| SMU-Union-China-iGEM 2025

Parts

We have designed and characterised high-quality BioBrick standardised components, which we believe will benefit future iGEM teams. Furthermore, we have experimentally validated the functionality of existing parts. The table below details the parts designed and validated by our team.

Our team completed a total of 21 new parts, including 9 basic parts and 12 composite parts. Additionally, we supplemented information or refined characterization for 4 used parts.

The most representative and core part of the project is scfv-MEDI8852(BBa_25Y94ZNW ). It encodes a single-chain fragment that binds to influenza HA's fusion peptide and groove, serving as both sensor identifier and neutralizing antibody secreted by our engineered bacteria. We have characterized and validated it by modeling, docking, colony PCR, ELISA, and hemagglutination inhibition assays. It multiple fields, including mucosal therapy, rapid diagnosis and 'antibody-drug' sequential therapy.

Sensing module

Part code	Title	Part type	Functional description
BBa_25HC5855	KxYKxGKxW signal peptide domain-containing protein	Basic Protein coding sequences	The signal peptide KxYKxGKxW is a protein-directed sequence that guides the directional transport of newly synthesized proteins to the cell membrane or extracellular environment.
BBa_25QF5YQ6	P59:Streptococcus cremoris promoter 59	Basic Promoter	This basic part functions as a potent constitutive promoter in this project, facilitating downstream gene expression in Lactobacillus rhamnosus (L. gg).
BBa_25Y94ZNW	scfv-medi8852	Basic Protein coding sequences	This basic part is our best new basic part.This basic part encodes the single-chain antibody fragment (scFv) of MEDI8852. This scFv binds to the fusion peptide and hydrophobic groove of HA, inhibiting HA0 cleavage and pH-induced conformational changes, thereby blocking viral membrane fusion with host cells and preventing viral entry into cells from the onset of infection.
BBa_25X1CPM9	Sensing Module: SP+EGFP+scfv-medi8852+PnpS	Composite Measurement	This composite part serves as the sensing module of our project. It employs the scFv-MEDI8852 derivative (see BBa_25Y94ZNW for details) to bind influenza A virus particles. Upon capture, it cooperates with the response module(see BBa_25O0BRWS and BBa_255Q4IZN for details) via a two-component signaling system to relay the detection signal and activate downstream anti-viral outputs, culminating in the secretion of neutralizing scFv molecules against the virus.
BBa_253YCPGX	two-component system histidine kinase PnpS	Basic Protein coding sequences	The two-component system histidine kinase, as a constitutive element in Lactobacillus rhamnosus, undergoes a conformational change and becomes strongly activated upon receiving an interfering signal transmitted through the hinge region. It then releases a phosphate group, transferring the signal to the downstream response regulator protein in the two-component system, thereby initiating downstream signaling pathways.

Response module

Part code	Title	Part type	Functional description
BBa_25Y94ZNW	scfv-medi8852	Basic Protein coding sequences	This basic part is our best new basic part.This basic part encodes the single-chain antibody fragment (scFv) of MEDI8852. This scFv binds to the fusion peptide and hydrophobic groove of HA, inhibiting HA0 cleavage and pH-induced conformational changes, thereby blocking viral membrane fusion with host cells and preventing viral entry into cells from the onset of infection.
BBa_252DK94H	scfv-1G01	Basic Protein coding sequences	This basic part retains the antigen-binding capacity and NA inhibitory activity of 1G01. It can inhibit viral release from infected cells and effectively counteract multiple subtypes of influenza A viruses.
BBa_25N1WI55	Signal Peptide [Lactobacillus rhamnosus]	Basic Protein coding sequences	This part is used to guide the newly synthesized scFv antibody protein through the bacterial cell membrane and transport it to the extracellular space.
BBa_255Q4IZN	phoB+RBS+Signal Peptide+1G01+mCherry	Composite Measurement	This part is used to ensure the synthesis and release of scfv-1G01 when viruses are detected.
BBa_25O0BRWS	phoB+RBS+Signal Peptide+medi8852+mCherry	Composite Measurement	This part is used to ensure the synthesis and release of scfv-medi8852 when viruses are detected.
BBa_25KBZIMR	Sensing & Response Modules for anti influenza A virus(scfv-medi8852)	Composite Device	This part is a complete circuit for anti-influenza A virus in our project design, sensing & reponse modules work together via a two-component signaling system to relay the detection signal and activate downstream anti-viral outputs, culminating in the secretion of neutralizing scFv molecules against the virus.

