AFMU China 2025 - Our synthetic biology journey from February to September
Project Timeline Overview
February and March: Concept Development
Project initiation, literature review, and establishing research direction for SynNotch-engineered macrophages.
April: Input Design
Development of liver-specific antibody targeting SLC17A2 for SynNotch system recognition domain.
May: Antibody verify and output Design
Verification of antibody specificity and design of P65 and SIRPα shRNA output components.
June and July: SynNotch system Construction
SynNotch system assembly, iterative optimization.
August: Construct the Syn-M and verify Dry lab
Construct the final Syn-M plasmid and sequencing data analysis and modeling.
September: Functional validation and LNP Development
P65 and SIRPαshRNA functional validation. lipid nanoparticle (LNP) vector design for therapeutic delivery.
Key Achievements
- Successfully constructed the input of liver-specific activation
- Validated functional output of P65 and SIRPα shRNA components
- Developed LNP delivery system for potential clinical translation
February and March: Concept Development
We solidified our project concept and established the core research direction. Our team conducted comprehensive literature reviews and identified the key challenges in liver cancer immunotherapy.
February 15-March 31 - Design SynNotch system and and outline approach
We defined the overall strategy for engineering macrophages using the SynNotch system and outlined the experimental approach. This phase involved intensive brainstorming sessions, resource planning, and establishing collaboration protocols with our advisors and partners.
April: Input Design - Liver-Specific Antibody
During April, we focused on designing the input component of our SynNotch system. We screened liver-specific antigens and developed software to identify cell membrane-specific antigens.
April 6-11 - Screening liver-specific antigens
We screened liver-specific antigens on the https://www.proteinatlas.org/humanproteome/tissue/liver website and developed software to further screen cell membrane-specific antigens. We searched the literature on SLC17A2 and compared it with the results of software analysis to verify the specificity of the antigen.
April 12-18 - Structuring prediction
We performed TMHMM-2.0 on mouse 17A2: protein transmembrane helix structure prediction and AlphaFold structure prediction to further clarify whether it has a transmembrane structure and whether it has an ideal extracellular segment.
April 19-May 20 - Preparing antibodies
The specific amino acid sequence was sent to Xi'an Haina Biotechnology Co., Ltd. to prepare Anti-slc17a2 svFV, and the company delivered three strains.
May: Verify the antibody and determine the output
In May, we designed the output component of our SynNotch system and verified the antibodies delivered by the company.
May 3-18 - Determining the output terminal
We searched the literature and visited clinicians, expert professors of molecular genetics and immunology, and determined that the output end was P65 and SIRPαshRNA.
May 21-30 - Verifying the antibody
We performed immunofluorescence staining of Anti-SLC17A2 scFV on mouse hepatoma cell line Hepa1-6 and mouse lung cancer cell line LLC after cell climbing. Screening three Anti-slc17a2 scFV strains delivered by Xi'an Haina Biotechnology Co., Ltd., and selecting high affinity and efficient antibodies.
June and July: Constructed the Syn-M System
In June, we designed and constructed the Syn-M-INPUT Test 1 to determine the input and output, and performed iterative verification.
In July, we designed and constructed the Syn-M-INPUT Test 2 to determine the input and output, and performed iterative verification.
June 1 -16 - Constructing the antibody SynNotch system
We designed the Syn-M-INPUT Test 1 plasmid system based on specific antibodies and carried out plasmid synthesis and lentivirus packaging by Xi'an Tsingke Biotechnology Co., Ltd.
June 17-31 - Input iteration
The packaged lentivirus was transfected to verify its liver-specific activation ability. In order to improve the effect, we continue to iterate.
July 1-16 - Input iteration
By consulting experts and consulting literature, we plan to replace the Hinge domain of the system from the SynNotch1 extracellular domain sequence to the CD8αhinge extracellular domain sequence and verify it. We designed the Syn-M-INPUT Test 2 plasmid system based on Anti-SLC17A2 scFV to verify the liver-specific activation ability again. Plasmid synthesis and lentivirus packaging were made by Xi'an Tsingke Biotechnology Co., Ltd.
July 17-30 - Input iteration
The packaged lentivirus was transfected and verified, and the Syn-M-INPUT Test 2 system based on Anti-SLC17A2 scFV was co-cultured with mCherry-AML12 cells for further verification. Anti-SLC17A2 scFV was co-cultured with mCherry-AML12 cells. After activation, Syn-m was sorted by magnetic beads and sent to Beijing Biomarker Biotechnology Co., Ltd. for transcriptome sequencing.
August: Construct the Syn-M and dry lab
In August, we construct the final Syn-M plasmid and performed sequencing data analysis and modeling.
August 1-17 - Construct the Syn-M and transcriptome sequencing
We identified the final version of the Syn-M plasmid and the plasmid synthesis and lentivirus packaging were made by Xi'an Tsingke Biotechnology Co., Ltd. The packaged lentivirus was transfected and verified.
In the meanwhile we performed principal component analysis to compare intra-group and inter-group differences between the experimental group and the control group. Then all the related genes were analyzed by mapping. Then the enrichment analysis of the cell pathway was carried out. Further analysis of related factors.
August 18-30 - Analysis of sequencing results and modeling
We analyzed the transcriptome sequencing data to identify key pathways and genes affected by our SynNotch system, providing insights for further optimization.
We simulated the liver microenvironment by modeling and constructed a mobile activation model to study whether Syn-M macrophages can move from the vascular area to the cancer area.
September: functional validation and LNP Construction
We verified its safety through animal experiments and iterated the delivery vector LNP.
September 1-14 - P65 and SIRPα functional verification
We co-cultured Syn-M with AML12 cells at a ratio of 1:2 for 24 hours and then performed immunofluorescence staining of F4/80 and P65. The concentrations of TNAFa, IL-6 and INFNr in the culture medium were detected by ELISA kit. P65 was overexpressed in Syn-M, and QPCR was used to detect downstream related inflammatory factors.
We co-cultured Syn-M with AML12 cells at a ratio of 1:2 for 24 hours and then performed immunofluorescence staining of F4/80 and Sirpα. Syn-M was co-cultured with AML12 for 24 h, and Syn-M was sorted out by magnetic bead sorting. The sorted Syn-M was co-cultured with CSFE-labeled liver cancer cells hepa1-6 and lung cancer cells LLC (co-incubation ratio macrophage to tumor ratio is 1:2). After two hours, the phagocytosis ratio was detected by flow cytometry.
September 15-30 - Designing and constructing LNP
We further iterate, design and construct lipid nanoparticle (LNP) by consulting experts and consulting literature. This work establishes the foundation for future development and potential clinical translation of our SynNotch-engineered macrophage therapy.
iGEM Project Notebook - AFMU China 2025
SynNotch-Engineered Macrophages for Liver Cancer Immunotherapy