Contribution
Practical lessons, parts, and methods we’re sharing with the iGEM community.
Our experience with the Asimov Mammalian Part Collection
During our project, we gained hands-on experience using the Asimov Mammalian Part Collection to design and assemble multi-unit genetic constructs. We found that while this library offers great flexibility and modularity, the repetitiveness of certain elements-such as promoters, UTRs, and poly(A) signals-introduces practical challenges that are not well underlined within the iGEM community.
Our contribution is to share this experience so that future teams can anticipate and address these issues early in their design process. Repetition of identical sequences across transcriptional units can complicate cloning and verification steps:
- Primer design becomes more difficult, leading to nonspecific amplification or mixed sequencing reads.
- Repetitive regions can cause incorrect recombination events during Gibson or other overlap-based assembly methods.
In addition, we reached out to both iGEM Headquarters and the Asimov team regarding several inconsistencies we identified in the distribution kit (see: Results → Distribution kit). By sharing these observations and recommendations, we hope to make the use of the Asimov collection more transparent and reliable for future iGEM teams.
Software
Our Software - built for iGEM teams
One-liner use: point the tool to your tumor/normal reads (or a curated targets.fasta), run once, and get
ranked, frame-correct eukaryotic toehold switches ready to clone and test - simple and reproducible.
The program streamlines the path from data to designs: it performs differential expression to find tumor-only transcripts, proposes overlapping k-mer triggers, assembles full eukaryotic toeholds with Kozak/AUG/linkers, and then optimizes and ranks candidates using structure/interaction scoring (ViennaRNA toolchain).
Why it supports safety & reliability: fewer subjective choices and fewer brittle, hand-picked targets improve provenance, auditing, and predictability in the wet lab. The pipeline caches references, enforces in-frame constructs, flags problematic motifs, and supports multi-trigger OR designs to reduce common failure modes.
What’s new here: an end-to-end flow (DE → design → export) tailored to eukaryotic toeholds, deterministic runs with sensible defaults, and two interfaces - a CLI for automation and a lightweight GUI for quick iteration and teaching. Outputs include clean FASTA/CSV/JSON plus optional structure previews.
More info & how-to: setup, examples, and instructions are in the Software tab.
Parts
As a part of our Contributions, we proudly present a well-curated set of biological parts, each characterized and documented, that significantly enhance the synthetic biology toolbox for future iGEM teams. You can find them in our Parts tab and in the iGEM Registry of Standard Biological Parts.
Functional elements
Our functional elements form a versatile foundation for mammalian expression systems. They include standardized reporter constructs, transcriptional units, and assembly modules optimized for compatibility and ease of cloning. These elements can be readily reused by future teams to build new expression cassettes, validate regulatory sequences, or integrate alternative coding regions.
Because they are designed for flexibility and common cloning methods, they serve as universal building blocks for constructing and testing novel genetic circuits in a variety of contexts.
Toehold switch library
We developed and characterized a set of AFP-sensing toehold switches that respond specifically to alpha-fetoprotein (AFP) mRNA - a biomarker for hepatocellular carcinoma (HCC). These switches can serve as a model for programmable RNA regulation, or a starting point for diagnostic designs targeting other transcripts.
Because trigger/switch sequences are easily adapted, the collection offers a modular framework that teams can modify and repurpose for biosensing or gene-control applications.
qPCR & cloning primers
We contribute a set of qPCR primers and cloning primers that, while simple, provide essential support for molecular validation and construct assembly. The qPCR primers target AFP, GSDMD, and reference genes such as GAPDH, enabling accurate quantification in toehold activation or general expression analysis.
The cloning primers, designed for colony PCR and sequencing verification, streamline the workflow and save time during construct validation.
More info: Parts tab.
Cell cultures
We worked with several mammalian cell lines - HEK293T, WI-38, A549, and HepG2 - for which protocols are not always standardized in the iGEM context. While HEK293T is relatively common, use of WI-38 (fibroblast), A549 (lung epithelial cancer), and HepG2 (hepatocellular carcinoma) is less frequent.
Our contribution is a comprehensive cell culture protocol (see: Experiments) covering passaging, transfection, seeding, and maintenance, adapted for these lines. By documenting these procedures, we aim to reduce trial-and-error for future teams working with non-canonical cell lines, saving time and improving reproducibility.
More info & experiments: Experiments tab.
Safety & Security
Another, yet extremely important in synthetic biology is the matter of safety and security. Hazard analysis is marked as one of critical steps in the design process of any synthetic biology product or project. Despite undeniable benefits coming from its inventions, the ability to engineer new forms of life may pose potential risks to societies and ecosystems, and is characterized by a high degree of uncertainty. Due to this fact the identification of threats and mitigation of risks is both a necessary and challenging activity.
We would like to share our experience gained during performance of System Theoretic Process Analysis (STPA) - an iterative hazard analysis tool developed by Massachusetts Institute of Technology professor Nancy Leveson. STPA is different from most techniques based on linear, chain-of-events accident causation models, which renders them ill-suited to be employed in the analysis of complex biological systems. However due to the uniqueness and novelty of the method, there are only a few sources that describe the technique. Therefore we prepared a simple guide (you can download it in the Safety and Security tab) as a complementary source for performing STPA by the future IGEM teams. It is broadened with examples more connected to the biological field and student life in hope that this will help with easier understanding of the method. Although STPA originally consists of three steps, we divided them into eight smaller steps to prevent overwhelm and confusion, due to the high complexity of the original three steps.
Due to limited, non-biological examples in available resources, we prepared a concise guide (see: Safety & Security) tailored to iGEM needs - with biology-adjacent examples and an 8-step breakdown of the original 3 steps to reduce cognitive load.
More info: Safety and security tab.