src/results/overview-of-the-cloning-strategy-for-a-spidroin-monomer-constructs-via-moclo.webp)

***Figure 20: Overview of the cloning strategy for a spidroin monomer constructs via MoClo.** C = Control; NTE = N-terminal element; mCTE = modified C-terminal element; SPM = spidroin monomer. A) Schematic of the expression construct design. B) SPM coding sequence assembly with fusion to a His6-tag in a preliminary MoClo level 0 reaction. C) Composition of the assembly of two transcriptional units with two different RBSs, st7 & st11, in a MoClo level 1 reaction. D) Schematic of an assembled MoClo level M vector harbouring a SPM construct. E) Integration of the SPM construct via homologous recombination into the *amyE* locus of *B. subtilis*. F) Verification of the correct transcriptional unit assembly level 1 of two SPM constructs with the same promoter, SPM CDS and terminator, but different RBS. Control digest of isolated vector with BbsI. 1) P*liaI*-st11-SPM-L3S1P47 (2786 bp); 2) P*liaI*-st11-SPM-L3S1P47 (2786 bp). C = control, empty pMMS1 (~1200 bp). G) Control digest verification of correct insertion and assembly of level M SPM constructs. 1) Construct with st11; 2) construct with st7 (4.1 kb, 2.6 kb, 1.4 kb, 1.1 kb, 591 bp & 124 bp). C = control, empty level M vector pMMSM1 (2.4 kb, 1.4 kb, 1.1 kb, 591 bp & 124 bp). Red boxes indicate barely visible bands. H) Verification of integration of level M SPM constructs in *B. subtilis* via Lugol starch hydrolysis assay. 1) Transformation with level M P*liaI*-st11-SPM-L3S1P47 construct; 2) Transformation with level M P*liaI*-st7-SPM-L3S1P47 construct; Red arrow indicates C = *B. subtilis* W168.*

Assessment of intracellular spidroin production in SDS-PAGE and Western Blot analysis

The resulting recombinant strains of *B. subtilis* harbouring the gene construct coding for the SPM were tested for the ability to produce the synthetic SPM. To this end, protein production cultures were inoculated to an OD600 = 0.05 and grown at 37°C in LB medium. Reaching mid-exponential phase, protein production was induced using 0.002% (w/v) of the peptide-antibiotic bacitracin. After 3 h, cultures were harvested and samples prepared.

Spidroin production has previously been shown to benefit from media supplementation with specific amino acids (Connor, Zha & Koffas, 2024). Furthermore, protein folding can be aided by growth temperature adjustments and thus enhance recombinant protein yields (Bhatwa *et al.*, 2021). For that reason, we tested the protein production additionally in 2xYT medium and lowered the growth temperature after induction to 18°C. Deviating from the conditions when grown at 37°C, cultures grown at 18°C were harvested after 18 h to allow for sufficient protein production time. The protein production was then analysed in SDS-PAGE and Western Blot using whole cell lysate (figure 21).

![figure 21](https://static.igem.wiki/teams/5673/src/results/spidroin-production-in-lb-and-2xyt-medium-was-not-detectable-in-sds-page-and-western-blot.webp)

***Figure 21: Spidroin production in LB and 2xYT medium was not detectable in SDS-PAGE and Western Blot.** Whole cell lysate from protein production cultures of recombinant *B. subtilis* carrying the SPM constructs grown in different conditions. Odd numbers: *B. subtilis* P*liaI*-st11-SPM-His6-L3S1P47. Even numbers: *B. subtilis* P*liaI*-st7-SPM-His6-L3S1P47. Culture conditions: samples 1 & 2: LB medium at 37°C; samples 3 & 4: LB medium at 18°C; samples 5 & 6: 2xYT medium at 18°C. Controls: WT = induced *B. subtilis* wild-type; C = control, mNeonGreen-His6. Green arrows indicate the expected migration of SPM, whereas the red arrows indicate the protein band of mNG-His6. SPM = spidroin monomer A) SDS-PAGE of *B. subtilis* carrying the SPM constructs grown in different conditions (see above) in a 12% SDS-gel. After electrophoresis, the SDS-gel was washed with ddH2O and stained with Coomassie blue after fixation. B) Western Blot of *B. subtilis* carrying the SPM constructs grown in different conditions (see above). A 12% SDS-gel was subjected to electroplotting onto an activated polyvinylidene fluoride (PVDF) membrane. Thereafter, the membrane was blocked and incubated with HRP-conjugated α-poly-histidine antibody. After washing, visualisation took place using TMB Enhanced One Component HRP Membrane Substrate.*

No significant differences in the band pattern of the whole cell lysates from *B. subtilis* strains carrying the SPM constructs grown in different conditions compared to the wild-type were obtained in SDS-PAGE (figure 21 A). However, in the growth condition in 2xYT medium at 18°C, a band at ~60 kDa was more pronounced compared to the wild-type and when grown in LB medium. In addition, spidroin production could not be detected in Western Blot (figure 21 B). The positive control, His6-tagged mNG showed an apparent band at approximately 34 kDa, corresponding to the expected molecular weight of ~26.6 kDa. To summarise, spidroin production was not detectable under the tested conditions in SDS-PAGE and Western blot, whereas the positive control mNG-His6 was observable, confirming the validity of the detection approach.

Nevertheless, the comparison of the optical densities at 600 nm after 18 h of growth revealed notable differences between *B. subtilis* cultures harbouring the SPM constructs when cultivated in LB or 2xYT medium, respectively (table 4). When grown in LB medium, both recombinant strains reached OD600 values comparable to the wild-type *B. subtilis* control. However, in the nutrient-rich 2xYT medium, the OD600 values for the SPM containing strains were reduced relative to growth in LB medium. Moreover, there were differences observed between the two constructs. The *B. subtilis* strain containing the RBS st7 grew to an OD600 of 4.12, whereas *B. subtilis* with RBS st11 grew to an OD600 of 5.05. This indicates that the nutrient-richer 2xYT medium led to an impaired growth in the *B. subtilis* strains with SPM construct with the effect being stronger in the *B. subtilis* strain carrying the RBS st7.

Tab. 4: Optical density values (600 nm) of recombinant *B. subtilis* harbouring the spidroin monomer constructs show a decrease in growth after induction in 2xYT medium. *B. subtilis* grown in LB and 2xYT medium at 18°C after induction. Induced *B. subtilis* wild-type served as a control. n = 1

| Condition | OD600 after 18 h cultivation |  |  |
| --- | --- | --- | --- |
|  | WT | P*liaI*-st7-SPM-L3S1P47 | P*liaI*-st11-SPM-L3S1P47 |
| LB | 6.4 | 6.14 | 6.34 |
| 2xYT | - | 4.12 | 5.05 |

Since the spidroin production failed or was undetectable in SDS-PAGE and Western Blot under the tested conditions, we decided to optimise the protein production protocol. Overly strong induction is known to potentially lead to cellular stress and protein degradation, often resulting in low or undetectable yields of heterologous proteins. Hence, carefully tuning the induction to achieve a lower, optimal expression rate can improve recombinant protein yields (Sosa-Carrillo *et al.*, 2023; Rosano & Ceccarelli, 2014). Based on these insights, we aimed to optimise the induction conditions in order to identify parameters that allow for successful spidroin production. 2xYT medium was selected for further cultivation of the *B. subtilis* cultures as an effect on growth was found in induced cultures compared to when grown in LB medium.

