We developed a compact, rapid, and orthogonal phosphorylation-based processing unit composed of modular composite parts: synKinase (ABL-bZipRR), synPhosphatase (PTPN1-bZipRR), the synSubstrate (CD3ζ-bZipRR), and a high-affinity SH2 synPhosphobinder. By using complementary ZipEE/ZipRR coiled-coils for proximity control, this collection implements fast phosphorylation and dephosphorylation logic. When kinase and substrate are brought into proximity, CD3ζ is phosphorylated. SH2 then engages phosphorylated synSubstrate to trigger downstream effector functions, such as gene expression and protein secretion. We also introduced a novel GPCR-PAGER sensing architecture that couples small molecule sensing with soluble protein target detection by an extracellular-fused nanobody. This is further fused to intracellular ABL activity via β-arrestin–ZipEE, enabling AND-style sensing of multiple ligand types and can be readily integrated into our signaling circuit.

Our phosphorylation-based circuits are orders of magnitude faster than transcriptional circuits and are straightforward to reconfigure. Other teams can fuse our kinase/phosphatase modules to alternative synthetic receptors or sensors to compute pairs of external signals. There is also the possibility to swap nanobodies to detect new ligands, or connect SH2:CD3ζ binding to split effectors delivered with our toolbox, such as splitTEV proteases or split transcription activators for gene expression and protein secretion. The framework we provide is a ready scaffold to build rapid cellular logic for applications from tumor-specific targeting in oncology to biosensing in clinical diagnostics, bioremediation and synthetic plant biology, enabling iGEM teams to build upon our parts, replace sensing domains, and scale toward new clinical or environmental use cases.

Furthermore, we tested and documented a recently published assay for the rapid and cost-efficient screening of protein binders (Capin et al., 2025). Based on cell-free expression, the two-hybrid assay avoids the need for cloning or cell transformation and requires only standard laboratory equipment. This allows future iGEM teams to easily validate in-sillico generated protein binders, with minimal time and effort before turning to more laborious methods. In combination with our dry lab model SPARC we hope this toolkit will enable the iGEM community to screen any binder-target complex in a high-throughput and reliable manner.

CF2H Assay Protocol - Click to Expand!

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With our dry lab efforts, we built a single, integrated computational framework that propels binder and circuit design from idea to validated candidates, dramatically accelerating the process while lowering the cost of experimental work. The pipeline starts with BindCraft, a deep-learning de novo binder design tool we adapted specifically to produce protein binders that are compatible with our GPCR and MESA sensing architectures. Top BindCraft designs can then be rapidly characterized using an automated MD-based prescreening workflow that ranks candidates within days and thereby cuts down on the need for expensive, high-throughput experimental screens.

To give mechanistic insight and predictive power, we developed coarse-grained membrane simulations to probe receptor dimerization dynamics and a full mathematical model of the phosphorylation circuit. This model is at the core of SPARC, our digital-twin environment, which includes an ultrasensitivity-based cell-optimization tool, enabling users to design synthetic cells computationally and explore parameter space before touching the bench. Our complete model is easily expandable, well-documented and complemented with a detailed SPARC user manual. This consists of example use cases so other iGEM teams can adapt BindCraft to new targets, reuse the MD prescreening for their candidates, apply membrane simulations to receptor studies, or employ our mathematical model to optimize phosphorylation-based circuits. Together these tools turn design, in silico testing, and subsequent experimental validation into a fast, accessible pipeline that other teams can pick up and build on.

SPARC User Manual - Click to Expand!

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We created two practical, shareable resources to strengthen science translation and synthetic biology education:

Regional Roadmap to Scientific Translation & Communication:
This is a compact guide of the regional innovation ecosystem, consisting of accelerators, contacts, prerequisites, and interviews with local founders and scientists. Students often don’t know such support networks exist, which is why this roadmap equips young scientists with the concrete tools and role models needed to build a supportive network, learn about entrepreneurship early in their academic journey, and turn their innovative ideas into impact. Although tailored to Heidelberg, its structure and templates are easy to adapt, so other iGEM teams can quickly build comparable regional guides for their own ecosystems.

Find our whole Regional Translational Roadmap here!

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Learning Platform & Summer School materials:
We created an interactive educational portal, consisting of lab protocols, calculators, module quizzes augmented with instructional videos that teach core lab techniques. The learning platform lowers technical barriers and supports scalable outreach and training, which can be suited to any curriculum and level of knowledge. The content was iteractively improved with teacher and student feedback and is ready for reuse and expansion by other iGEM teams and educators.