Results
Introduction
Genome editing faces three key barriers: low efficiency, instability, and complexity.
Traditional methods like CRISPR/Cas9 or λ-Red are effective but often slow, expensive, and inefficient for large fragments (>10 kb).
GenOMe aims to overcome these limitations by creating a modular E. coli platform where simple PCR fragments can be stably integrated into the genome.
It enables single-copy, inheritable integrations without unwanted antibiotic markers and supports multi-gene assembly directly in the chromosome — providing a clean, scalable, and user-friendly framework for genome engineering.
Experimental Overview
To demonstrate GenOMe’s performance, we followed the Build stage of our Engineering Cycle step by step:
- Design — ssDNA was designed to install attB sites, and a multifunctional Landing Pad was constructed with attP sites, Bxb1 integrase, and selectable markers.
- Construct — Using the Two-to-Two mechanism, the Landing Pad was integrated into the E. coli genome and verified through antibiotic selection, blue-white screening, and junction PCR.
- Validate — attP-flanked DNA payloads were introduced to confirm integration efficiency via single-gene insertion, dual-gene insertion, and complete gene replacement.
1. Design of ssDNA and the Landing Pad
1.1 ssDNA targeting oligonucleotide for attB1/attB6 integration
Our ssDNA (TargetingOligo_B1_B6) was inspired by ORBIT[1] and redesigned to contain two attB cores (attB1/attB6) separated by a short spacer, enabling dual-end recombination for the Two-to-Two mechanism. This 150-nt ssDNA replaces a 38-bp segment in lacZ (positions 366,238–366,277), simultaneously creating two docking sites for Bxb1-mediated integration.
The synthetic 150-nt ssDNA (TargetingOligo_B1_B6) installs attB1 and attB6 at the lacZ locus, using lacZ-derived homology arms (blue) and a short spacer (yellow) between the two attB cores to create dual docking sites for Landing Pad integration.
1.2 Landing Pad DNA construct for genomic integration at attB3/attB2/attB5 sites
The LandingPad_B3B2B5 fragment, flanked by attP1/attP6, recombines with chromosomal attB1/attB6 through Bxb1 integrase, achieving site-specific, stable insertion.
Inside the Landing Pad, additional attB3/attB2/attB5 sites serve as expandable genomic slots, while KanR enables selection and bxb1 provides autonomous recombination.
The LandingPad_B3B2B5 fragment recombines with attB1/attB6 on the chromosome, embedding attB3/attB2/attB5, KanR, and bxb1, forming the GenOMe strain.
2. Constructing the GenOMe Strain
At the beginning of our design, we considered the Two-to-Two recombination strategy to be highly challenging, and we expected only a low probability of success. However, after applying GenOMe Navigator — Guiding Genome Integration Decisions, our dry-lab team was able to provide precise parameter recommendations that directly improved wet-lab performance.
The Navigator simulated the kinetics of the recombination process and analyzed how the induction time and concentration of SSAP and Bxb1 integrase affected overall integration efficiency. It predicted that extending SSAP induction to about 45 minutes would significantly enhance attB site installation, while Bxb1 integrase required only a short active window of around 30 minutes once the attB sites became available.
Simulation of SSAP expression dynamics showed that a 45-minute induction markedly improved recombination efficiency compared to 30 minutes.
Additionally, the Navigator indicated that Bxb1 protein levels reached a stable plateau after approximately 4 hours, suggesting that prolonged induction would not further increase yield.
Predicted accumulation curves showed that Bxb1 integrase levels stabilized after around 4 hours of induction
Guided by these insights, our wet-lab team fine-tuned induction parameters for the construction of the GenOMe strain. Using these optimized conditions, TargetingOligo_B1_B6 and LandingPad_B3B2B5 were co-electroporated into E. coli MG1655 harboring the helper plasmid pHELP_TS_V2_ampR, which expresses SSAP, MutL_E32K, and Bxb1 integrase. After induction, the Landing Pad was successfully integrated into the lacZ locus through the Two-to-Two reaction. Remarkably, despite our initial doubts, the integration achieved an efficiency of approximately 80%, demonstrating the predictive power and practical value of GenOMe Navigator in guiding real experimental outcomes.
