Contribution

GenOMe — Our Contribution to the iGEM Community

We’re a group of young scientists who love building things in the lab and exploring new ideas.
When we joined iGEM, we wanted to create something meaningful — something that future teams could actually use and build upon.

That’s how GenOMe began. It’s a next-generation platform that brings BioBrick design from plasmids into the genome, making genome integration faster, more stable, and easier for any team to reproduce. Our wet-lab team developed the GenOMe workflow and created reusable strains and integration cassettes, while our dry-lab team built a predictive modeling software that helps optimize the Two-to-Two recombination process. Together, we turned GenOMe into more than just a project — it’s a foundation that other iGEM teams can use, modify, and expand. We hope it becomes a shared platform where creativity keeps growing, connecting ideas and people across the iGEM community.

GenOMe — the genome LEGO baseplate

We developed GenOMe, a next-generation genome engineering platform that extends the BioBrick modular philosophy from plasmids to chromosomes. By integrating attB docking sites, Bxb1 recombinase, and a cyclic Two-to-Two recombination mechanism, GenOMe enables E. coli to achieve stable, marker-free, single-copy genome integration within 48 hours, achieving an average efficiency of ~80%.

Unlike traditional plasmid systems that require constant antibiotic pressure and suffer from copy-number variation, GenOMe provides a predictable and inheritable genomic framework that can be reused and expanded by any iGEM team. The platform consists of two standardized components:

Cassette BioBrick (Slot Cassette) — a plug-and-play module carrying user-designed DNA fragments flanked by attP sites.
GenOMe Host Strain — an E. coli chassis pre-installed with attB docking sites and expressing Bxb1 integrase.

**Figure 1**. Components of the GenOMe integration platform.

The Cassette BioBrick carries user-designed DNA fragments flanked by Bxb1 attP sites (attPₓ and attPᵧ) and includes standard BioBrick restriction sites (EcoRI, XbaI, SpeI, PstI), maintaining compatibility with iGEM assembly. The GenOMe strain is an engineered E. coli chassis containing chromosomal attB sites (attBₓ and attBᵧ), which serve as genomic docking slots. Together, they form the core of the GenOMe platform, enabling precise, marker-free, and inheritable genome integration through Bxb1-mediated attP–attB recombination.

To insert a target gene, users simply clone their DNA fragment into the Cassette BioBrick and co-electroporate it with the GenOMe strain. Within two days, the desired sequence is stably integrated into the chromosome — a simple, one-step workflow accessible to any iGEM team.

**Figure 2**. Workflow of genome integration using the GenOMe platform.

The GenOMe system allows rapid, marker-free integration of user-designed DNA into the E. coli chromosome. 1. Insert your DNA fragment into the Cassette BioBrick. 2. PCR amplify the insert using VF2/VR primers to generate attP-flanked fragments. 3. Co-electroporate the fragment into the GenOMe host strain containing chromosomal attB sites and Bxb1 integrase. 4. Bxb1 recombinase catalyzes precise attB–attP recombination, permanently integrating the DNA payload into the genome.

Engineering Success

Using a consistent colony-forming-unit–based definition (CFU_antibiotic / CFU_LB × 100%), our GenOMe / Two-to-Two workflow delivered 79% integration efficiency for dual-gene insertion and 80% efficiency for full landing-pad replacement — effectively a near-saturation, single-round outcome.Mechanistically, this performance arises from two coordinated design features:

Two-to-Two design: each Cassette BioBrick carries two attP sites that pair with two attB sites on the chromosome. This synchronized dual-end recombination ensures correct orientation, reduces intermediate loss, and enhances integration precision.
Bxb1-mediated unidirectional recombination: the reaction is RDF-independent, forming irreversible attL/attR junctions that permanently lock the inserted DNA, preventing excision and ensuring long-term stability.

**Figure 3**. The Two-to-Two recombination mechanism of GenOMe.

Two attP sites on the cassette recombine with two attB sites on the chromosome through Bxb1 integrase, precisely integrating the DNA payload and forming stable attL/attR junctions for high-efficiency, marker-free genome editing.

