Genetic engineering in bacteria is foundational to synthetic biology, but current methods remain complex, unstable, and poorly modular. Traditional plasmid-based systems require constant selection pressure, are prone to segregation, and often impose metabolic burdens on the host. Integrating DNA into the genome offers more stability, yet conventional genome editing approaches—such as lambda Red recombineering or CRISPR-assisted homologous recombination—are time-consuming and technically demanding, especially for kilobase-scale modifications. This limits the accessibility and scalability of genome engineering for synthetic biology applications.
ORBIT (Oligonucleotide-mediated Recombineering followed by Bxb1 Integrase Targeting) presents a streamlined alternative. Originally developed in Mycobacteria and recently adapted for E. coli, ORBIT enables high-efficiency, kilobase-scale genomic modifications through a simple workflow. A short oligonucleotide introduces an attB site into the chromosome, which then serves as the target for site-specific integration of a non-replicating plasmid via Bxb1 integrase. Compared to classical lambda Red methods, ORBIT has demonstrated up to 1000-fold higher efficiency and supports stable, scarless insertions of fragments up to 11 kb. Its design removes the need for cloning long homology arms and offers a host-independent, plug-and-play genome editing strategy (Saunders & Ahmed, 2024; Murphy et al., 2018).
To make ORBIT more compatible with iGEM-style modular design, our team adapted and simplified the system using insights from prior iGEM work (Yao et al., 2017). Instead of inserting entire plasmids—including unwanted vector sequences such as origins of replication—we implemented a dual-attB/attP strategy that enables clean, precise integration of only the functional DNA payload. We preserved the use of ORBIT’s original helper system for attB site installation via oligo recombineering, while modifying the design of integration plasmids to ensure only the intended coding regions are inserted. This hybrid approach allows for greater modularity, efficient pathway assembly, and cleaner genome edits aligned with synthetic biology standards.
Our system begins with the design of a synthetic oligonucleotide encoding a Bxb1 attB site flanked by ~40 bp homology arms. Upon electroporation, the ORBIT helper plasmid facilitates RecT-mediated annealing of the oligo into the lagging strand during replication. Once the attB site is installed in the genome, a second electroporation introduces a non-replicating plasmid containing the complementary attP site and the functional payload. Bxb1 integrase then catalyzes site-specific recombination between attB and attP, irreversibly inserting the DNA cassette into the chromosome. The dual attP design ensures that only the payload (not the plasmid backbone) is integrated, yielding clean, stable insertions. This process requires no PCR, no long homology arms, and no host DNA repair machinery (Baer et al., 2022).
To validate our system, we begin by inserting a GFP expression cassette into the genome—a visible and quantifiable reporter. Our long-term goal is to support metabolic pathway testing by enabling stable, stepwise integration of multi-gene modules. Because the attB site can be pre-installed in standardized loci, our chassis strains can function as "landing pads" for future teams to rapidly test constructs without relying on plasmids. This toolbox enables iGEM teams to focus on design and function, rather than construction, and is especially valuable for projects involving long operons, biosensor libraries, or fermentation engineering.
We envision a future where genome engineering in E. coli is as accessible and modular as using plasmids—only more stable. By distributing strains with attB landing pads and providing integration-ready plasmids, we aim to establish a plug-and-play genome engineering platform that allows teams to treat the bacterial genome as a biological motherboard. This shift from transient plasmid expression to stable genome integration empowers more reliable long-term studies, scalable pathway engineering, and foundational tools for synthetic biology at large.