On the left, wild-type E. coli MG1655 colonies appear blue due to intact lacZ expression.
On the right, GenOMe strain colonies appear white, indicating successful chromosomal integration of LandingPad_B3B2B5, which disrupts the lacZ locus.
3. Colony PCR — Colonies grown on kanamycin plates were analyzed by PCR using primers spanning the integration junction: the forward primer was located within the Landing Pad (Bxb1) region, and the reverse primer on the chromosomal lacZ region. Only correctly integrated clones produced the expected ~741 bp amplicon on the gel, confirming precise site-specific integration of the Landing Pad at the designed lacZ locus through the Two-to-Two mechanism.
Red arrows indicate the locations of the forward and reverse primers.
3. Functional Validation of the GenOMe System
We validated GenOMe in three escalating scenarios—single-gene insertion (TestVer1), dual-gene insertion (TestVer2), and complete landing pad replacement (TestVer3)—to demonstrate that the platform supports stable, efficient, and clean genome engineering, leaving no unnecessary antibiotic markers in the final genome. All tests were performed through Bxb1-mediated Two-to-Two recombination, with integration confirmed by GFP fluorescence, antibiotic selection (when applicable), and junction-spanning PCR.
TestVer1 — Single-gene insertion (attP2/attP3 → attB2/attB3)
To verify the integration capability of GenOMe, we first carried out a single-gene insertion test using the construct IntTest_P3_GFP_P2. This fragment, flanked by attP2/attP3, was integrated into chromosomal attB2/attB3 sites embedded within the Landing Pad through Bxb1-mediated recombination. GFP-positive colonies were observed under blue/UV light, confirming successful genomic expression of the inserted reporter gene.
Integration of the IntTest_P3_GFP_P2 fragment (1,039 bp), flanked by attP2/attP3, into chromosomal attB2/attB3 sites.
TestVer2 — Dual-gene insertion (attP2/attP3 → attB2/attB3)
To evaluate whether GenOMe supports multi-gene payloads, we introduced IntTest_P3_GFP_genR_P2 carrying GFP and gentamicin resistance (GenR), flanked by attP2/attP3. Through Bxb1-mediated recombination, the fragment was integrated into attB2/attB3 within the Landing Pad. Colonies grew on gentamicin plates and showed GFP fluorescence, confirming functional integration.
The IntTest_P3_GFP_genR_P2 fragment (1,605 bp) was integrated into attB2/attB3 via Bxb1 integrase, resulting in stable chromosomal insertion of GFP and GenR.
Left: colony morphology under UV light. Right: GFP-positive, gentamicin-resistant colonies visualized using a fluorescence microscope (Olympus CKX53, 40×).
TestVer3 — Landing-pad replacement (attP3/attP5 → attB3/attB5)
To test full module replacement, we performed integration of the IntTest_P3_GFP_genR_P5 fragment carrying GFP and GenR, flanked by attP3/attP5, targeting attB3/attB5 to replace the original LandingPad_B3B2B5. This integration removed KanR and bxb1, leaving 43 bp attL and attR scars flanking the inserted fragment.
Although Bxb1 was replaced and no longer present in the genome, our model predicted that only a small amount of Bxb1 expression is sufficient to complete integration — and the experiment confirmed it. These results demonstrate that GenOMe achieves efficient and precise genome replacement, leaving only two clean 43 bp att scars on both sides of the inserted DNA.
The IntTest_P3_GFP_genR_P5 fragment (1,613 bp) carrying GFP and GenR, flanked by attP3/attP5, was integrated into attB3/attB5 via Bxb1 integrase, replacing the original LandingPad_B3B2B5. This replacement removed KanR and Bxb1, leaving 43 bp attL and attR scars flanking the inserted fragment.
Left: colony morphology under UV light. Right: GFP-positive, gentamicin-resistant colonies visualized using a fluorescence microscope (Olympus CKX53, 40×).
Integration Efficiency Assay
To evaluate the performance of GenOMe, integration efficiency was quantified by comparing colony counts on selective and non-selective LB agar plates. Electroporated cells were plated on antibiotic-containing (selection) and plain LB agar (non-selection) plates, and the ratio of colony-forming units (CFU) was used to calculate the proportion of cells in which the DNA fragment was successfully integrated into the chromosome.