Modeling further guided the optimal attB/P orientation and timing of Bxb1 induction, minimizing off-target or partial recombination events. Together, these principles make GenOMe’s Two-to-Two system a fast, precise, and inherently stable genome-integration platform, advancing synthetic biology beyond the plasmid era.

GenOMe Navigator — Guiding Genome Integration Decisions

**Figure 4**. GenOMe Navigator main interface. Users can choose between Mode A (Initial Integration, site selection) and Mode B (Production Mode) to simulate and predict integration success rates under different experimental parameters.

GenOMe Navigator is a predictive software tool developed to support iGEM teams using the GenOMe system. It transforms a multi-stage modeling framework into an intuitive interface that helps users predict integration success and make data-driven decisions in genome engineering.

Background: Modeling Framework (M1–M5)

This software is built upon a five-stage modeling framework (M1–M5) that connects theoretical simulations with biological experimentation.

M1 established baseline equations describing the rate of attB site formation, defining the theoretical limit for individual recombination events.

M2 introduced stochastic variation to capture colony-to-colony heterogeneity observed in experiments.

**Figure 5**. 1% × 1h vs 4h, showing Bxb1 & SSAP with separate production rates. M3 and M4 modeled coordination between two genomic loci (attB₁ and attB₆), revealing that synchronized recombination markedly enhances efficiency and stability.

**Figure 6**. Locus dependence of attB formation success (Cycle 5).M5 generated a predictive relationship between DNA length, att-site spacing, and integration probability, forming the computational basis for the software interface.

Software Functions

Mode A – Site Selection (DNA Layer):
Predicts integration success based on genomic distance from oriC and GC content, visualized as a heatmap.
This mode identified optimal loci and helped our wet-lab team adjust protein induction time and concentration, turning failed integrations into successful ones.
Mode B – Production Mode (Protein Layer):
For second-round two-to-two integrations, insert fragment length (1–2 kb) was found to be the dominant factor, with ≥ 80 % predicted success, and the Navigator also estimates the number of plates needed to reach a target positive colony count.
Population Estimator (Population Layer): Converts probabilities into expected positive colonies and SOP recommendations, helping teams plan screening effort quantitatively.

Output and Impact

GenOMe Navigator produces clear visual outputs (heatmaps, curves, and lookup tables) that enable researchers to plan experiments rationally.
During our project, its predictions guided wet-lab parameter changes and directly improved integration outcomes.
The software is open source and easily recalibrated for new integrase systems, making it a general decision-support tool for synthetic biology projects.

GenOMe Navigator turns complex models into actionable design rules, empowering future teams to engineer genomes with precision and confidence.

Best New Composite Part — LandingPad_B3B2B5

LandingPad_B3B2B5 is the core composite part of the GenOMe system — a multifunctional DNA module that equips E. coli with modular, marker-free, and reusable genome integration capability.
This part establishes a reusable genomic docking platform, the first of its kind under iGEM BioBrick standards, enabling multi-round, site-specific integrations directly within the chromosome.
It has been experimentally characterized and officially submitted to the iGEM Registry under the part number BBa_25XNR8U7C

Design Concept

The LandingPad_B3B2B5 fragment is a compact, self-contained DNA construct carrying attP and attB sites, a kanamycin resistance marker (KanR), and the bxb1 integrase gene. These components together enable autonomous recombination, modular expansion, and marker replacement, forming the genomic foundation of the GenOMe system.

**Figure 7**. Structure of LandingPad_B3B2B5. The Landing Pad contains attP1 and attP6 for recombination with chromosomal attB1/attB6, additional attB3, attB2, and attB5 sites reserved for future integrations, a kanamycin resistance marker (KanR) for selection, and the Bxb1 integrase gene for autonomous recombination. PCR primers VF2 and VR are positioned for amplification of the entire fragment.

Best Part Collection — GenOMe Parts Collection

The GenOMe Parts Collection is a modular and expandable toolkit that brings the BioBrick concept from plasmids into the chromosome. It enables stable, inheritable, and marker-reversible genome integration under the Two-to-Two recombination system, creating a closed-loop workflow from attB site installation to multi-round expansion. For iGEM teams, it provides a ready-to-use foundation to directly move their designs into the genome with precision and reproducibility.