To this end, cultures of the *B. subtilis* strains carrying the SPM constructs were inoculated to an OD600 = 0.05 and grown at 37°C in 2xYT medium. To investigate the effect of induction timing on protein production, protein production was induced at early-exponential phase (OD600 = 0.3) and mid-exponential phase (OD600 = 0.5) using 0.002% (w/v) bacitracin. Additionally, to assess the impact of a lower inducer concentration, protein production was induced at mid-exponential phase (OD600 = 0.5) with a lower bacitracin concentration (0.0003% (w/v) bacitracin). Following induction, cultures were incubated at 18°C for 18 h, and the cells harvested and processed as described in the methods section. The protein production was then analysed in SDS-PAGE and Western Blot using whole cell lysate (figure 22).

![figure 22](https://static.igem.wiki/teams/5673/src/results/spidroin-production-under-different-induction-conditions-was-undetectable-in-sds-page-and-western-blot.webp)

***Figure 22: Spidroin production under different induction conditions was undetectable in SDS-PAGE and Western Blot.** Whole cell lysate from protein production cultures of *B. subtilis* strains carrying the SPM constructs grown in 2xYT medium at 18°C under different induction conditions were analysed. Induction conditions: samples 1 & 2: at OD600 = 0.3 addition of 0.002% (w/v) bacitracin; samples 3 & 4: at OD600 = 0.5 addition of 0.002% (w/v) bacitracin; samples 5 & 6: at OD600 = 0.5 addition of 0.0003% (w/v) bacitracin. Odd numbers: *B. subtilis* P*liaI*-st11-SPM-His6-L3S1P47. Even numbers: *B. subtilis* P*liaI*-st7-SPM-His6-L3S1P47. Controls: WT = induced *B. subtilis* wild-type; C = control, mNeonGreen-His6. Green arrows indicate the expected migration of SPM, whereas red arrow indicate the protein band of mNG-His6. SPM = spidroin monomer A) SDS-PAGE of *B. subtilis* strains carrying the SPM constructs under different induction conditions (see above) in a 12% SDS-gel. After electrophoresis, the SDS-gel was washed with ddH2O and stained with Coomassie blue after fixation. B) Western Blot of *B. subtilis* strains carrying the SPM constructs under different induction conditions (see above). A 12% SDS-gel was subjected to electroplotting onto an activated polyvinylidene fluoride (PVDF) membrane. Thereafter, the membrane was blocked and incubated with HRP-conjugated α-poly-histidine antibody. After washing, visualisation took place using TMB Enhanced One Component HRP Membrane Substrate.*

Like the previous tested conditions, SDS-PAGE analysis (figure 22 A) showed no notable differences in the band patterns of whole cell lysates from *B. subtilis* strains harbouring SPM constructs compared to the wildtype. In addition, spidroin production remained undetectable in Western Blot (figure 22 B). In contrast, the positive control, mNG-His6, revealed a band at 34 kDa, confirming the functionality of the experiment.

The observed growth effect in 2xYT medium on *B. subtilis* strains harbouring the SPM construct was reproducible (table 5). Interestingly, when cultures were induced with a lower inducer concentration (0.0003% (w/v) bacitracin), the effect abolished and the strains reached an OD600 comparable to the wildtype control. Additionally, the induction timing, namely induction in early-exponential compared to the mid-exponential phase, using the same inducer concentration did not affect the final optical density measured after 18 h of growth. Due to time constraints, no further attempts were made to optimise the protein production protocol or investigate alternative strategies for spidroin production.

Tab. 5: Effects on bacterial growth of different induction conditions on cultures of *B. subtilis* harbouring the spidroin monomer constructs. *B. subtilis* grown in 2xYT medium at 18°C after induction. Induction took place at either early- or mid-exponential phase (OD600 = 0.3 or 0.5) with either 0.0003 or 0.002% (w/v) bacitracin.

| Induction conditions: |  | OD600 after 18 h cultivation |  |  |
| --- | --- | --- | --- | --- |
|  |  | WT | P*liaI*-st7-SPM-L3S1P47 | P*liaI*-st11-SPM-L3S1P47 |
| At OD600 = 0.5 | 0.002% (w/v) | 6.89 | 4.16 | 4.85 |
|  | 0.0003% (w/v) | - | 6.76 | 6.84 |
| At OD600 = 0.3 | 0.002% (w/v) | - | 4.26 | 4.93 |

Multi-copy spidroin production attempt and secretory production

## 🕸️ Multi-copy spidroin production attempt and secretory production

Since attempts at intracellular spidroin production failed to yield detectable amounts of protein in SDS-PAGE and Western blot analyses, we shifted our focus towards assessing the secretory production of spidroins in *B. subtilis*. A comparable observation was reported by Connor *et al.*, who found no detectable protein during intracellular expression but achieved measurable yields when production occurred via the secretory pathway in *Bacillus megaterium*. These findings motivated us to investigate whether a secretory expression strategy would similarly improve the production of our synthetic spidroins. Indeed, Connor *et al.* had already demonstrated that secretory production can yield detectable protein levels in SDS-PAGE and Western blot, providing a strong rationale for our approach (Connor, Zha & Koffas, 2024).

Another possible explanation for the absence of detectable intracellular spidroins is stress-induced activation of intracellular proteases, which may degrade newly synthesised spidroins before they accumulate to measurable levels. Furthermore, low overall expression levels could also contribute to failed detection. To address this limitation, we employed a multi-copy expression strategy, hypothesising that the increased gene dosage would elevate expression and potentially yield detectable amounts of spidroin.

*B. subtilis* W168 is widely regarded as an ideal host for heterologous protein production and secretion. The secretion of target proteins not only simplifies purification and downstream processing but also enables direct application of the expressed proteins in biotechnological processes without requiring prior isolation. However, exploiting the secretion system of a host organism requires careful consideration, including the use of a native signal peptide that is both recognised by the host’s secretion machinery and functional when fused to the protein of interest.

To meet these requirements, the *yoaW* secretion signal peptide was used in combination with a StrepII-SUMO tag, which has previously been shown to facilitate efficient secretion of recombinant proteins (Heinrich *et al.* 2019). This N-terminal fusion was introduced into the spider silk CDS sequences via the standard MoClo hierarchy as lvl -1 parts. In a later engineering cycle, a C-terminal pep86 tag was fused to the constructs to enable detection using the Nano-Glo® HiBiT Extracellular Detection System (Promega). The pep86 tag is an 11 amino acid long peptide that binds with high affinity to a large fragment of luciferase called LgBiT, thereby reconstituting a functional enzyme (Dixon *et al.* 2016; Pereira *et al.* 2019). As shown in Figure 23, this system can be used to detect secreted recombinant proteins tagged with the pep86 fragment. By adding the LgBiT fragment and the luciferase substrate to the culture supernatant, luminescence can be measured if the tagged recombinant protein is secreted (Dixon *et al.* 2016; Pereira *et al.* 2019).

![figure 23](https://static.igem.wiki/teams/5673/src/results/schematic-illustration-of-the-nano-glo-hibit-extracellular-detection-system.webp)

***Figure 23: Schematic illustration of the Nano-Glo® HiBiT Extracellular Detection System.** A genomically integrated transcription unit encoding a recombinant protein fused to the small pep86 tag (also called HiBiT, 11 amino acids) is expressed in bacterial cells. If the fusion protein is secreted into the extracellular medium, pep86 can associate with the larger LgBiT fragment (156 amino acids), which is added to the supernatant along with the luciferase substrate, furimazine. Upon complementation of pep86 and LgBiT, an active luciferase enzyme is reconstituted, producing a luminescent signal proportional to the amount of secreted protein.*

*pysp1* expression attempt in multi-copy

A multi-copy strategy is useful in heterologous protein production since an increase in the number of gene copies encoding the target protein can boost overall protein yield and therefore enables high-demand applications.
The basis for this multi-copy approach was the *E. coli* – *B. subtilis* shuttle vector pBSMuL1 constructed by Brockmeier *et al.* (2006). This is a suitable expression vector for producing heterologous proteins in *B. subtilis*. To adapt it for the use within the MoClo system and to meet the specific needs of this study, a MoClo-compatible version of the vector was created based on the pBSMuL1 backbone. Additionally, the *mrfp1*ΔlacO cassette was inserted. Upon successful cloning in the future, the cassette would be replaced by the target insert, resulting in white clones on the plate, while clones carrying the parent vector still show red colourisation.
A pBSMuL1-like level 1 vector was constructed to provide the flexibility to change the coding sequence as well as the promoter, ribosomal binding site, secretion signal peptide sequence (sslipA; in case of ssyoaW in combination with a StrepII-tag, SUMO protein and a His6-tag), a C-terminal His6-tag and terminator. This was achieved by implementing MoClo overhangs starting with GGAG and ending with CGCT. After Gibson assembly and validation via restriction digest and sequencing, this vector has been named pLIMO1 (LI: Lilli, MO: Modular Cloning) for simplification. The functionality of the created vector pLIMO1 was assessed by cloning of two reporter constructs harbouring the promoters P*xylA* and P*hpaII* used earlier in the promoter screening, and by assessing the *sfgfp* expression (figure 24).

