Parts

Overview

During our project, we designed, engineered, assembled, and tested a collection of standard biological parts to construct the GenOMe system — a modular platform for chromosome-level BioBrick integration.

Our work features two major contributions:

Best New Composite Part — LandingPad_B3B2B5 (BBa_25XNR8U7C), a multifunctional DNA module that enables precise and marker-reversible genome integration.
Best Part Collection — GenOMe Parts Collection, a complete toolkit that allows modular, multi-round, and inheritable BioBrick integrations directly in the E. coli genome.

Together, these parts extend the BioBrick philosophy from plasmids to chromosomes, creating a stable and reusable foundation for next-generation synthetic biology.

Best New Composite Part — LandingPad_B3B2B5 (BBa_25XNR8U7C)

LandingPad_B3B2B5 LandingPad_B3B2B5 is the core composite part of the GenOMe system — a multifunctional, self-contained DNA module that equips E. coli with modular and marker-reversible genome integration capability under the iGEM BioBrick standard.

It integrates four functional layers:

attP1/attP6 for recombination with chromosomal attB1/attB6;
Bxb1 integrase for catalyzing recombination;
a temporary KanR marker for selection;
internal attB3/B2/B5 sites reserved for future insertions.

This multi-layer design transforms a single BioBrick into a self-contained genome integration machine. Guided by synthetic ssDNA (TargetingOligo_B1_B6), the Landing Pad integrates precisely into the lacZ locus and achieves ~79% efficiency, validated by fluorescence, antibiotic selection, and junction PCR. Compared with λ-Red or CRISPR/Cas9 systems (1–5%), this represents a one to two orders of magnitude improvement.

LandingPad_B3B2B5 converts plasmid-based BioBrick circuits into stable genomic modules — ensuring inheritance without antibiotic dependence. It serves as the engine of the GenOMe system, being the first reusable, marker-reversible integration BioBrick under the iGEM standard.

**Figure 1**. ssDNA-guided attB installation and Landing Pad integration. A synthetic ssDNA (TargetingOligo_B1_B6) carrying attB1/attB6 and homology arms pairs with chromosomal DNA during replication, embedding two attB docking sites. LandingPad_B3B2B5 then integrates precisely through Bxb1-mediated recombination, validated by fluorescence and PCR (~79% efficiency).

Best Part Collection — GenOMe Parts Collection

The GenOMe Parts Collection is a modular and expandable toolkit that enables stable, inheritable, and marker-reversible genome integration — extending the BioBrick standard from plasmids into the E. coli chromosome. Together, these parts establish a closed-loop system for genome engineering — from installing attB docking sites to cyclic, marker-free expansion. For iGEM teams, it provides a ready-to-use framework to move any BioBrick circuit directly into the genome.

Category 1. Genome Integration Core

Defines the molecular foundation of GenOMe — orthogonal, predictable, and plug-and-socket design. It includes Bxb1 integrase and a complete set of orthogonal attB/attP recombination sites (attB1–attB6 / attP1–attP6), each operating independently to ensure precise, unidirectional integration without cross-talk.

Name	Link	Type	Purpose
Bxb1	BBa_25UYZJG5	Basic	Encodes a codon-optimized Bxb1 serine integrase for E. coli with RFC[10] compatibility. The integrase mediates site-specific recombination between attB and attP sites, enabling precise DNA insertion, deletion, or inversion.
attB1	BBa_25REVO5U	Basic	Recognized exclusively by Bxb1 integrase and recombines specifically with Bxb1 attP1 site, generating attL1/attR1 depending on orientation.
attB2	BBa_25T79QB4	Basic	Recognized exclusively by Bxb1 integrase and recombines specifically with Bxb1 attP2 site, generating attL2/attR2 depending on orientation.
attB3	BBa_257GTPAM	Basic	Recognized exclusively by Bxb1 integrase and recombines specifically with Bxb1 attP3 site, generating attL3/attR3 depending on orientation.
attB5	BBa_25X46UCH	Basic	Recognized exclusively by Bxb1 integrase and recombines specifically with Bxb1 attP5 site, generating attL5/attR5 depending on orientation.
attB6	BBa_2523JBB0	Basic	Recognized exclusively by Bxb1 integrase and recombines specifically with Bxb1 attP6 site, generating attL6/attR6 depending on orientation.
attP1	BBa_25Q81FDA	Basic	Recognized exclusively by Bxb1 integrase and recombines specifically with Bxb1 attB1 site, generating attL1/attR1 depending on orientation.
attP2	BBa_25E8Q9SD	Basic	Recognized exclusively by Bxb1 integrase and recombines specifically with Bxb1 attB2 site, generating attL2/attR2 depending on orientation.
attP3	BBa_25O8Z8RP	Basic	Recognized exclusively by Bxb1 integrase and recombines specifically with Bxb1 attB3 site, generating attL3/attR3 depending on orientation.
attP5	BBa_25GXN0L1	Basic	Recognized exclusively by Bxb1 integrase and recombines specifically with Bxb1 attB5 site, generating attL5/attR5 depending on orientation.
attP6	BBa_25MGSJ8Q	Basic	Recognized exclusively by Bxb1 integrase and recombines specifically with Bxb1 attB6 site, generating attL6/attR6 depending on orientation.

**Figure 2**. Orthogonal Bxb1-mediated recombination system. Each attB–attP pair (1–1, 2–2, 3–3, 5–5, 6–6) recombines specifically under Bxb1 integrase, forming attL and attR junctions without cross-talk. The reaction is irreversible without RDF, ensuring stable, one-way integration and multi-locus compatibility.