Composition and Categories

Category 1. Genome Integration Core

Establishes the molecular foundation of GenOMe with orthogonal attB/attP pairs (1–6) and Bxb1 integrase. Each attB–attP pair functions independently, ensuring unidirectional, stable recombination without cross-talk. This provides predictable performance for multi-site genome editing.

**Figure 8**. Orthogonal Bxb1 recombination system for one-way, multi-locus integration.

Category 2. Integration Platform

Implements the integration workflow using TargetingOligo_B1_B6 (ssDNA template for attB1/attB6 installation) and LandingPad_B3B2B5, the first functional genomic slot that allows expansion via embedded attB3/B2/B5 sites.

**Figure 9**. Figure P2 | Installation of attB sites and Landing Pad integration into the E. coli genome.

Category 3. Cyclic Integration and Expansion System

Alternating Slot A (GenR) and Slot B (KanR) cassettes enable continuous, marker-free rounds of integration. Each cycle removes the previous selection marker and generates new att sites, allowing stepwise genome assembly.

**Figure 10**. Cyclic integration workflow enabling iterative and marker-free genome expansion.

Construction and Validation

LandingPad_B3B2B5 was integrated into the E. coli genome through the ssDNA-guided Two-to-Two recombination system. The 150-nt ssDNA carrying attB1/attB6 contained homology arms that guided precise Bxb1-mediated integration at the designed chromosomal locus. Functional validation confirmed correct integration by antibiotic selection, blue-white screening, and junction PCR, yielding the expected ~741 bp product and verifying precise chromosomal insertion.

By designing a synthetic ssDNA with sequence homology to the target locus, we can guide the Landing Pad to precisely integrate into the designated site on the genome. The ssDNA acts as a “commander,” directing the Landing Pad’s landing process for accurate chromosomal embedding.

Performance and Application

The Landing Pad enables efficient and precise genome editing through the Bxb1 recombination system. It supports antibiotic marker replacement and iterative integrations via its embedded attB3/B2/B5 docking sites, allowing continuous expansion without plasmid dependence. These results demonstrate that GenOMe achieves clean and accurate genome replacement, leaving only two 43 bp att scars flanking the inserted DNA.

Impact and Community Contribution

LandingPad_B3B2B5 embodies the vision of the GenOMe project — turning genome integration into a standardized, reusable, and scalable process. By embedding new integration sites directly within the genome, this part provides a sustainable framework for iterative synthetic biology design. It extends the BioBrick concept beyond plasmids, allowing future iGEM teams to construct stable, inheritable systems at the chromosomal level. By sharing this composite part, we aim to provide the iGEM community with a universal genomic platform that supports creativity, precision, and long-term collaboration in synthetic biology.

What We Provide

GenOMe offers the iGEM community an open, reusable, and expandable platform for genome engineering — not just a single part, but a complete system connecting design, experiment, and modeling in one framework.

Chassis Strains

E. coli MG1655 strains pre-installed with attB1 / attB6 docking sites form the genomic foundation for integration. These validated chassis strains are currently under deposit application at the Food Industry Research and Development Institute (FIRDI, Taiwan). Once approved, they will be distributed under a Research Use Only (RUO) license. Until then, teams may contact us directly for academic access.
GenOMe Handbook & Protocols

A practical and user-friendly guide containing detailed experimental protocols, troubleshooting tips, and modeling notes. It enables teams to reproduce and extend the GenOMe workflow — from ssDNA installation to Landing Pad integration — with minimal barriers. Together, these resources form an accessible foundation that allows future iGEM teams to transform genome engineering into a reproducible, open, and collaborative process.
Standardized Parts & Modeling Toolkit
- LandingPad_B3B2B5 (BBa_25XNR8U7C): Core composite part for chromosomal docking and integration.
- TargetingOligo_B1_B6: ssDNA template for attB installation.
- Cassette BioBrick Series: Standardized vectors and PCR templates for modular DNA insertion.
- Modeling Toolkit: Five-layer simulation (M1–M5) predicting integration efficiency based on recombination parameters (Bxb1, SSAP, att-site distance, and timing).
Educational & Collaborative Resources

Open-source illustrations, integration animations, and workshop materials designed for high school and undergraduate education. These materials are freely shared for outreach and training across the synthetic biology community.