![figure 24](https://static.igem.wiki/teams/5673/src/results/bacterial-growth-and-fluorescence-signals-under-constitutive-promoter-regulation.webp)

***Figure 24: Bacterial growth and fluorescence signals under constitutive promoter regulation, P*xylA* and P*hpaII*.** In A, the growth curves of *B. subtilis* W168 harbouring the P*xylA*/P*hpaII*-m*sfgfp*-assemblies in MCSE-medium are shown. In B, promoter activities expressed as fluorescence/OD600 (WT-corrected). The OD600 graph is a representative of a total of two replicates. The fluorescence graph shows the mean and standard deviation of two biological replicates.*

*B. subtilis* W168 cells were transformed with the pLIMO1 P*liaI*-ssyoaW/sslipA-spider silk monomer (one single repeat) and tetramer (four repeats) constructs, respectively. After validation via an analytical restriction digest with BsaI, bacteria carrying the correct assemblies were used to inoculate LB medium cultures to a starting OD600 of 0.05. Figure 25 shows the bacterial growth of the respective cultures with the WT as a reference.

![figure 25](https://static.igem.wiki/teams/5673/src/results/bacterial-growth-of-the-b-subtilis-w168-cultures-carrying-spider-silk-construct.webp)

***Figure 25: Bacterial growth of the *B. subtilis* W168 cultures carrying spider silk constructs with either one repeat (monomer) or four repeats (tetramer) in LB medium.** A WT culture was also induced with 20 µg/mL bacitracin to serve as a control for later protein detection. The respective cultures were induced with 20 µg/mL bacitracin (line). n=1.*

Initially all cultures grew almost identical until induction. Then a clear difference between the cells carrying a spider silk construct to the ones with the WT were visible, resulting in an end-OD600= 1-1.74 compared to the WT values of OD600= 3.32-3.76 three hours post-induction. From all grown cultures, three samples for SDS-PAGE followed by Western Blot analysis were takes 3 h post-induction. This allowed us to examine five different fractions, i.e. whole cell lysate, secreted proteins, lysate after bead beating, cytoplasmic proteins and cell debris, to ensure a complete analysis of spider silk production. The corresponding protein gels with one representative example of a Western Blot are shown in Figure 26. The protein gels display the plethora of intracellular and secreted proteins. Although the spider silk proteins are labelled with secretion signals, it was expected, if production occurred, that some protein is still within the cell. But no gel showed clear bands for neither the monomer spider silk protein, which would be expected at 85 kDa (plus 15 kDa for ss*yoaW*-construct in all fractions except the supernatant or plus 8 kDa for sslipA), nor for the tetramer spider silk protein, which would be likely at 135 kDa (plus ss*yoaW*-construct or ss*lipA*). A GFP-His6 functioned as a positive control and showed a distinct band just above 26 kDa, corresponding to the expected molecular weight of 26.8 kDa (green arrow). As only the control, the GFP-His protein, was detected in the Western Blot, only one representative image is shown. Since no bands corresponding to the expected molecular weights of the synthetic spider silk constructs were visible in neither the protein gels nor in the Western Blots, the conclusion was that the proteins were either not produced or were present at levels too low to be detected by the methods used.

![figure 26](https://static.igem.wiki/teams/5673/src/results/protein-gels-of-five-different-fractions-of-the-b-subtilis-w168-wt-and-b-subtilis-w168.webp)

***Figure 26: Protein gels of five different fractions of the *B. subtilis* W168 WT and *B. subtilis* W168 carrying spider silk constructs showed no clear band for spider silk proteins, nor did Western Blot.** For each fraction (whole cell lysate, secreted proteins, lysate after bead beating, cytoplasmic proteins and cell debris) two SDS-PAGEs were run followed by performing with one a Western Blot, utilising a monoclonal anti-poly-histidine peroxidase antibody, 1:2000, and a substrate solution for colorimetric protein detection. The purple arrow indicates the height of the spidroin-tetramer, the blue one the height of the spidroin monomer and the green one the GFP-His positive control. L= molecular weight marker, 1= non-induced WT, 2= induced WT, 3= ss*yoaW*-monomer, 4= ss*yoaW*-tetramer, 5=ss*lipA*-monomer, 6=ss*lipA*-tetramer and C= control protein with a His-tag. 10% SDS gel.*

In summary, the production of synthetic spider silk proteins was tested using two different secretion signal peptides that have previously been tested for *msfgfp* secretion, namely ss*lipA* and ss*yoaW*. In addition, the spider silk sequences were assembled as a monomer (one repeat) and as a tetramer (four repeats) to test different lengths of the protein. After several transformation attempts, it was apparent that no colonies could be obtained from any of the assemblies carried out with constitutive promoters such as P*xylA* and P*hpaII*. Only after a week of incubation at 30°C small colonies were visible on the LB agar plate. When these were inoculated into liquid LB media, they did not grow. This suggests that a constitutive transcriptional control probably causes such a high level of stress to the cell that it will lyse. Therefore, the inducible P*liaI* was used for each spidroin assembly. Colonies were visible after one day of incubation under non-inducing conditions. When *B. subtilis* W168 was grown with the P*liaI*-ss*lipA*/ss*yoaW*-monomer/tetramer-constructs in LB medium, all cells grew almost identically pre-induction, suggesting tight control of P*liaI*, which was the reason for selecting this promoter. Post induction, all cells carrying a spidroin construct showed a strong reduction in growth rate compared to the WT strain, suggesting that the cells are at least trying to produce the synthetic spider silk proteins. However, no distinct (over-) production protein bands were seen in any fraction of the SDS-PAGE, neither for a monomer construct (85 kDa; if not secreted plus 15 kDa for the ss*yoaW* construct or plus 8 kDa for sslipA), nor for the tetramer spider silk protein, which would be expected to be visible at 135 kDa (plus signal peptide). Instead, a similar pattern was observed for almost all protein gels, except for the cell debris fraction and the secreted protein fraction precipitated by TCA. However, in the spidroin fractions of total cell lysate, cytoplasmic proteins and lysate after bead beating, a distinct band at around 41 kDa was visible, which could be DnaJ (40.7 kDa), a molecular chaperone that ensures protein folding and activates DnaK (65.8 kDa). In the lysate after bead beating, a distinct band at around 40 kDa was seen in all lanes except the non-induced WT sample, which could be LiaS (40.5 kDa), the sensor histidine kinase of the *liaIHGFSR* operon that senses the inducer bacitracin (Jordan *et al.*, 2006). The other components such as *liaI* (13.2 kDa), LiaH (25.5 kDa), LiaG (25 kDa), LiaF (26.9 kDa), LiaR (23 kDa) cannot be clearly detected as the protein gel only shows distinct bands up to about 26 kDa.

