Key Achievements

Through our work, we developed a comprehensive PySp1 Part Collection and associated tools that enable the modular engineering and expression of pyriform silk genes in Bacillus subtilis.

• Implementation of a regulatory toolbox for B. subtilis in the RFC1000 standard and their characterisation with sfgfp.
• Construction of Araneus ventricosus pysp1 repeat elements, ranging from one to eight repeats
• Implementation of two N- and C- terminal pysp1 elements enabling flexible in-frame fusions of tags
• Assembly of multiple CDS variants, both tagged and untagged, incorporating different combinations of repeat modules.
• Creation of pep86-tagged constructs compatible with the split NanoLuc assay for extracellular detection and secretion studies.
• Development of diverse transcription units with a range of promoters and regulatory elements for expression tuning
• With this standardised and expandable part collection, we introduce pyriform silk, a novel spider silk type, to the iGEM community, paving the way for its exploration as a next-generation biomaterial.

Introduction

The PySp1 Part Collection provides a modular, standardised platform for the engineering of synthetic pyriform silk genes. Pyriform silk is a highly specialised adhesive silk produced by orb-weaving spiders, and its underlying genes (pysp1 and pysp2) are among the largest and most repetitive protein-coding sequences known. Despite their biotechnological potential, only two complete pysp1 coding sequences have been annotated to date, from A. ventricosus (MH376748, Wang et al., 2019) and Argiope argentata (KY398016.1, Chaw et al., 2017).

Because of their extreme length (up to 17.3 kb) and repetitive domain structure, pysp1 genes are notoriously difficult to synthesise and clone. Traditional approaches fail due to instability during amplification and the high cost of synthesis, limiting progress in the synthetic biology community.

Design and Strategy

To overcome these challenges, we designed a hierarchical assembly system that enables efficient construction of pysp1 genes in modular fashion. The native gene sequence was analysed, codon-harmonised for bacterial expression and systematically divided into three Level -1 modules, each representing key repetitive and non-repetitive regions.

These Level -1 parts can be iteratively combined to generate Level 0 constructs of increasing repeat numbers, all fully compatible with the RFC[1000] Modular Cloning (MoClo) standard. This design allows flexible and rapid assembly of transcriptional units and easy adaptation to different host systems.

Our assembly workflow preserves compatibility with standard Type IIS restriction cloning and supports one-pot MoClo reactions, ensuring seamless build-up of highly repetitive sequences that are otherwise challenging to manipulate.

Our Part Categories:

At the core of the PySp1 system lies a four-tiered modular hierarchy:

Disclaimer: Some parts are technically a composite part, however the implementation and annotation via the composite part option did not work as the sequences changed. Therefore we submitted these part as basic part and will change this as soon as possible.

Application and Reusability

While the system was designed with B. subtilis in mind, both the Level -1 and Level 0 modules are host-independent and can be repurposed for transcription unit assembly in other chassis. Maintaining the RFC[1000] standard ensures that all parts can be easily integrated into existing iGEM workflows and plasmid backbones.

Through this effort, we provide the community with a fully RFC[1000]-compatible toolkit for the modular engineering of synthetic pyriform silk genes. These parts form a foundation for further exploration of spider silk design, heterologous expression, and materials innovation.

Modified MoClo Entry Vectors

To further streamline the assembly process, we developed a set of custom MoClo entry vectors that simplify screening and verification of correct transformants aligning with the standard entry vectors of RFC1000. These plasmids are derived from standard RFC[1000] backbones but include an mrfp1 reporter cassette lacking lacO sequences for easy visual identification of successful cloning events.

Our toolkit includes:

• pRPAV: a dedicated Level -1 entry vector optimised for the assembly of base modules.
• pMMS0: derived from pICH41308-mrfp1-ΔlacO, serving as the Level 0 assembly vector.
• pMMS1: derived from pICH47742-mrfp1-ΔlacO, functioning as the Level 1 expression vector.
• pMMSM1: based on pAGM8301-mrfp1-ΔlacO, acting as the Level M/ 2 assembly vector.
• pLIMO1: pBSMuL1-like level 1 vector for multi-copy plasmid approaches in B. subtilis

The inclusion of the mrfp1 drop-out marker enables rapid and reliable red/white screening of correct constructs at every assembly level, significantly reducing time spent on verification and improving overall cloning efficiency.