Licenses

CC BY 4.0 – for text, figures, and educational materials.
MIT License – for modeling software and scripts.

All GenOMe resources are open-access and documented in the iGEM Registry and on our official Wiki. These open licenses ensure that every iGEM team can freely reuse, modify, and expand the GenOMe system — promoting transparency, collaboration, and the continued growth of open synthetic biology.

Integrated Human Practices

HIA experiment 1 — figure 11. collaberation with HIA.

HIA experiment 2 — figure 11. collaberation with HIA.

Our project, GenOMe, was built through close collaboration with experts in both wet and dry lab fields.
We sincerely thank Dr. Yinling Chiang (NYCU), Prof. Paul Lin (NYCU), Prof. I-Son Ng (NCKU), and Prof. Tzong-Yi Lee (NYCU) for their invaluable guidance.

Dr. Chiang reminded us of the issue of antibiotic accumulation and inspired us to refine our genomic design for a cleaner and safer system.
Prof. Lin guided us to focus on the large-scale integration feature of GenOMe, which later became our major advantage.
Prof. Ng helped us rethink the role of antibodies and suggested our antibody-replacement mechanism, ensuring both stability and flexibility.
Prof. Lee supported our modeling by identifying an additional variable (p3) in the Two-to-Two equation, improving our model’s predictive accuracy.

Their advice not only shaped the scientific framework of GenOMe but also strengthened our ability to connect theory and experiment. We deeply appreciate their mentorship, which made our contribution more complete and meaningful for future iGEM teams.

Education & Public Engagement

University Outreach:
We joined the admission interview day at National Yang Ming Chiao Tung University (NYCU DBT) to introduce iGEM and synthetic biology to high school students. After our explanation, survey results showed a significant improvement in understanding (p < 0.001).
Collaboration with HIA-Taiwan:
We worked closely with Team HIA-Taiwan for a month, sharing experimental experience, providing sequence design guidance, and recording a joint podcast to summarize our learning journey.
STAR Kindergarten Program:
Together with HIA-Taiwan, we designed a storytelling and interactive game activity to teach young children about the benefits and risks of bacteria.

We sincerely thank NYCU DBT for giving us the chance to inspire future scientists, and HIA-Taiwan for their collaboration and creativity. Through these experiences, we not only shared knowledge but also learned the power of curiosity and imagination from every age group.

Looking Forward

GenOMe was created with one simple goal — to stay alive within the iGEM community.
We want it to be a platform that anyone can easily use, expand, and improve.
Our hope is that future iGEM teams will keep building on this foundation, making genome engineering more collaborative, more standardized, and easier for everyone to explore.

To make this possible, we’ve made every part of GenOMe — the Cassette BioBrick, the host strain, and the modeling toolkit — open and well documented.
The GenOMe chassis strain is currently under deposition at the Food Industry Research and Development Institute (FIRDI), Taiwan, and will be available under Research Use Only (RUO)access.This ensures that future teams can safely access, reproduce, and extend our work.

Through standardized BioBrick assembly logic and a modular Two-to-Two integration system,
GenOMe provides a reusable, stable, and flexible platform for genome design.
Users simply insert their target DNA into the Cassette BioBrick, amplify it by PCR, and co-electroporate it with the GenOMe host strain, achieving precise, marker-free chromosomal integration within 48 hours.
This transforms genome engineering from a multi-step, expert-only procedure into a hands-on, reproducible, and shareable tool for every iGEM team.

We look forward to seeing future iGEM teams expand GenOMe — through new att-site designs, improved workflows, or applications in new organisms. Together, we can keep GenOMe growing as an open, evolving platform for everyone in synthetic biology.