Therefore, the lack of detection in this study may be due to the DNA copy number and therefore the tRNA pool being too low for the translation of these repetitive long proteins, despite the sequences being codon harmonised. Since there is still a huge knowledge gap in the area of pyriform silk sequences, there were not many data available for such a harmonisation process. Therefore, an additional vector with genes encoding tRNAs for the commonly required amino acids, such as glutamine, glycine and proline, could provide increased levels of these tRNAs and reduce the risk of shortages. Yang *et al.* (2016) showed that with a higher glycyl tRNA pool, the biosynthesis of glycine-rich silk proteins in *E. coli* was successful. Another bottleneck could be that the amino acid pool was too low and a targeted supplementation of amino acids could (0.3 g/L of each Gln, Glu, His, Ile, Phe, Pro, Tyr, Lys, Met) increase production, as shown by Connor *et al.* (2024). This resulted in a 135% increase in the production of a 16.1 kDa silk protein relative to both rich and minimal media, yielding secretory titres of approximately 100 mg/L in flask cultures. Further ideas for optimising the heterologous production of synthetic spider silk proteins are discussed later.

Synthetic *pysp1* could not be detected via SDS-PAGE and Western blot but via split NanoLuc assay in single-copy expression attempts

As the multi-copy expression attempt appeared to impose increasing cellular stress and did not yield detectable amounts of protein. We returned and focussed our optimisation steps towards the secretory expression of the synthetic *pysp1* in a single-copy approach.

Following integration of the level M *pysp1* constructs into the *B. subtilis* W168 genome, the protein production capabilities of the resulting strains were evaluated. Initially, the goal was to confirm protein production before optimising the setup, harvest timepoints or media composition. Production cultures were grown in LB medium, inoculated to an OD600 of 0.05, and gene expression induced by the addition of 0.3% (w/v) xylose at an OD600 of 0.4 0.5. Incubation was carried out at 37 °C under aerobic conditions. Cultures were harvested after 3 h of induction, and protein production was analysed by SDS-PAGE and Western blot across different cellular fractions. In parallel, OD600 readings were taken to assess whether production of the synthetic silk proteins affected *B. subtilis* growth.

Accordingly, the TUC14 (monomeric RPE, [BBa\_255CP6QO](https://registry.igem.org/parts/bba-255cp6qo)), TUC16 (tetrameric RPE, [BBa\_25VXKHEZ](https://registry.igem.org/parts/bba-25vxkhez)) and TUC18 (octameric RPE, [BBa\_25AE0AHE](https://registry.igem.org/parts/bba-25ae0ahe)) strains, which all have a His6-tag within their *yoaW* secretion signal peptide, were cultured. Their corresponding growth curves, alongside those of controls strains, are presented in Figure 27.

![figure 27](https://static.igem.wiki/teams/5673/src/results/growth-curves-of-b-subtilis-w168-strains-harbouring-pysp1-constructs.webp)

***Figure 27: Growth curves of *B. subtilis* W168 strains harbouring pysp1 constructs and single tags.** Optical density was measured at λ = 600 nm using a cuvette spectrophotometer with 1:10 dilutions. The system was blanked with 0.85% (w/v) NaCl. Four timepoints were measured at T0 = 0 min, T1 = 120 min, T2 = 150 min and T3 = 330 min. Cultures were induced with 20 µg/mL bacitracin after measurements of T2 (150 min, dashed line), at an OD600 of 0.4 0.5. Y-axis is on a logarithmic scale. No replicates were measured.*

Initially, all constructs and the wild types showed similar growth. Following induction after the measurements of T2 (150 min) the uninduced TUC14 and wild-type control strains demonstrated a steeper rise in their final OD600 compared to the induced production strains. Notably, the most pronounced impact on growth was seen in the TUC14 strain, which carried the shortest protein construct. In contrast, TUC16 and TUC18 reached final OD600 values comparable to the induced wild-type control. However, both TUC16 and TUC18 already displayed slightly elevated OD600 values at the point of induction, corresponding to a smaller increase in OD600 post-induction compared to the controls.

This moderate growth inhibition in the induced production strains, relative to the uninduced controls, suggested active production of the synthetic pyriform silk proteins. Due to limited data points and a lack of biological replicates, these observations remain preliminary and require further validation. As time constraints prevented additional tests, direct protein detection via SDS-PAGE and Western blotting was proceeded with.

Cell pellets from the cultures were lysed using the previously described method. The whole cell lysate fraction was analysed first. Despite the inclusion of secretion signal peptides in the constructs, intracellular retention of the proteins was expected if production occurred. Figure 28A presents the SDS-PAGE gel of the whole cell lysates. Due to insufficient sample loading, no bands were observed in the uninduced wild-type control. Nevertheless, no significant differences were detected across all lanes. A His-tagged msfGFP served as a positive control (ctrl.) for the Western blot and produced a distinct band just below 34 kDa, corresponding to the expected molecular weight (yellow arrow, 26.8 kDa). Faint bands above 250 kDa were visible in both TUC16 and TUC18 samples but were absent in the TUC14 lane. However, these high-molecular-weight bands were also present in the induced wild-type control, making specific detection of synthetic spidroins in this region unlikely. No visible bands were detected at the expected molecular weights of the target proteins (green arrow: TUC14/SD4 – 102 kDa; pink arrow: TUC16/SD5 – 165 kDa; blue arrow: TUC18/SD6 – 249 kDa).

Given the high background observed in the whole cell lysate gel, the same samples were subjected to SDS-PAGE again, followed by electroblotting onto a polyvinylidene fluoride (PVDF) membrane for Western blot analysis. After blocking, the membrane was incubated with a conjugated anti-hexahistidine-peroxidase-antibody and treated with the colorimetric TMB Enhanced One Component HRP Membrane Substrate for specific detection of His-tagged proteins. The resulting blot is shown in Figure 28B.

![figure 28](https://static.igem.wiki/teams/5673/src/results/synthetic-spider-silk-proteins-could-not-be-detected-via-sds-page-or-western-blot.webp)

***Figure 28: Synthetic spider silk proteins could not be detected via SDS-PAGE or Western blot.** Whole cell lysate samples from different production strains (TUC14/16/18) were tested. Additionally, an uninduced (unind.) and an induced (ind.) wild-type control were tested as well. Also, His-tagged m*sfgfp* was used as control (ctrl.) for the Western blot. A) SDS-PAGE of the different strains using a 10% SDS gel. Color Prestained Protein Standard, Broad Range (10-250 kDa) was used as ladder. Samples were normalised to a fixed OD600 prior to preparation, and equal volumes were loaded into each well. However, only one fifth of the volume was loaded for the His-tagged m*sfgfp* control due to its high protein concentration. Samples were run in 1× running buffer (25 mM Tris-HCl, 192 mM Glycine, 3.5 mM SDS) at 120 V for 10 min. Then, the voltage was increase to 180 V until the running front started leaking out of the gel. Afterwards, the gel was washed in deionised water prior to staining in SimplyBlue™ SafeStain. B) A non-stained 10% SDS gel was used for electroblotting in 1× running buffer supplemented with 20% (v/v) methanol at constant 75 mA for 70 min onto an activated (100% (v/v) methanol) polyvinylidene fluoride membrane. Afterwards, the membrane was blocked, followed by incubation with an anti-hexahistidine-peroxidase-antibody. The colorimetric TMB Enhanced One Component HRP Membrane Substrate was used for visualisation. Images were taken 10 min after the addition of the substrate. The arrows indicate the expected heights of the different constructs from the different transcription units (green arrow TUC14/SD4 – 102 kDa, pink arrow TUC16/SD5 – 165 kDa, blue arrow TUC18/SD6 – 249 kDa) and the His-tagged m*sfgfp* control (yellow arrow, 26.8 kDa).*

Unfortunately, none of the synthetic protein constructs could be detected via Western blot, with the exception of the His-tagged msfGFP positive control, which produced a distinct and prominent band just below 34 kDa - corresponding to its expected molecular weight of 26.8 kDa (yellow arrow).

