BioBricks Parts Collection

Probiotic Dual-Immunization Platform (Passive + Active)

Overview

Modularity is at the heart of synthetic biology, enabling the rapid adaptation of vaccine platforms to emerging viral threats. The AvianGuard active immunity system represents a highly modular oral vaccine platform that converts probiotic bacteria (Lactococcus lactis) into live antigen-delivery vehicles capable of stimulating robust mucosal and systemic immune responses in avian species.

The power of this platform lies in its genetic modularity: conserved viral antigens can be swapped and reconfigured to target different avian pathogensfrom highly pathogenic avian influenza (H5N1) to Newcastle disease virus (NDV), infectious bursal disease (IBD), and beyond. By anchoring these antigens to the bacterial cell surface using different anchor domains (SpaX and CWm6), we enable customizable immune activation strategies suited to different contexts and target species.

With AvianGuard, our team has created a foundational toolkit for rapid-response vaccine development against avian viral diseases. We envision exciting applications beyond H5N1: protecting poultry from emerging variants, developing region-specific vaccines for endemic diseases, and establishing vaccine platforms that can be deployed without cold-chain infrastructure in resource-limited settings.

We wanted to democratize access to this technology by assembling a well-documented Parts Collection that allows iGEM teams and researchers worldwide to harness the modularity of the AvianGuard system. We're excited to see what the community can build with these BioBrick Parts!

Parts Collection Structure

We have organized our BioBricks into two major subcollections corresponding to the dual mechanisms of AvianGuard:

Collection 1: Active Immunity Components (Lactococcus lactis-based)

These parts enable the construction of surface-displayed antigen systems using engineered probiotic bacteria. They include:

Nisin-inducible expression system (PnisA promoter, NisR/NisK regulatory cassette)
Conserved viral antigens (M2e, LAH, NA stalk)
Cell wall anchors (SpaX, CWm6)
Signal peptides and linkers (Usp45, flexible GS/GGGGS linkers)
Fluorescent reporters (GFP-SpaX, RFP-CWm6) for validation

Collection 2: Passive Immunity Components (E. coli-based cyclobodies)

These parts enable production of cyclized, thermostable nanobodies for direct viral neutralization:

Anti-H5N1 nanobodies targeting hemagglutinin and neuraminidase
Intein cyclization domains (Npu DnaE Split Intein)
Purification tags (His-tag, Strep-tag)
Expression optimization elements for E. coli BL21
Signal peptides and linkers (Usp45, flexible GGGGS linkers)
Non-native exteins for inteins optimization (CWN, GGH)

Active Immunity: Basic Parts

Part Number	Type	Description	Category
BBa_K2978211	Regulatory	Spacer + Terminator: Non-coding spacer region (76 bp) followed by a transcriptional terminator sequence. Provides physical separation between the CWm6 anchor domain and downstream elements while ensuring proper transcription termination and preventing read-through into adjacent genetic regions.	Basic part
BBa_25KXKP82	Regulatory	PnisA + RBS: Nisin-responsive promoter from L. lactis that activates transcription when phosphorylated NisR binds to its operator sequence. The integrated ribosome binding site enables efficient translation of downstream genes. This cassette provides tight, dose-dependent control of gene expression in response to nisin concentration.	Basic part
BBa_25BEV0RT	Device	Nisin Induction System: Two-component regulatory system enabling nisin-inducible gene expression. Consists of NisR response regulator and NisK histidine kinase sensor under constitutive promoter, followed by nisin-inducible PnisA promoter. Upon nisin binding, NisK phosphorylates NisR, activating transcription from PnisA for controlled downstream gene expression.	Basic part
BBa_255XOW4U	Assembly	BioBrick Prefix + Assembly Scar: Standard BioBrick prefix containing EcoRI and XbaI restriction sites for RFC10 assembly, followed by BsaI recognition site and assembly scar sequence (CTCGGGAG). Enables standardized cloning and integration into the iGEM Registry. Essential for modular assembly and part compatibility.	Basic part
BBa_25NSOPR0	Assembly	BioBrick Suffix + Assembly Scar: Standard BioBrick suffix containing SpeI and PstI restriction sites for RFC10 assembly, preceded by BsaI recognition site and assembly scar sequence (TCGCT). Essential for modular assembly, part concatenation, and compatibility within the BioBrick framework.	Basic part
BBa_251BZQC8	Coding	Tandem M2e + Linker: Four tandem copies of the conserved influenza A M2e epitope separated by flexible GS linkers. Enhances immunogenicity through epitope multimerization, promoting B-cell activation and broadly neutralizing antibody production. C-terminal linker enables fusion to additional functional domains.	Basic part
BBa_25IP5UUG	Coding	Tandem M2e + SpaX: Four tandem copies of highly conserved influenza M2e epitope fused to SpaX cell wall anchor. M2e repeats enhance immunogenicity through epitope multimerization, eliciting broadly cross-protective antibodies. SpaX domain (LPXTG motif) enables covalent surface display via sortase-mediated anchoring.	Basic part
BBa_25ULJY8T	Coding	LAH + Linker: Conserved Lateral Amphipathic Helix (LAH) from influenza hemagglutinin that elicits broadly neutralizing antibodies. C-terminal flexible linker enables fusion to downstream domains while maintaining epitope accessibility and structural independence.	Basic part
BBa_25NYARXY	Coding	LAH + SpaX: Fusion of LAH domain from influenza hemagglutinin with SpaX cell wall anchor. LAH is a highly conserved membrane-proximal region serving as neutralizing antibody target. SpaX contains LPXTG motif enabling covalent cell wall anchoring via sortase-mediated transpeptidation. Provides dual functionality for immune enhancement and stable surface display.	Basic part
BBa_25JUKAFO	Coding	NA stalk + CWm6: Highly conserved neuraminidase (NA) stalk domain fused to CWm6 cell wall binding domain. NA stalk remains structurally conserved across influenza subtypes, serving as target for broadly neutralizing antibodies providing heterosubtypic protection. CWm6 enables non-covalent peptidoglycan binding for surface display.	Basic part
BBa_25H6A0LY	Coding	GFP + SpaX: GFP-SpaX fusion protein for cell surface display of fluorescence. SpaX anchor domain (containing LPXTG sortase recognition motif) covalently attaches GFP to bacterial cell wall via sortase-mediated transpeptidation. Enables visualization of surface-displayed proteins and whole-cell fluorescent biosensing.	Basic part
BBa_25GATJWP	Coding	RFP + CWm6: RFP-CWm6 fusion protein for cell surface display of red fluorescence. CWm6 domain non-covalently binds to peptidoglycan in bacterial cell wall. Enables membrane-localized red fluorescent detection, compatible with dual-reporter systems in Gram-positive bacteria.	Basic part