Integration Efficiency Formula
Integration Efficiency is calculated as:
$$ \text{Integration Efficiency} = \frac{\text{CFU}_{\text{antibiotic}}}{\text{CFU}_{\text{LB}}} \times 100\% $$
Or equivalently:
$$ \text{Integration Efficiency} = \frac{ \left( \dfrac{\text{Colonies}_{\text{Abx}}}{\text{Dil}_{\text{Abx}} \times V_{\text{Abx}}} \right) }{ \left( \dfrac{\text{Colonies}_{\text{LB}}}{\text{Dil}_{\text{LB}} \times V_{\text{LB}}} \right) } \times 100\% $$
Where:
- ColoniesAbx = number of colonies on antibiotic plate
- ColoniesLB = number of colonies on non-selective LB plate
- Dil = dilution factor
- V = plated volume (mL)
Based on this analysis, GenOMe achieved consistently high integration rates:
- TestVer2 – IntTest_P3_GFP_genR_P2: 79% integration efficiency.
- TestVer3 – IntTest_P3_GFP_genR_P5: 80% integration efficiency.
Integration Efficiency — A Step Beyond Existing Methods
The GenOMe / Two-to-Two workflow achieved 79% efficiency in dual-gene insertion and 80% in full landing-pad replacement within a single experimental round. This near-saturation efficiency reflects both the optimized Two-to-Two recombination design and the use of ssDNA oligonucleotides inspired by the ORBIT system[2], which demonstrated efficient Bxb1-mediated integration in E. coli.
In contrast, ORBIT typically reports ~0.1–1% single-locus efficiency under equivalent CFU-based calculations[2], while classical λ-Red dsDNA recombineering performs at only 10⁻⁹–10⁻³ per cell. Even CRISPR/Cas9 + λ-Red systems, such as that described by Pyne et al.[3], achieved 39–47% correct clones for chromosomal replacements in E. coli but did not report CFU-normalized values.
When normalized by CFU, GenOMe’s ~80% integration efficiency represents a single-step, near-saturation outcome that enables precise, efficient, and marker-free genome modification within two days — a major advance over previous E. coli genome integration frameworks.
Validation and Insights
Our experiments confirmed that GenOMe enables stable, single-copy, and inheritable genome integrations through Bxb1’s unidirectional recombination, which locks each edit as attL/attR and maintains stability over generations. Each replacement cycle removed selection markers such as KanR and bxb1, resulting in a clean, marker-free genome with only minimal att scars. By alternating att sites (e.g., attB2/3/5), GenOMe supports scalable multi-gene assembly, achieving integration efficiencies of approximately 80% in both multi-gene and full-replacement tests.
Through careful optimization of competent-cell preparation and induction timing, we turned early integration failures into consistent success. Dual-marker screening using GFP and antibiotic resistance effectively minimized false positives, while validation across TestVer1–3 demonstrated GenOMe’s versatility in single-gene integration, dual-gene insertion, and complete locus replacement.
In summary, GenOMe transformed simple PCR products into stable, inheritable genomic edits, establishing a robust and modular platform for fast, reliable, and scalable genome engineering in E. coli.
[1] Saunders, H. & Ahmed, A. (2024). ORBIT in Escherichia coli: Oligonucleotide recombineering with Bxb1 integrase targeting enables kilobase-scale genome editing.
Nucleic Acids Research, 52(5), 227. https://doi.org/10.1093/nar/gkae227
[2] Saunders, S. H., & Ahmed, A. M. (2024). ORBIT for E. coli: kilobase-scale oligonucleotide recombineering at high throughput and high efficiency. Nucleic Acids Research, 52(8), e43. https://doi.org/10.1093/nar/gkae227
[3] Pyne, M. E., Bruder, M. R., Moo-Young, M., Chung, D. A., & Chou, C. P. (2015). Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli. Applied and Environmental Microbiology, 81(15), 5103–5114.
https://doi.org/10.1128/AEM.01248-15
