Description

GenOMe: the genome LEGO baseplate for iGEM teams.

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A next-generation platform that turns BioBricks into stable, inheritable genomic arrays with ~80% integration efficiency in just two days, making genome editing as intuitive as building with LEGO bricks.

BioBrick fragments can be integrated into the E. coli chromosome within 2 days, achieving an integration efficiency of ~80%, demonstrating the speed and reliability of the GenOMe platform.

Why We Built GenOMe

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In iGEM, most gene circuits are built on plasmids — convenient for testing, but not for scaling. Plasmids suffer from unstable copy numbers, expression drift, and metabolic burden[1], making it difficult to obtain reliable data and accurate parameters for modeling.

Moving circuits into the chromosome removes the need for antibiotics and provides single-copy, stable expression, offering a reliable baseline for quantitative studies and predictive modeling. Such stability making chromosomal integration well suited for industrial production, medical applications, and long-term research designs where consistent performance is essential[2].

However, traditional methods like CRISPR/Cas9 or λ-Red are slow, expensive, and inefficient — large insertions (>10 kb) may take weeks, require costly DNA synthesis, and need extensive screening[3].

That’s why we developed GenOMe — a plasmid-free, modular integration platform that transforms genome editing into a fast, reliable, and plug-and-play process.

How GenOMe Works

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GenOMe consists of two core components:

• Cassette BioBrick (Slot_cassette) – a plug-and-play vector carrying user payloads flanked by attP sites for genome integration.
• GenOMe Host Strain – an engineered E. coli containing attB docking sites and chromosomally expressed Bxb1 integrase.

The GenOMe workflow is simple and standardized:
Users load their DNA payload into the Cassette BioBrick, amplify it with VF2/VR primers, and obtain an attP-flanked fragment ready for integration.
The purified PCR product is then introduced into the GenOMe chassis by electroporation. Inside the cell, chromosomally expressed Bxb1 integrase catalyzes site-specific recombination between attP and attB sites, precisely locking the payload into the genome.

Within two days, the DNA fragment is cleanly and stably integrated, leaving only minimal att scars — producing a plasmid-free, inheritable genome ready for further design cycles.

The DNA payload is inserted into the Cassette BioBrick, amplified with VF2/VR primers, and electroporated into the GenOMe Host Strain. Dark-colored attB sites (chromosomal) recombine with light-colored attP sites (cassette), showing precise and efficient Bxb1-mediated genome integration — stable, marker-free, and completed within two days.

*Note: After XbaI/SpeI digestion, treating the cassette vector with calf intestinal phosphatase (CIP) helps prevent self-ligation and improves cloning efficiency, especially for large inserts.

Fast and Efficient Genome Integration

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To validate the performance of GenOMe, we quantified its integration efficiency using the formula below.

Electroporated cells were plated on both selective and non-selective LB agar plates.
Colony counts revealed a consistent integration efficiency of approximately 80% across tests.

• TestVer2 –IntTest_P3_GFP_genR_P2: 79% efficiency, confirming dual-gene (GFP + GenR) insertion in a single step.
• TestVer3 –IntTest_P3_GFP_genR_P5: 80% efficiency, demonstrating complete replacement of the Landing Pad with only minimal attL/attR scars remaining.

Together, these results confirm that GenOMe achieves precise, marker-free, and highly efficient genome integration within just two days.

Beyond Single Insertions: Cyclic Integration

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Through the precise design of attB/P sites, GenOMe enables stepwise DNA integration — each round introduces a new insertion site, allowing new DNA fragments to be added like LEGO bricks to build stable, inheritable, and extensible genomes.

GenOMe is a cyclic and modular platform that supports sequential DNA integrations into the genome.
The system alternates between two cassettes:

• Slot A cassette (Slot_A_cassette_GFP_genR, odd slots)
• Slot B cassette (Slot_B_cassette_mCherry_kanR, even slots)

Each integration round replaces the previous marker and opens a new site for the next fragment, enabling continuous stacking of genetic modules. Through this iterative process, GenOMe constructs stable, inheritable, and expandable genomic arrays within the bacterial chromosome.

GenOMe enables sequential DNA insertions through alternating cassette integrations.
Each round introduces a new DNA fragment into the chromosome while replacing the previous selection marker — switching between genR and kanR to maintain single-antibiotic selection. By repeating this cycle, multiple gene modules (1, 2, 3...) can be integrated in order, forming a stable, inheritable, and expandable genomic array.

Why It Matters for iGEM

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GenOMe provides iGEM teams with a reliable, scalable, and user-friendly framework for genome engineering. It enables single-copy, inheritable integrations without leaving unwanted antibiotic markers, and supports multi-gene assembly directly in the chromosome.
With standardized cassettes and adaptors, even beginners can easily perform integrations.
Ultimately, GenOMe acts as a genome “LEGO baseplate”, empowering teams to build more ambitious, creative, and high-impact designs in synthetic biology.