Active Immunity: Composite Parts

Part Number	Type	Description	Category
BBa_254NT33L	Device	Active_Ind._spaX_GFP: Complete nisin-inducible expression system with GFP-SpaX surface display. Proof-of-concept construct for validating inducible expression and covalent cell wall anchoring. Contains: Terminator-NisR-NisK-Constitutive Promoter-PnisA-RBS-Usp45-GFP-SpaX-Terminator.	Composite part
BBa_25QWRKS9	Device	Active_Ind._GFP-SpaX_RFP-M6: Dual-reporter system demonstrating simultaneous expression and surface display via two different anchoring mechanisms (SpaX and CWm6). Enables validation of co-display strategies. Contains both GFP-SpaX and RFP-CWm6 fusion constructs under nisin-inducible control.	Composite part
BBa_25XO2DJQ	Device	LAH-M2e-SpaX-NA-M6: Complete H5N1 vaccine construct targeting multiple conserved epitopes with dual-anchor strategy. Combines LAH-4xM2e-SpaX (covalent anchoring) and NA stalk-CWm6 (non-covalent anchoring) for comprehensive immune activation against hemagglutinin and neuraminidase.	Composite part
BBa_25CXLT13	Device	M2e-LaH-SpaX-NA-M6: Alternative epitope configuration for comparative immunogenicity studies. Tests effect of antigen order (M2e-LAH vs LAH-M2e) on immune responses. Contains 4xM2e-LAH-SpaX + NA stalk-CWm6.	Composite part

Passive Immunity: Basic Parts

Part Number	Type	Description	Category
BBa_R0062	Promoter	pLux - AHL-inducible promoter activated by the LuxR-AHL complex. Enables controlled gene expression in response to 3OC6-HSL. The new characterization of this part is our contribution to iGEM	Basic part
BBa_251MHLD6	Coding	Usp45 signal peptide - Directs the nascent cyclobody precursor toward the periplasm for intein-mediated cyclization.	Basic part
BBa_25MCVNPK	Protein Domain	Npu DnaE N-intein - N-terminal fragment of split intein for autocatalytic protein splicing.	Basic part
BBa_25XEDBNX	Protein Domain	Npu DnaE C-intein - C-terminal fragment of split intein completing cyclization reaction.	Basic part
BBa_25UZWIB2	Coding	R1a-A5 nanobody - Binds HA2 domain of hemagglutinin (first 59 aa from fusion peptide).	Basic part
BBa_25HV150K	Coding	Nb10 nanobody - Targets receptor-binding sites in HA1 domain of hemagglutinin.	Basic part

