Contribution | JGU-Mainz

Introduction

Welcome to our Contributions page, where we showcase the innovative tools, methodologies, and educational resources developed during this year’s iGEM competition. Among our key achievements is a standardized Bacillus subtilis parts collection containing multiple regulatory elements - promoters, RBS variants, and terminators. Each element was characterized with sfGFP in both plasmid and genome-integration contexts to provide reliable data for gene expression optimization in B. subtilis. All DNA in this collection has been formatted according to the RFC1000 standard, as our goal was to produce components that are immediately usable by future teams and easy to expand.

We also present the first parts collection for a newly established spider silk type in the iGEM Registry - pyriform silk. This library includes multiple preassembled synthetic pyriform silk genes and basic parts for rapid redesign, allowing teams to tailor constructs to their specific needs, including the option to test different production hosts. In addition, we developed a novel cloning strategy called Pyricloning, which enables efficient and stable assembly of highly repetitive DNA sequences to assemble these new constructs.

Beyond our experimental work, we created several educational and public engagement resources that we are excited to share with the iGEM community for future use and development. Building on the work of previous iGEM teams (iGEM Lund 2022, iGEM GO Paris-Saclay 2022), we expanded the Synthetic Biology Dictionary, a resource designed to support newcomers and students by explaining key terms, concepts, and laboratory techniques in clear, accessible language. We also created an HTML & CSS cheat sheet for teams that find wiki construction challenging. Finally, we compiled our presentation materials from school visits and workshops, featuring schematics and animations, all available for future teams to adapt and use.

Parts Collection: Regulatory elements for Bacillus subtilis in the RFC1000 framework

At the core of our regulatory parts collection for B. subtilis are the genetic regulatory elements we characterized in various combinations for B. subtilis W168: the promoters PxylA, PliaI, PhpaII, and PbceA; the RBS variants st7, st11, st4, and wk8; and the synthetic terminator L3S1P47. Each regulatory element was tested through genomic integration at the amyE locus to ensure consistent, single-copy expression. In addition, a subset of these parts was also cloned and characterized in a plasmid backbone (our RFC1000-compatible derivative of pBSMuL1) to allow comparison of gene expression between genomic and multicopy plasmid contexts. This enables future users to evaluate copy-number effects when data are available and select the setup best suited to their project.

To facilitate adoption, every regulatory element is provided with an RFC1000-formatted sequence and a functional description. Experimental data can be found in our Results section, and all protocols are available in our Notebooks. Practical notes are included for each promoter: PxylA for xylose-titratable expression, PhpaII as a strong constitutive option, and PliaI and PbceA for bacitracin-responsive regulation. The terminator L3S1P47 serves as a reliable insulation element for multi-gene designs.

Section1-contribution1 — **Figure 1: 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 PxylA, PbceA, PliaI or PhpaII, respectively. C) Set of level 0 parts for the assembly of reporter constructs in evaluating translational efficiency of different RBSs. The promoter PliaI, 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-(PhpaII, 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*

**Figure 2: The *liaI* promoter is the strongest among the promoters tested.** The promoter strength of the inducible promoters PxylA, PbceA and PliaI as well as the constitutive promotor PhpaII 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.

This toolbox provides a straightforward and effective method for assessing optimal gene expression levels in B. subtilis strains, for example, in our case, identifying the ideal expression strength for synthetic pyriform silk production.

Parts Collection: Modular Pyriform Silk Library

Complementing our regulatory toolbox, we developed a modular pyriform silk library designed for rapid combinatorial testing. To maximize flexibility, we introduced an additional hierarchy level below the RFC1000 level 0, enabling the design of diverse silk coding sequence (CDS) constructs from basic domain sequences - an ideal setup for exploring how sequence changes influence biomaterial properties, particularly for protein-based materials like spider silk.

The silk library is composed of repeat modules (monomeric, dimeric, trimeric, tetrameric, hexameric, and octameric), which can be expanded using our Pyricloning strategy, and terminal modules that allow users to control tagging and secretion directly. Specifically, we include two N-terminal modules (with and without a start codon) and two C-terminal modules (with and without a stop codon), enabling either standalone expression or in-frame fusions to N- or C-terminal tags.

Functional tags provided in the collection include a pep86 tag for split NanoLuc assays (allowing easy and sensitive detection of secreted spidroins), a His₆ tag for IMAC purification and Western blot detection, and a Strep-II–SUMO fusion downstream of the ssyoaW signal sequence to facilitate secretion and affinity purification. We also include the alternative secretion signal sslipA for teams that prefer different secretion kinetics or require an alternative if ssyoaW is not optimal. All silk modules and tags are compatible across the various RFC1000 framework levels.

section2-contribution — **Figure 3: *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.

This toolbox is designed to be easily expandable for teams interested not only in testing pyriform silk constructs but also in creating chimeric silk genes, for example, by combining domains from multiple silk genes into a single CDS to screen for novel silk types with potentially improved mechanical properties.

Pyricloning Strategy

Highly repetitive sequences pose significant challenges to synthetic biologists due to the technical difficulties they introduce. Traditional synthesis and cloning approaches often fail to handle iterative assemblies and flexible frameworks, frequently leading to recombination or slippage events that result in incorrect constructs.

To overcome this, we developed Pyricloning, a modular, stepwise assembly strategy that separates repeat unit construction from the formation of complete CDS or transcription units. This approach combines elements from the RFC25 standard for iterative repeat assembly with the RFC1000 standard for creating full CDSs using the terminal elements from our parts collection.

Pyricloning is not limited to our project, it is fully transferable to other repetitive sequence designs, providing the synthetic biology community with a new, flexible method for assembling repetitive sequences without compromising accuracy or modularity. Our wiki features detailed diagrams and step-by-step protocols for Pyricloning to help future teams easily reproduce or adapt the method for their own applications.

Educational Resources and Tools

Alongside our experimental contributions, we developed several educational resources to support future iGEM teams and newcomers to synthetic biology.

Our Synthetic Biology Dictionary (2nd Edition) provides concise explanations of essential concepts, methods, and terminology commonly encountered in iGEM projects—from basic terms like promoter and ribosome binding site to more advanced topics such as DARPins and strain engineering.

To complement this, we created an HTML & CSS Cheat Sheet - a concise yet comprehensive reference summarizing the most useful HTML tags, CSS properties, and layout principles we learned during our wiki-building process.

We encourage future teams to expand these resources and contributed public engagement materials (infographics, posters, workshop handouts) to promote a broader understanding of synthetic biology.

Summary of Contributions

In summary, our contributions include:
(1) a standardized, RFC1000-formatted regulatory toolkit for B. subtilis characterized with sfGFP in plasmid and genomic contexts; (2) a modular pyriform silk library with repeat-, tag-, and secretion-compatibility; (3) the Pyricloning strategy for robust assembly of repetitive sequences; and (4) educational resources, including a Synthetic Biology Dictionary, a wiki development cheat sheet, and public engagement materials, designed to foster knowledge sharing and community growth within iGEM.