Category 2. Integration Platform

Implements the recombination logic into a functional framework. It includes TargetingOligo_B1_B6 (ssDNA that installs attB1/attB6) and LandingPad_B3B2B5 , which integrates via these sites to establish the first functional genomic slot. Once integrated, the Landing Pad provides new attB3/B2/B5 sites for future rounds, creating a self-expanding, marker-reversible platform.

Name	Link	Type	Purpose
TargetingOligo_B1_B6	BBa_25BIMNOC	Basic	This oligonucleotide is designed for homologous recombineering to disrupt the lacZ locus.
LandingPad_B3B2B5	BBa_25XNR8U7	Composite	The LandingPad_B3B2B5 cassette establishes genomic docking sites in E. coli for future integrations.

Category 3. Cyclic Integration and Expansion System

Alternating Slot A and Slot B cassettes (GenR and KanR) enable multi-round genome integrations. Each cycle replaces the previous marker and generates new att sites, allowing continuous, marker-free expansion with only two 43 bp scars per cycle.

Name	Link	Type	Purpose
Slot_A_cassette_GFP_genR	BBa_25GCF7TB	Composite	The Slot_A_cassette_GFP_genR is designed to replace the kanR marker on LandingPad_B3B5 (BBa_250I7ZHC).
Slot_B_cassette_mCherry_kanR	BBa_25H9ZHNI	Composite	The Slot_B_cassette_kanR is an empty integration cassette carrying an attB-flanked kanamycin resistance gene (kanR).
SlotCassette	BBa_25DOPQXF	Composite	The SlotCassette is an integration cassette designed to target LandingPad_B3B5 (BBa_250I7ZHC) for a single-round genome insertion.
Slot_A_cassette_genR	BBa_2553SA2K	Composite	The Slot_A_cassette_genR is an empty integration cassette carrying an attB-flanked gentamicin resistance gene (genR).
Slot_B_cassette_kanR	BBa_25NNS05G	Composite	The Slot_B_cassette_kanR is an empty integration cassette carrying an attB-flanked kanamycin resistance gene (kanR).

**Figure 3**. Cyclic integration mechanism for iterative genome assembly. Alternating Slot A (GenR) and Slot B (KanR) cassettes enable multi-round genome integrations. Each round replaces the previous marker while generating new att sites for the next insertion, achieving clean, marker-free assembly.

Additional Supporting Modules

Although not part of the official Best Part Collection, these modules ensure experimental reproducibility and system reliability.

Utility & Marker Modules

Provide codon-optimized antibiotic markers and test constructs for validating recombination accuracy and selection stability.

Name	Link	Type	Purpose
kanR_codonOpt	BBa_25TAXEAT	Basic	This part encodes a codon-optimized kanamycin resistance gene, adapted for E. coli.
genR_codonOpt	BBa_253FXF55	Basic	This part encodes a codon-optimized gentamicin resistance gene, adapted for E. coli.
IntTest_P3_GFP_P2	BBa_25HI62V8	Composite	The IntTest_P3_GFP_P2 cassette is designed to integrate into the LandingPad_B3B2B5 (BBa_25XNR8U7) docking site and express GFP.
IntTest_P3_GFP_genR_P2	BBa_25K9R4DU	Composite	The IntTest_P3_GFP_genR_P2 cassette is designed to integrate into the LandingPad_B3B2B5 (BBa_25XNR8U7) docking site.
IntTest_P3_GFP_genR_P5	BBa_25Z1J2ZG	Composite	The IntTest_P3_GFP_genR_P5 cassette integrates into the LandingPad_B3B2B5 (BBa_25XNR8U7) locus.
JunctionPrimer_LandingPad_B3B2B5_FWD	BBa_25PKZQIK	Basic	This forward primer anneals to the lacZ genomic locus upstream of the integration site.
JunctionPrimer_LandingPad_B3B2B5_REV	BBa_253LD85E	Basic	This reverse primer anneals within the LandingPad_B3B2B5 construct (in the Bxb1 region).
JunctionPrimer_IntTest_FWD	BBa_25CPB2V1	Basic	This forward primer anneals within the GFP region of the IntTest cassette.
JunctionPrimer_IntTest_REV	BBa_25MGE8FG	Basic	This reverse primer anneals to the lacZ genomic sequence outside the integration site.

Supporting and Assembly Tools

Include primers and prototype constructs used during overlapping PCR, cloning, and validation — assisting GenOMe system optimization.

Name	Link	Type	Purpose
LandingPad_B3B5	BBa_250I7ZHC	Composite	The LandingPad_B3B5 cassette is an upgraded version of LandingPad_B3B2B5 (BBa_25XNR8U7), designed to extend its functionality by supporting cyclic integration.
OverlappingPCRPrimer_attP3_VF2	BBa_25UT6ABN	Composite	This primer combines the iGEM VF2 universal primer with an attP3 sequence.
OverlappingPCRPrimer_attP2_VR	BBa_25QJ1YOY	Basic	This primer combines the iGEM VR universal primer with an attP2 sequence (presented in 5′→3′ notation, though used in reverse orientation during PCR).

**Figure 4**. Construction of the GenOMe chassis via Landing Pad integration. The LandingPad_B3B2B5 fragment recombines with chromosomal attB1/attB6 via Bxb1, embedding into the E. coli genome to form a stable, reusable chassis. This confirms the entire workflow — ssDNA-guided attB installation, Landing Pad integration, and marker verification.

Summary and Conclusion

Together, LandingPad_B3B2B5 and the GenOMe Parts Collection form the world’s first modular, reusable, and validated platform for chromosomal BioBrick integration — bridging plasmid design and genome engineering. This contribution provides iGEM teams with a stable, inheritable, and scalable chassis for ambitious synthetic biology designs.