Subsequently, additional cellular fractions obtained during sample preparation were analysed to improve protein separation and increase the chances of detecting faint and distinct bands. Secreted proteins were isolated by precipitating them with TCA from the culture supernatant and resuspending the precipitate in SDS sample buffer. Furthermore, the whole cell lysate was fractionated by centrifugation to separate cytoplasmic proteins from the cell debris/membrane-associated protein fraction. However, SDS-PAGE and Western blot analyses of all these fractions yielded results comparable to those of the whole cell lysate.

Since no bands corresponding to the expected molecular weights of the synthetic protein constructs were visible in either the SDS-PAGE gels or Western blots, it was concluded that the pyriform silk proteins were either not produced or at levels too low to be detected by these methods. Due to time limitations, it was not possible to optimise the production protocol, which might have improved protein yields, or to test different constructs with alternative promoters. Instead, protein production was tested using the highly sensitive Nano-Glo® HiBiT Extracellular Detection System, which is capable of detecting proteins produced at low abundance (Hall *et al.*, 2012; Miao, Zhou & Wang, 2024).

### Detection via NanoLuc assay

The luminescence measurements are summarised and shown in Figure 29. Due to time constraints, most samples were measured in single replicates, however, two biological replicates were obtained for the induced wild type and TUC14 samples.

![figure 29](https://static.igem.wiki/teams/5673/src/results/endpoint-luminescence-measurements-of-production-cultures.webp)

***Figure 29: Endpoint luminescence measurements of production cultures.** Measurements were performed in TecanSpark plate reader with 1000 ms integration time. Data accompanied by error bars are based on two replicates, while those without error bars represent single measurements. For the TUC18 cell pellet no data was obtained due to technical reasons. Samples were blanked with a LB medium control. Values were normalised to an OD600 of 1. Strains were induced with 20 µg/ml bacitracin.*

Induced TUC16 exhibited the strongest cell associated luminescence signal at endpoint, accompanied by a clear increase in supernatant luminescence, indicating robust *pysp1* expression with measurable extracellular presence under 20 µg/mL bacitracin after LB blanking and normalisation to OD600 = 1 with 1000 ms integration time. WT and TUC14 showed low signals in both fractions, with only modest induction effects, consistent with minimal reporter activity under these conditions and emphasising the contrast with TUC16. TUC18 displayed an elevated supernatant signal upon induction, suggesting expression and potential secretion, but the corresponding cell pellet measurement was unavailable due to a technical issue, so intracellular accumulation for TUC18 cannot be directly compared to TUC16 in this dataset. Measurements with error bars reflect two replicates and appear most consistent for induced TUC16, whereas single bars without error bars represent single measurements and should be interpreted with caution regarding variance.

Discussion - Translational Bottlenecks, Secretion Stress, and Proteolytic Degradation Constrain Synthetic Spidroin Yield

Producing native spidroins through the farming or milking of spiders is unfeasible due to their solitary lifestyle and cannibalistic behaviour (Vierra *et al.*, 2011; Ramezaniaghdam, Nahdi & Reski, 2022). For this reason, recombinant spidroin production has emerged as the only feasible industrial route to obtain spider silk. Yet despite decades of research, recombinant production has encountered major obstacles, with consistently low yields generally attributed to inefficient transcription and translation as well as the metabolic burden imposed on the host cell (Fahnestock & Irwin, 1999; Xia *et al.*, 2010; Connor, Zha & Koffas, 2024).

Even though the coding sequence of *pysp1* was codon harmonised and the intracellular and secretory *pysp1* production attempted, and extensive optimisation, no detectable protein was observed by SDS–PAGE or Western blot. Only very low production signals were detected by the highly sensitive Nano-Glo® HiBiT luminescence assay (Hall *et al.*, 2012; Miao, Zhou & Wang, 2024). The poor yields were hypothesised to result from secretion blockage via the SecYEG complex, possibly caused by premature folding of the synthetic *pysp1* during translocation (Jiang, Wynne & Huber, 2021).

Despite employing promoter–RBS combinations that demonstrated strong reporter activity, spidroin production remained undetectable in SDS–PAGE and Western blot regardless of growth medium (LB or 2xYT) or induction temperature (37°C or 18°C). This aligns with recurring challenges in heterologous spidroin production: translational errors, metabolic burdens, or outright construct toxicity (Ramezaniaghdam, Nahdi & Reski, 2022). Similarly, Connor, Zha & Koffas were unable to detect spidroins in *Bacillus megaterium*, again pointing to translation and folding incompatibilities when expressed intracellularly. The authors of that work even speculated that certain spider silk proteins may intrinsically require a secretory environment for productive folding. Accordingly, the authors could show the secretory production of the same spidroins construct (Connor, Zha & Koffas, 2024).

The possible reasons for the lack or extremely low level of spidroin synthesis centre on transcriptional and translational challenges associated with the long length and highly repetitive composition of *pysp1* (Connor, Zha & Koffas, 2024). Prokaryotic systems can suffer from premature ribosomal pausing and termination when tRNA pools become depleted in response to overused codons or amino acid requirements (Fahnestock & Irwin, 1999; Xia *et al.*, 2010; Snoeck, Guidi & De Mey, 2024). Polyproline-rich motifs exacerbate this by inducing ribosome stalling (Park *et al.*, 2024; Hong *et al.*, 2024). When stalling occurs, rescue pathways recognise incomplete peptides and transcripts, targeting both for degradation (Buskirk & Green, 2017). While the synthetic spidroin coding sequence here had been codon-harmonised to better match *B. subtilis* codon usage, spidroin length and repetitive content still made premature terminations and stalling likely. Due to time and resource constraints, the harmonised versus original sequences were not directly compared, leaving unresolved how effective harmonisation truly was in avoiding these issues. Altogether, truncation, misfolding, and low-level expression appear plausible outcomes under these translational stresses (Connor *et al.*, 2023; Xia *et al.*, 2010; Snoeck, Guidi & De Mey, 2024).

![figure 30](https://static.igem.wiki/teams/5673/src/results/secretion-stress-schematic.webp)

***Figure 30: Schematic summary of the recombinant pyriform silk production in B. subtilis W168 and potential resulting stresses.**  After expression of the synthetic pyriform silk gene (synpysp1), multiple stresses could have occurred, leading to protein degradation and cellular impairments. Blue arrows indicate concentration changes. 1) Translation of the repetitive constructs can lead to depletion of tRNA pools (A), disrupting global protein biosynthesis. 2) The signal peptide of the translated protein interacts with SecB, targeting the unfolded spidroin for secretion through the SecYEG complex. At high production levels, this may result in blockage of the pore and secretion stress (B). This prevents the secretion of vital proteins and the incorporation of essential lipoproteins into the membrane. The resulting stress response induces the upregulation of extracellular and membrane-associated proteases, which degrade (misfolded) proteins. 3) Chaperones assist with proper folding and attempt to refold potentially misfolded proteins resulting from translational stress. If large amounts of synthetic pyriform silk proteins misfold during translation, they occupy a significant portion of chaperone and proteasome capacity. As a result, native proteins that require chaperones cannot fold properly, and even more misfolded proteins accumulate (C). If these systems are overwhelmed, misfolded proteins eventually aggregate (D), as proteasome capacity is also limited (E). This, in turn, again leads to the upregulation of proteases and protein degradation.*

The hypothesis that aggregates might explain the lack of SDS–PAGE or Western blot detection seems less likely, given that whole-cell lysates were analysed and no spidroin signals were present. Instead, stress responses and proteolytic degradation are more consistent with the data. Previous studies have similarly reported spidroin misfolding in intracellular hosts followed by degradation (Snoeck, Guidi & De Mey, 2024; Jin *et al.*, 2022).