Passive Immunity: Composite Parts

Part Number	Type	Description	Category
BBa_25EOLTL5	Intermediate	pLux-GFPmut3b transcriptional unit with RBS B0032 - AHL-inducible transcriptional unit consisting of the pLux promoter (BBa_R0062), RBS BBa_B0032, GFPmut3b reporter (BBa_E0040), and terminator BBa_B0015. It functions as the reporter module in the device BBa_253I2NAK, where GFP expression is activated by the LuxR–AHL complex. The LuxR regulator is constitutively expressed from BBa_K2656114, which complements this construct. The use of RBS BBa_B0032 reduces translation efficiency, resulting in lower GFP output, ideal for fine-tuned quorum-sensing systems. This part was designed through a DBTL engineering cycle, using modeling and experimental data from BBa_K3893028 to predict and verify promoter performance under reduced translation strength. Experimental validation confirmed the predicted low-expression profile, validating the model’s predictive accuracy.	Composite Part
BBa_253I2NAK	Device	AHL-induced GFP low expression device (with RBS B0032) - This device consists of an AHL-inducible transcriptional unit that drives the expression of GFP, and a constitutive transcriptional unit expressing the LuxR regulator. It was used to characterize the pLux promoter (BBa_R0062). It is the result of our Engineering DBTL cycle. We performed the DBTL cycle to improve the function of the promoter from device BBa_K3893028.	Composite Part
BBa_25VS95MM	Device	pLux- Inducible Anti-Hemagglutinin R1aA5-Nb10 Cyclobody Secretion: This construct enables quorum-sensing-regulated expression of a bispecific cyclobody under the control of the pLux promoter. The circuit includes luxI, responsible for producing acyl-homoserine lactone (AHL), which activates the pLux promoter through the LuxR-AHL complex once a threshold concentration is reached. Upon induction, transcription of the cyclobody precursor is initiated. The sequence downstream of the RBS includes the Usp45 signal peptide for secretion toward the periplasmic space, where intein-mediated protein splicing is expected to cyclize the molecule. The resulting cyclobody consists of two nanobodies, R1a-A5 targeting the HA2 domain and Nb10 targeting receptor-binding sites within the HA1 domain of hemagglutinin, providing dual and high-affinity recognition.	Composite Part
BBa_25ZP9BYH	Device	pBAD-Inducible Anti-Hemagglutinin R1aA5-Nb10 Cyclobody Secretion: This construct enables the regulated production of a bispecific cyclobody under the arabinose-inducible pBAD (BBa_I0500) promoter. Upon induction with arabinose, the system expresses a precursor containing inteins and exteins that undergo protein splicing in the periplasm, guided by the Usp45 secretion signal. The resulting cyclobody combines two nanobodies with distinct binding specificities: R1a-A5, which recognizes the conserved HA2 domain of hemagglutinin, and Nb10, which targets receptor-binding sites within the HA1 domain. This design enables tunable expression and dual antigen recognition, supporting modular applications in passive immunization platforms.	Composite Part
BBa_25G482PI	Device	J23101 Anti-Hemagglutinin R1aA5-Nb10 Cyclobody Secretion: This construct contains the BioBrick parts required to assemble a cyclobody precursor under the constitutive promoter J23101. Once expressed, it produces a precursor composed of inteins and exteins that have not yet undergone splicing. Downstream of the RBS, the Usp45 signal peptide directs the product to the periplasm, where protein splicing occurs to form the cyclobody. The final molecule combines two nanobodies: R1a-A5, which targets the HA2 domain, and Nb10, which binds to the receptor-binding sites in HA1, enabling dual recognition of hemagglutinin.	Composite Part

Design Principles: Modularity, Abstraction & Standardization

Together, this parts collection allows researchers to mix and match components to create functional oral vaccine devices capable of:

Targeting multiple avian viral diseases through antigen swapping
Operating in different bacterial chassis and systems (L. lactis, Lactobacillus, E. coli)
Providing tunable immune activation via dose-dependent nisin induction
Combining active and passive immunity for comprehensive protection
Enabling rapid prototyping of vaccines against emerging threats

Our BioBricks adhere to the core principles of synthetic biology:

Modularity

Antigens, anchors, nanobodies, and regulatory systems can be independently swapped

Abstraction

Each module has well-defined inputs/outputs (e.g., nisin → antigen expression; cyclobody → viral neutralization)

Standardization

All parts use BioBrick assembly standards for seamless integration

Applications Beyond H5N1

While we validated this platform with H5N1 influenza, the modular design enables rapid adaptation:

Target Pathogen	Antigen Swap Strategy	Cyclobody Retargeting
Newcastle Disease Virus (NDV)	Replace with NDV F or HN proteins	Generate anti-F/HN nanobodies
Infectious Bursal Disease (IBD)	Display VP2 capsid protein	Target VP2 neutralizing epitopes
Infectious Bronchitis Virus (IBV)	Use S1 spike protein	Anti-S1 blocking nanobodies
Avian Metapneumovirus (aMPV)	Display G protein	Develop fusion-inhibiting VHHs