Safety module

Part code	Title	Part type	Functional description
BBa_25R32UQ7	sppK	Basic Coding	This part encodes a histidine kinase (sppK), which typically participates in signal transduction processes within bacteria. Histidine kinases are enzymes capable of transmitting signals from the extracellular environment into the cell; they commonly respond to environmental changes and regulate cellular behaviour. In our project, it acts synergistically with sppR and psppA to respond to stimulation by the peptide IP673, thereby inducing transcription of genes downstream of the sppA promoter. sppK perceives an extracellular signal and autophosphorylates. It then transfers the phosphate to sppR. The phosphorylated sppR binds to the sppA promoter (psppA), activating it and initiating transcription of genes downstream.
BBa_25PHUFTR	sppR	Basic Regulatory	This part encodes a response regulator (sppR), which typically functions in conjunction with histidine kinases to participate in the regulation of signal transduction and cellular behaviour. Upon receiving signals, response regulators alter their activity, thereby modulating the expression of other genes. In our project, it acts synergistically with sppK and psppA to respond to stimulation by the peptide IP673, thereby inducing transcription of genes downstream of the sppA promoter. sppK perceives an extracellular signal and autophosphorylates. It then transfers the phosphate to sppR. The phosphorylated sppR binds to the sppA promoter (psppA), activating it and initiating transcription of genes downstream.
BBa_25W263MB	psppA	Basic Promoter	This encodes sppA promoter, which is a DNA sequence found in lactic acid bacteria that initiates transcription of sakacin P upon receiving specific extracellular signals peptide signal IP-673. In our project, it acts synergistically with sppR and sppK to respond to stimulation by the peptide IP673, thereby inducing transcription of genes downstream. sppK perceives an extracellular signal and autophosphorylates. It then transfers the phosphate to sppR. The phosphorylated sppR binds to the sppA promoter (psppA), activating it and initiating transcription of genes downstream.
BBa_25MWMH98	pTlpA + RBS + eGFP + Double Terminator	Composite Measurement	This part is created to characterize the influence of TlpA on the expression of recombinant genes in Lactobacillus rhamnosus GG in different temperature conditions.
BBa_25S96O1L	pldhL+RBS+TlpA+rrnB T1 terminator	Composite Measurement	This part incorporates the TlpA regulator, which is sensitive to temperature changes, allowing for control of gene expression in response to environmental temperature shifts. This part is desined to work synergistically with BBa_25MWMH98, modulating the expression of recombinant genes in Lactobacillus rhamnosus GG under varying temperature conditions. Together, these parts enable the characterization of how temperature-induced changes in TlpA activity affect the expression levels of the recombinant gene(s), providing insights into the potential for temperature-based regulation in Lactobacillus rhamnosus GG.
BBa_25SO3V2Z	lacIq+YF1+FixJ+FixK2+eGFP	Composite Measurement	This part is used to test the expression of the blue light system.
BBa_25G1GAN9	SppK+SppR+PsppA+MazF	Composite Measurement	This part was designed to test the killing effect of the MazF toxin on the Lactobacillus Igg strain.SppK affects SppR through external signal transduction, and then SppR regulates the PsppA promoter to drive the transcription of downstream genes.
BBa_25ZEUIHZ	Blue light-activated kill switch	Composite Device	This part is a complete circuit for blue light activated kill switch in our project design via pdawn system.
BBa_252KOYX4	SppK+SppR+PsppA+λrepressor+λ PR promoter+eGFP+SsrA	Composite Measurement	This part serves as a functional checkpoint for the λ repressor/λ PR promoter cassette--the core of the pDawn photorelay--within our blue-light-activated kill switch. Upon assembly, it reports the system's ON/OFF fidelity by toggling eGFP fluorescence in response to blue-light presence or absence.
BBa_251Z8AWF	pLdhL+RBS+eGFP + Double Terminator	Composite Measurement	This part was created to characterize the constitutive promoter of the lactate dehydrogenase (ldhL) in Lactobacillus rhamnosus GG.
BBa_25OME7TE	SpldhL + RNA thermometer (ROSE) + eGFP + Double Terminator	Composite Measurement	This part was created to characterize the influence of an RNA thermometer on the expression of recombinant genes in Lactobacillus rhamnosus GG.

Used parts

Part code	Title	Contribution type	Description	Modification
BBa_K115017	RNA thermometer (ROSE 32℃)	Informational	Components of the temperature self-destruct circuit within the safety module	Discovery & "Zipper" Mechanism We added information from literature: the ROSE RNA thermometer originates from rhizobia, functioning as a zipper-like thermal sensor: at low temperatures, the stem loop closes to shield the ribosomal binding site (RBS); upon warming, the stem gradually unravels to release the RBS, thereby initiating translation.
BBa_K2500004	TlpA	characterization	A part of our temperature induced kill switch, TlpA works synergistically with pTlpA, modulating the downstream expression	TlpA: Temperature induced eGFP We engineered a plasmid containing an inserted reporter gene sequence to monitor fluorescence intensity at varying temperatures, thereby characterising TlpA. This approach provides insights into the potential for temperature-based regulation within Lactobacillus rhamnosus GG.
BBa_K2500003	pTlpA	characterization	A part of our temperature induced kill switch, TlpA works synergistically with pTlpA, modulating the downstream expression	TlpA-pTlpA: eGFP induction We engineered a plasmid containing an inserted reporter gene sequence to monitor fluorescence intensity at varying temperatures, thereby characterising TlpA. This approach provides insights into the potential for temperature-based regulation within Lactobacillus rhamnosus GG.
BBa_K1075044	Promoter(const.)-RBS34-YF1-FixJ-FixK-LambdaC-pC (pDawn)	characterization	A part of our blue light induced kill switch, CI works synergistically with lacIq Promoter, modulating the downstream expression	CI-pR eGFP Assay We engineered a plasmid containing an insert reporter gene, employing fluorescence intensity to monitor eGFP expression, thereby validating the functionality of the CI-pR system within Lactobacillus rhamnosus.
BBa_K1075044	Promoter(const.)-RBS34-YF1-FixJ-FixK-LambdaC-pC (pDawn)	characterization	A part of our blue light induced kill switch	FixJ-YF1-FixK2 Assay We engineered a plasmid containing an insert reporter gene, employing fluorescence intensity to monitor eGFP expression, thereby validating the functionality of the FixJ-YF1-FixK2 circuit within Lactobacillus rhamnosus.