Growth analysis provided parallel evidence for translational burden. Under intracellular production in LB medium, *B. subtilis* strains carrying spidroin constructs grew comparably to wild-type. However, in nutrient-rich 2xYT medium, recombinant strains showed clear growth impairments, strongly suggesting translational errors visibly impacted physiology (Connor, Zha & Koffas, 2024). However, the secretory production of spidroin constructs similarly led to growth impairments in LB medium. Overproduction of silk proteins is documented to retard bacterial cell growth due to errors in translation (Heidebrecht & Scheibel, 2013). Intriguingly, spidroin expression driven by the st7 RBS caused stronger growth defects than st11, despite st11’s higher predicted initiation rate. Since RBS function depends strongly on downstream coding context (Mutalik *et al.*, 2013), and stress burdens result from multiple interacting processes, the data suggest a nonlinear and complex relationship between translational initiation and cellular stress.

The regulatory framework contributed further complications. The *liaI* promoter driving expression is controlled by the LiaRS two-component system, which responds to bacitracin (Toymentseva *et al.*, 2012; Radeck *et al.*, 2013). Upon sensing bacitracin, LiaS activates LiaR, which induces via P*liaI* *liaI*H expression. Its promoter activity is tightly regulated, concentration-dependent, and transient, falling after about two hours due to native stress response dynamics (Castillo-Hair *et al.*, 2019). Kesel *et al.* showed that reducing bacitracin from 20 µg/mL to 3 µg/mL halved expression rates, and in this study, lowering the inducer indeed improved growth of recombinant strains. This suggests that spidroin-induced growth inhibition was directly proportional to expression levels. Timing of induction, whether in early or mid-exponential phase, made no difference, further supporting a direct toxicity effect. As production in this study was shifted to 18°C post-induction, prolonged promoter activity was hypothesised, though unconfirmed empirically.

Another major explanation for failure lies in proteolysis. *B. subtilis* maintains protein quality through multiple cytosolic and extracellular chaperones, including DnaK, GroEL, and trigger factor (Moliere & Turgay, 2009), and at least seven AAA+ proteases (Matavacas & von Wachenfeldt, 2022). These systems prevent misfolded protein accumulation but may degrade misfolded spidroins. Moreover, spiders employ specialised chaperones in their silk glands for spidroin folding (Miserez, Yu & Mohammadi, 2023). In recombinant contexts, without such chaperones, disordered spidroins may overwhelm folding machinery, induce stress, and undergo degradation (Li, Zhou & Lu, 2004; Tjalsma *et al.*, 2004). Notably, in 2xYT medium at 18°C, recombinant *B. subtilis* showed an SDS–PAGE band at ~60 kDa, more pronounced than in control strains. This band likely corresponded to the cytosolic chaperone DnaK (66 kDa; UniProt: P17820), confirming chaperone upregulation during stress. While nutrient-rich media provided more amino acids favouring spidroin translation, they simultaneously worsened protein folding stress responses.

The final part of the study investigated constructs of different repeat lengths, monomer (single repeat) and tetramer (four repeats), under secretion signals ss*lipA* and ss*yoaW*, each controlled by either constitutive promoters (P*xylA*, P*hpaII*) or the inducible P*liaI*. No colonies formed with constitutive constructs, indicating lethal stress from unregulated expression. After prolonged incubation at 30°C, a few tiny colonies appeared but failed to grow in liquid culture. By contrast, colonies with P*liaI* constructs grew normally pre-induction, consistent with tight promoter regulation. Once induced, growth rates declined sharply in all recombinant strains compared to wild-type, implying that cells attempted spidroin expression but could not sustain it. Expected protein bands (~85 kDa for monomer constructs with signal peptides, ~135 kDa for tetramers) were absent from SDS–PAGE. Instead, distinct bands at ~41 kDa likely reflected DnaJ (40.7 kDa) or LiaS (40.5 kDa), direct evidence of molecular chaperone or cell envelope stress responses (Jordan *et al.*, 2006). Bands near 32 kDa may be PrsA, a foldase anchored to the membrane where it refolds proteins post-translocation (Neef *et al.*, 2020). This strongly suggests folding and secretion stress induction but degradation of spidroins before detection. Quantitative RT PCR of stress-related genes would be useful in future to verify upregulation of these networks (Livak & Schmittgen, 2001).

Western blot experiments confirmed these observations: GFP-His6 or mNG-His6 controls were detected, confirming the experimental validity, but neither monomer nor tetramer spidroins produced bands. Possible explanations include (i) low expression below detection limits, (ii) instability and degradation due to absent protease inhibitors during sample prep, or (iii) losses during fractionation. Mass spectrometry was proposed as a more sensitive detection method capable of confirming extremely low expression by quantifying peptide fragments (Connor *et al.*, 2023).

Switching our detection strategy to the more sensitive HiBiT pep86 luminescence tag system, showed that spidroins were indeed produced at very low levels in P*liaI*-driven monomer, tetramer, and even octamer constructs, expressed from single-copy chromosomal integrations. Increased luminescence in both cell pellet and supernatant fractions suggested limited secretion also occurred (Dixon *et al.*, 2016; Pereira *et al.*, 2019). Accordingly, the lack of detection in SDS–PAGE or Western blot here may be due to low copy number, insufficient translation resources, or unstable expression.

The translational resource limitations are highly plausible. Even codon-harmonised constructs continued to rely heavily on glutamine, glycine, and proline. High demand for tRNAs corresponding to these amino acids likely overwhelmed natural pools. Consistent with this, Yang *et al.* (2016) showed that supplementing glycyl-tRNA enabled *E. coli* to produce glycine-rich silk proteins, and Connor *et al.* reported that supplementing amino acids improved silk production by 135%, achieving titres of ~100 mg/L for small silk proteins. In the present study, neither tRNA supplementation nor amino acid feeding was employed, making these limitations critical bottlenecks.

In conclusion, the results from this study show that recombinant *B. subtilis* initiates spidroin gene expression, as evidenced by growth impairments and stress protein upregulation, but fails to produce detectable spidroins in gels or blots. Both intracellular and secretory approaches produced consistent outcomes: severe cellular stress, growth inhibition, chaperone upregulation, and either degradation or non-accumulation of spidroins. These findings mirror other unsuccessful attempts in prokaryotic systems and confirm that full-length spidroins present extraordinary translational and folding challenges. The combined data strongly suggest that yield was limited by translational stalling, tRNA depletion, amino acid resource constraints, premature folding during secretion, and subsequent proteolytic degradation. Sensitive reporter assays confirmed trace expression, but titres were orders of magnitude below the levels required for downstream applications. The findings underscore the importance of translational resource engineering, codon and amino acid alignment, co-expression of chaperones, and alternative detection methods. Until these limitations are addressed, large repetitive spidroins like *pysp1* remain unsuitable for reliable recombinant production in *B. subtilis*.

Outlook - Optimisation of Spidroin production

## Outlook - Optimisation of Spidroin production

Optimising spidroin production in *B. subtilis* will benefit from a tighter transcriptional control, careful process handling to minimise proteolysis and stress while enabling stable, sustained expression. A practical starting point is to adopt the IPTG-inducible P*spank* promoter for a tunable, continuous induction also over longer fermentation times with lowered temperatures, due to the trait that IPTG is not metabolised by the cell (Castillo-Hair *et al.*, 2019; Rokop, Auchtung & Grossman, 2004; Donovan, Robinson & Glick, 1996). Furthermore, the expression induction can be titrated to concentration that balances metabolic burden and optimal expression rates rather than driven at maximal rates that risk translational or secretion stress. Another way of optimisation could be the media optimisation. The supplementation with certain amino acids highly represented in *pysp1* is such an example to reduce metabolic bottlenecks during high-demand expression of *pysp1* (Connor, Zha & Koffas, 2024).

