We provide a set of clear and easy-to-follow protocols for RPA, DNA hybridization, and LbCas12a purification, as well as assays. These methods are becoming increasingly more important in synthetic biology, yet they are still less standardized than techniques such as PCR or cloning. Most of the information we found was either too broad or spread out across different papers, so we put it together into clear workflows that other iGEM teams can follow directly.
Our RPA protocols offer detailed guidance on setting up reactions with the correct reagent ratios and incubation conditions for both symmetrical and asymmetrical RPA, including protocols adapted for lower incubation temperatures. Symmetrical RPA forms the foundation for successful LbCas12a assays, while asymmetrical RPA is important for DNA hybridization experiments.
Our DNA hybridization protocols describe the conditions where probes bind reliably, including practical notes on salt concentration, temperature, and incubation time. We wrote step-by-step instructions for single-probe hybridization on PCR products as well as on products from symmetrical and asymmetrical RPA. We also made a protocol for double DNA hybridization with two different fluorophores, so that two probes can bind at the same time. To make sure the probes are specific, we included strategies to test against non-target pathogens. In addition, we set up a protocol for DNA hybridization on a lateral flow strip with two probes.
Our protocols for LbCas12a protein purification offer a clear starting point for teams interested in purifying proteins themselves. They cover the full process, from cell transformation and sequencing preparation to nickel affinity purification. Each step is documented in detail to ensure reproducibility and make the workflow accessible even to teams with limited experience in protein work.
Our LbCas12a assay protocols demonstrate how to set up and optimize a cleavage assay using quenched fluorescent reporters. They include instructions on preparing reactions, adjusting ssDNA reporter concentrations, and achieving signal outputs that are both visually detectable and quantifiable with a fluorescence reader. These protocols are designed to be followed step by step, but they are also flexible enough to be adapted to different DNA targets or detection platforms by future teams.
Have a look at our Protocols.
Other teams can use these protocols to repeat our results or adapt them for their own targets and setups.
In addition to protocols, we share a collection of primers designed and validated for both PCR and RPA. Designing primers can be time-consuming, since they require the right balance of melting temperature, GC content, and compatibility with the chosen amplification method. Check out our primer collection.
Our dry lab work was designed not just to support our own project, but to create a powerful, open-source Modeling Toolkit for future iGEM teams to accelerate, de-risk, and contextualize their own diagnostic projects. We are proud to contribute each of our models as distinct frameworks, which are available in our repository.
We provide the complete code for our ODE-based kinetic model, which simulates a coupled RPA-CRISPR/Cas12a system. A key feature of this contribution is that its core RPA amplification module has been experimentally validated against wet lab data. This provides a crucial anchor of confidence in the model's foundational predictions. The model's demonstrated ability to predict complex negative interactions, such as the "cis-cleavage deadlock" we identified, makes it a powerful tool for other teams to de-risk their own novel diagnostic designs in silico before committing to expensive and time-consuming lab work.
Furthermore, our documentation provides a valuable case study on navigating parameter uncertainty, from using sensitivity analysis for proprietary "black box" kits to adapting with literature data when experiments are inconclusive.
We contribute a complete, integrated framework for forecasting the real-world performance and shelf-life of a diagnostic. This includes our Enzyme Stability Model, a ready-to-use tool based on the Arrhenius equation. We transparently document our pragmatic approach to parameterization in the face of data scarcity, providing a methodology for using size-matched proxy proteins that other teams can adopt.
The most powerful aspect of this contribution is the integration of the stability model with the kinetic model. This creates an end-to-end simulation that produces outputs like a time-to-threshold heatmap, providing a direct, quantitative link between storage conditions and diagnostic performance. This is an invaluable tool for any team interested in the practical, logistical challenges of developing a deployable product.
While our model is specifically parameterized for STI transmission, its true value for future iGEM teams lies in its modular code and its implementation of advanced epidemiological concepts. We provide a well-documented framework that includes several powerful, reusable features:
Future teams can adapt this framework to other diseases by replacing the STI-specific transmission module with their own mechanics while reusing the core engine for population dynamics, network formation, and behavioral responses. This makes our work a valuable starting point for any team looking to build a rich, agent-based simulation to justify their project's real-world relevance.
We have documented the codes for our wiki as clearly as possible, to provide helpful guidance for future teams. Especially features such as our HP timeline could be of interest to future iGEM teams. The code is available on the iGEM gitlab repository.
To support future iGEM teams in structuring their Human Practices work, we created practical templates and tools that make the process more systematic and transparent. These resources help teams move beyond ad hoc conversations by documenting their reasoning, planning, and follow-up in a structured way. Our contributions include:
We believe these tools will help future teams by making their Human Practices process more rigorous, transparent, and easier to share with both judges and the wider iGEM community.
To support future educational efforts, all of the educational material is available on our Education page. This contains a full PCR and gel electrophoresis workshop as well as school presentations following the goal of introducing synthetic biology at different educational levels.
Have a look at the results of our survey.