Strain engineering may further stabilise the spidroin production, for instance, attempting the co-expression of tRNAs matching enriched *pysp1* codons (for example, for Ser, Ala and Gln) complements the deliberate codon harmonisation strategy and helps prevent tRNA pool depletion that often limit repetitive silk proteins (Simmons, Xu & Rainey, 2019; Zhu *et al.*, 2020; Xia *et al.*, 2010). Conversely, in the secretory route, deletion of major extracellular proteases and attenuation of stress-induced intracellular proteases lowers degradation risk, while the co-expression of chaperones such as DnaK/DnaJ/GroEL for intracellular expression and PrsA for secreted proteins supports co- and post-translocation folding and reduces quality-control–triggered proteolysis (Wu *et al.*, 1998; Widmaier *et al.*, 2009). Lastly, protein production protocol refinements should include adding protease inhibitors during harvest, lysis, and sample preparation to avoid artifactual losses that can obscure low-abundance products; moderating translation initiation via weaker RBSs or adjusted spacer lengths complements lower induction temperatures to improve folding and reduce aggregation. Finally, the detection should pair standard SDS-PAGE/Western with sensitive luminescent tagging such as pep86/LgBiT complementation, which has already revealed low-level production when gels and blots were negative. Pursuing an iterative optimisation should begin from single-copy genomic expression to define tolerable induction and temperature windows before scaling to multi-copy plasmids, thereby reducing toxicity and proteolysis while establishing a robust baseline for subsequent yield improvements.

Proof of Concept: Biomineralisation and Prototyping - Xanthan

## 🏗️ Proof of Concept: Biomineralisation and Prototyping

Introduction

Biopolymers can serve multiple functions in MICP-based applications: they retain moisture, offer nucleation sites for calcite formation, bridge gaps between particles, and improve the distribution of bacterial cultures within the substrate (Jang, 2020; Soldo *et al.*, 2020). Moreover, biopolymer-treated materials showed improved resistance to environmental conditions through improved compressive strength, tensile strength, and elasticity (Soldo *et al.*, 2020).

Xanthan gum, in particular, is known for its ability to increase cohesion in soil, fill sand pores, and improve erosion resistance by enhancing water retention, all of which contribute to improved mechanical properties such as compressive and tensile strength (Gioia and Ciriello 2006; Jang 2020). Its gel-like matrix helps bridge gaps between particles, which is particularly valuable in bio-consolidated sand, where large pore spaces can weaken the material (Gioia & Ciriello, 2006; Lai *et al.*, 2024). By supporting bacterial attachment and offering nucleation sites for calcite, xanthan may help overcome this limitation and improve the overall effectiveness of MICP (Jang, 2020; Lai *et al.*, 2024).

Evaluation of the influence of xanthan gum on biomineralisation

As a proof of concept, the impact of xanthan, a widely used biopolymer produced by the bacterium *Xanthomonas campestris* (Bhat *et al.* 2022), on the biocementation of sand bricks via MICP, facilitated by the *Solibacillus silvestris* isolate CGN12, was investigated.

Initially, xanthan was produced from production cultures of *X. campestris*, which were incubated for over a week. Following incubation, xanthan was purified and subsequently lyophilised. The purified xanthan was then added to the biocementation medium (YAC medium) at varying concentrations.

A fixed volume of sand was added to individual wells of a silicone mold and mixed with a defined number of CGN12 cells resuspended in 5 mL of YAC medium, supplemented with xanthan concentrations ranging from 0 1% (w/v). The experimental setup is shown in Figure 31A. Over time, the sand bricks began to dry, as shown in Figure 31B. Each sand brick received at least one additional round of YAC medium (without xanthan) before being allowed to dry completely.

![figure 31](https://static.igem.wiki/teams/5673/src/results/xanthan-micp-coupled-biocementation-set-up.webp)

***Figure 31: Xanthan-MICP coupled biocementation set up.** The wells measured 4.9 cm in length, 2.6 cm in width and 1.1 cm in depth. Each well was filled with 12.5 g of quartz sand (particle size 0.1–0.5 mm) supplemented with 5 mL of a fresh S. silvestris cell suspension in YAC medium (OD600 = 2.7, 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). All xanthan concentrations shown are (w/v). A) Initial preparation of the wells (t = 0). The image was taken immediately after the last well was supplemented with medium. B) Sand bricks after 6 days of biocementation.*

As the experimental setup was intentionally non-sterile, to better reflect potential real-world applications where sterile conditions cannot be guaranteed, microbial contaminations began to emerge after several days. This is visible in the right wells of conditions 2 and 3 in Figure 31B.

In the initial biocementation experiment, two conditions were compared: pure YAC medium and YAC medium supplemented with 1% (w/v) xanthan. Both conditions were inoculated with *Solibacillus silvestris* CGN12 cells to a final OD600 of 2.7. Following inoculation, the sand bricks were left uncovered to dry overnight. On the following day, 3 mL of fresh YAC medium (without xanthan) was added to each well, just enough to form a thin liquid film. The bricks were again allowed to dry completely.

Once fully dried, the sand bricks were removed from the silicone molds for qualitative assessment of their mechanical properties. As shown in Figure 32A and B, the sand brick formed in the presence of xanthan was noticeably thicker and more structurally cohesive than the one formed without xanthan. Notably, the non-xanthan brick fractured during removal, indicating lower mechanical robustness. In contrast, the xanthan-containing brick retained its integrity, displayed sharper edges, and appeared more consolidated.

These observations were further supported by the residual sand left in the wells, as shown in Figure 32C. While the cylindrical parts of the bricks appear to be filled more in the well of the xanthan brick, the majority of the loose sand on the images in Figure 32 comes from the non-xanthan brick. Additionally, the solidified cylinders from the xanthan condition were sufficiently hardened to resist removal, whereas most of the sand from the cylinders of the non-xanthan condition disintegrated and fell out during extraction from the mold.

![figure 32](https://static.igem.wiki/teams/5673/src/results/bioconsolidated-sand-bricks-after-biocementation.webp)

***Figure 32: Bioconsolidated sand bricks after biocementation.** Initial attempt of biocementation with S. silvestis in YAC medium (OD600 = 2.7, 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). Following the initial inoculation, one additional 3 mL portion of fresh YAC medium was added the next day, after which the sand bricks were left to dry. No foil was used to prevent media evaporation or contamination during incubation. Images were taken 17 days after the initial inoculation. All xanthan concentrations shown are (w/v). A) Side view of the bioconsolidated sand bricks. B) Top view of the bioconsolidated sand bricks. C) Wells of the removed sand bricks.*

These results suggest that the addition of xanthan at the tested concentration enhanced the biocementation of sand bricks. However, it remained unclear whether the observed improvement was primarily due to increased MICP or simply the precipitation of salts from the medium in the presence of xanthan.

To differentiate between these effects, an additional experiment was conducted including a control condition in which only YAC medium supplemented with 1% (w/v) xanthan was added to the sand, without the addition of CGN12 cells. Replicates of the other conditions were also prepared, and the effect of a lower xanthan concentration (0.5% (w/v)) was tested as well. To emphasise the MICP-related effects over salt precipitation, this experiment involved covering the sand bricks with cling film after the medium was added, thereby reducing evaporation. After two days of incubation, the cling film was removed, and 4 mL of fresh YAC medium (without additional xanthan) was added. The bricks were then again covered with cling film for two days before being left to dry completely.

The resulting bricks are shown in Figure 33. Due to the modified incubation protocol, microbial contaminations had time to establish before the medium had dried, as indicated by the orange arrows in Figure 33A. Upon removal of the bricks, visible contaminations were also observed within the bricks themselves, as seen in the red dots in Figure 33B. Despite this, biocementation appeared to have taken place across all conditions.

Unexpectedly, the sand brick generated using only YAC medium supplemented with 1% (w/v) xanthan and no CGN12 cells (condition 1) was among the thickest and most consolidated, along with one of the replicates containing 0.5% (w/v) xanthan and CGN12 cells (condition 3, upper brick on the right). Notably, both of these conditions also showed strong signs of contamination. In contrast, the bricks produced using CGN12 cells but no xanthan (condition 2) were the weakest, with two breaking during removal from the mold and appearing the thinnest overall. The sand bricks produced with CGN12 cells and xanthan (0.5% or 1% (w/v)) were visually similar to each other and displayed greater mechanical integrity compared to those formed without xanthan.

![figure 33](https://static.igem.wiki/teams/5673/src/results/biocementation-of-sand-bricks-with-varying-amounts-of-xanthan.webp)

***Figure 33: Biocementation of sand bricks with varying amounts of xanthan.** The wells measured 4.9 cm in length, 2.6 cm in width and 1.1 cm in depth. Each well was filled with 12.5 g of quartz sand (particle size 0.1–0.5 mm) supplemented with 5 mL of a fresh S. silvestris cell suspension in YAC medium (OD600 = 2.7, 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). After three days, 5 mL of fresh YAC medium (without cells or xanthan) was added. This step was repeated once more after another three days. Samples were then left to dry. To prevent rapid evaporation, cling foil was used to cover the silicon mold during the first two days following each medium addition. All xanthan concentrations shown are (w/v). Images were taken after 12 days of incubation. A) Orange arrows indicate contaminations formed during the biocementation process. B) Bioconsolidated sand bricks after removal from the mold. The arrangement of the bricks is flipped vertically compared to A).*

These results further reinforce the findings of the initial experiment, indicating that xanthan enhances the biocementation of sand bricks in YAC medium at the tested concentrations. Furthermore, they proved the concept of introducing a biopolymer to enhance the mechanical properties of a biocemented sand brick.

Discussion: Xanthan influences durability of bio-consolidated sand bricks

Microbially induced calcite precipitation (MICP) is a promising alternative to cement-based binders for sustainable consolidation of granular materials (Achal & Mukherjee, 2015). However, MICP-treated materials often show limited mechanical performance (Castro-Alonso *et al.*, 2019; Gebru *et al.*, 2021). To address this, recent studies have explored biopolymer additives to improve matrix cohesion, bacterial immobilisation, and precipitation uniformity (Dubey *et al.*, 2022). In this study, xanthan gum was evaluated as a model biopolymer additive in a non-sterile proof-of-concept MICP setup. Xanthan was chosen as a substitute for pyriform silk, which could not yet be obtained in sufficient quantities for testing, but which we ultimately aim to explore for biocementation applications.

Sand bricks formed with xanthan (0.5–1% w/v) were visibly stronger and more cohesive than those without it (Figure 32, Figure 33). Interestingly, even samples treated with 1% xanthan but no bacteria were among the most consolidated. Biopolymers such as xanthan are thought to enhance MICP products by distributing bacteria more evenly, filling interstitial spaces, and providing nucleation sites (Lai *et al.*, 2024; Li *et al.*, 2022; Xu *et al.*, 2019). In this case, however, the observed consolidation may have been largely abiotic, possibly due to salt crystallisation or polymer crosslinking during drying. As this was only a single qualitative experiment, replication and quantitative testing are needed. Still, the results agree with previous studies showing that biopolymers can significantly improve compressive strength and cohesion in MICP-treated soils (Chang *et al.*, 2016; Dubey *et al.*, 2022).

The chemical properties of xanthan provide additional mechanistic insights. Its carboxyl groups interact with divalent cations like Ca2+ (Groves & Chaw, 2015; Maity & Sa, 2014), dynamically altering viscosity (Fjodorova *et al.*, 2022) and forming a gel-like matrix (Figure 34A). Such a matrix increases contact between bacteria, substrates, and soil particles, promoting localized supersaturation (Mendonça et al. 2021). Xanthan gels have also been used for bacterial entrapment in industrial processes (Dzionek *et al.*, 2021) and shown to enhance bacterial adhesion during MICP (Li *et al.*, 2022). Furthermore, calcite precipitation could shift equilibrium to release bound Ca2+, enabling slow ion release and more uniform mineralization. Binding Ca2+ may also buffer against inhibitory concentrations for strain CGN12 (Seidel *et al.*, 2025). These mechanisms support more efficient nucleation and crystal growth (Hoffmann *et al.*, 2021; Lai *et al.*, 2024).

At higher xanthan levels (>1% w/v), however, negative effects are likely. Excessive Ca²⁺ sequestration can reduce free ion availability, while blocked pores may prevent nutrient diffusion and bacterial migration (Lai *et al.*, 2024; Rebata-Landa, 2007). Thus, xanthan’s role appears concentration-dependent, with benefits at moderate levels but inhibition at higher ones (Figure 34B).

![figure 34](https://static.igem.wiki/teams/5673/src/results/schematic-representation-of-xanthan-s-theorized-effects-on-micp-driven-sand-biocementation.webp)

***Figure 34: Schematic representation of xanthan’s theorized effects on MICP-driven sand biocementation** All xanthan concentrations shown are (w/v). Elements are not drawn to scale. A) MICP-driven biocementation of sand improves with the addition of xanthan through the provision of a gel-like matrix. At high xanthan concentrations (>1% (w/v) the beneficial effect of xanthan on MICP is reduced. B) Bacteria may utilize Ca2+ ions close to the xanthan (0.5-1% (w/v) resulting in increased calcite precipitation. At higher concentrations (>1% (w/v) xanthan may block the pores between the sand particles and reduces the free Ca2+ concentration, preventing bacterial migration and a strong uniform MICP.*

The non-sterile experimental setup introduced another layer of complexity. Because MICP is widespread among soil microbes (Lowenstam, 1981), contaminating organisms may have altered calcite precipitation. Still, even the most consolidated bricks showed contamination (Figure 32), suggesting that MICP biocementation is resilient against microbial competition.

The non-sterile experimental setup introduced another layer of complexity. Because MICP is widespread among soil microbes (Lowenstam, 1981), contaminating organisms may have altered calcite precipitation. Still, even the most consolidated bricks showed contamination (Figure 32), suggesting that MICP biocementation is resilient against microbial competition.

While xanthan served here as a stand-in for pyriform silk, parallels with other protein-based polymers are promising. For example, silk fibroins from *Bombyx mori* cross-linked with genipin enhanced both biomineralisation and compressive strength in MICP-treated sand (Li & Achal, 2023). Likewise, spider silk proteins have proven effective substrates for calcite biomineralisation in biomedical studies (Dmitrovic *et al.*, 2016; Kiseleva *et al.*, 2020; Mehta & Hede, 2008). These findings strengthen the rationale for testing pyriform silk once sufficient quantities are available.

Overall, xanthan addition improved sand biocementation and highlighted the importance of biopolymer chemistry in shaping MICP processes. Optimising polymer concentration, ion dynamics, and microbial activity will be crucial to maximise performance. Having established xanthan as a functional stand-in for pyriform silk and demonstrated its potential to enhance MICP-driven biocementation, we next turned to curdlan, a structurally distinct β-(1,3)-glucan. Unlike xanthan, curdlan forms strong thermo-reversible gels and has been proposed as a stabilising matrix in composite materials.

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